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Sample records for archaeal rna polymerase

  1. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten; Dalgaard, Jacob; Lykke-Andersen, Jens; Phan, Hoa T.N.; Trevisanato, Siro; Østergaard, Laust; Larsen, Niels; Leffers, Henrik

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows is a...

  2. Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS

    Science.gov (United States)

    Nagy, Julia; Grohmann, Dina; Cheung, Alan C. M.; Schulz, Sarah; Smollett, Katherine; Werner, Finn; Michaelis, Jens

    2015-01-01

    The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

  3. Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter.

    OpenAIRE

    Wettach, J; Gohl, H P; Tschochner, H; Thomm, M

    1995-01-01

    TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show...

  4. Transcription by Methanothermobacter thermautotrophicus RNA Polymerase In Vitro Releases Archaeal Transcription Factor B but Not TATA-Box Binding Protein from the Template DNA

    OpenAIRE

    Xie, Yunwei; Reeve, John N.

    2004-01-01

    Transcription initiation in Archaea requires the assembly of a preinitiation complex containing the TATA- box binding protein (TBP), transcription factor B (TFB), and RNA polymerase (RNAP). The results reported establish the fate of Methanothermobacter thermautotrophicus TBP and TFB following transcription initiation by M. thermautotrophicus RNAP in vitro. TFB is released after initiation, during extension of the transcript from 4 to 24 nucleotides, but TBP remains bound to the template DNA. ...

  5. Direct Modulation of RNA Polymerase Core Functions by Basal Transcription Factors

    OpenAIRE

    Werner, Finn; Weinzierl, Robert O. J.

    2005-01-01

    Archaeal RNA polymerases (RNAPs) are recruited to promoters through the joint action of three basal transcription factors: TATA-binding protein, TFB (archaeal homolog of TFIIB), and TFE (archaeal homolog of TFIIE). Our results demonstrate several new insights into the mechanisms of TFB and TFE during the transcription cycle. (i) The N-terminal Zn ribbon of TFB displays a surprising degree of redundancy for the recruitment of RNAP during transcription initiation in the archaeal system. (ii) Th...

  6. Methanobacterium thermoautotrophicum RNA Polymerase and Transcription In Vitro

    OpenAIRE

    Darcy, Trevor J.; Hausner, Winfried; Awery, Donald E.; Edwards, Aled M.; Thomm, Michael; Reeve, John N.

    1999-01-01

    RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum ΔH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and com...

  7. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    OpenAIRE

    Tom eKillelea; Celine eRalec; Audrey eBosse; Ghislaine eHenneke

    2014-01-01

    International audience DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased perfor...

  8. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  9. RNA-dependent RNA polymerases of plants

    OpenAIRE

    Fraenkel-Conrat, H

    1983-01-01

    The existence of RNA-dependent RNA polymerases (EC 2.7.7.48) in plants has been definitely proven by their isolation in pure form from cucumber and tobacco in our laboratory and from cowpea at Wageningen. These enzymes are single-chain proteins of 100-130 kilodaltons. They show clear physical and biochemical differences characteristic for a given plant species, even when their amounts in the plants were greatly increased prior to isolation by infection with the same virus. The role of these e...

  10. Ribonucleoproteins in Archaeal Pre-rRNA Processing and Modification

    Directory of Open Access Journals (Sweden)

    W. S. Vincent Yip

    2013-01-01

    Full Text Available Given that ribosomes are one of the most important cellular macromolecular machines, it is not surprising that there is intensive research in ribosome biogenesis. Ribosome biogenesis is a complex process. The maturation of ribosomal RNAs (rRNAs requires not only the precise cleaving and folding of the pre-rRNA but also extensive nucleotide modifications. At the heart of the processing and modifications of pre-rRNAs in Archaea and Eukarya are ribonucleoprotein (RNP machines. They are called small RNPs (sRNPs, in Archaea, and small nucleolar RNPs (snoRNPs, in Eukarya. Studies on ribosome biogenesis originally focused on eukaryotic systems. However, recent studies on archaeal sRNPs have provided important insights into the functions of these RNPs. This paper will introduce archaeal rRNA gene organization and pre-rRNA processing, with a particular focus on the discovery of the archaeal sRNP components, their functions in nucleotide modification, and their structures.

  11. Zinc'ing down RNA polymerase I

    OpenAIRE

    Chanfreau, Guillaume F.

    2013-01-01

    Most RNA polymerases contain zinc, yet the precise function of zinc and its influence of polymerases stability are unknown. A recent study provides evidence that zinc levels control the stability of RNA polymerase I in vivo and that the enzyme might serve as a zinc reservoir for other proteins.

  12. RNA-Based Assessment of Diversity and Composition of Active Archaeal Communities in the German Bight

    Directory of Open Access Journals (Sweden)

    Bernd Wemheuer

    2012-01-01

    Full Text Available Archaea play an important role in various biogeochemical cycles. They are known extremophiles inhabiting environments such as thermal springs or hydrothermal vents. Recent studies have revealed a significant abundance of Archaea in moderate environments, for example, temperate sea water. Nevertheless, the composition and ecosystem function of these marine archaeal communities is largely unknown. To assess diversity and composition of active archaeal communities in the German Bight, seven marine water samples were taken and studied by RNA-based analysis of ribosomal 16S rRNA. For this purpose, total RNA was extracted from the samples and converted to cDNA. Archaeal community structures were investigated by pyrosequencing-based analysis of 16S rRNA amplicons generated from cDNA. To our knowledge, this is the first study combining next-generation sequencing and metatranscriptomics to study archaeal communities in marine habitats. The pyrosequencing-derived dataset comprised 62,045 archaeal 16S rRNA sequences. We identified Halobacteria as the predominant archaeal group across all samples with increased abundance in algal blooms. Thermoplasmatales (Euryarchaeota and the Marine Group I (Thaumarchaeota were identified in minor abundances. It is indicated that archaeal community patterns were influenced by environmental conditions.

  13. Structural Biology of Bacterial RNA Polymerase

    Directory of Open Access Journals (Sweden)

    Katsuhiko S. Murakami

    2015-05-01

    Full Text Available Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477–42485, an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP. In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank, describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  14. The X-ray Crystal Structure of RNA Polymerase from Archaea

    Energy Technology Data Exchange (ETDEWEB)

    Hirata,A.; Klein, B.; Murakami, K.

    2008-01-01

    The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits3, 4. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 Angstroms resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 Angstroms ) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

  15. Structure of human mitochondrial RNA polymerase

    OpenAIRE

    Ringel, Rieke; Sologub, Marina; Morozov, Yaroslav I.; Litonin, Dmitry; Cramer, Patrick; Temiakov, Dmitry

    2011-01-01

    Transcription of the mitochondrial genome is performed by a single-subunit RNA polymerase (mtRNAP) that is distantly related to the RNAP of bacteriophage T7, the pol I family of DNA polymerases, and single-subunit RNAPs from chloroplasts1, 2, 3, 4. Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA5, 6, 7. TFAM is an abundant protein that binds and bends promoter DNA 15–40 base pairs upstream of the transcripti...

  16. Promoter clearance by RNA polymerase II

    OpenAIRE

    Luse, Donal S.

    2012-01-01

    Many changes must occur to the RNA polymerase II (pol II) transcription complex as it makes the transition from initiation into transcript elongation. During this intermediate phase of transcription, contact with initiation factors is lost and stable association with the nascent transcript is established. These changes collectively comprise promoter clearance. Once the transcript elongation complex has reached a point where its properties are indistinguishable from those of complexes with muc...

  17. Intrinsic transcript cleavage activity of RNA polymerase.

    OpenAIRE

    Orlova, M; Newlands, J; Das, A; Goldfarb, A; Borukhov, S

    1995-01-01

    The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage cons...

  18. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D;

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation...... the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that......, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA...

  19. The structure and role of RNA polymerases in Plasmodium.

    Science.gov (United States)

    Bzik, D J

    1991-08-01

    During the past few years the characterization of several Plasmodium falciparum RNA polymerase subunits has revealed potentially significant differences between the corresponding subunits of the host and parasite enzymes(1-3). The largest subunits of P. falciparum RNA polymerase II and III contain enlarged variable domains that separate conserved domains in these subunits. The partially characterized beta and beta '-like subunits of an organellar P. falciparum RNA polymerase also appear to be distinct from the host RNA polymerases. In this review David Bzik discusses the structure and role of RNA polymerases in Plasmodium. PMID:15463499

  20. Baculovirus RNA Polymerase: Activities, Composition, and Evolution

    Institute of Scientific and Technical Information of China (English)

    A.Lorena Passarelli

    2007-01-01

    Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA replication. It is composed of four subunits, making it the simplest multisubunit RNAP known. Two subunits contain motifs found at the catalytic center of other RNAPs and a third has capping enzyme functions. The function of the fourth subunit is not known. Structural studies on this unique RNAP will provide new insights into the functions of this enzyme and the regulation of viral genes and may be instrumental to optimize the baculovirus gene expression system.

  1. TAF1B is a TFIIB-like component of the basal transcription machinery for RNA polymerase I.

    Science.gov (United States)

    Naidu, Srivatsava; Friedrich, J Karsten; Russell, Jackie; Zomerdijk, Joost C B M

    2011-09-16

    Transcription by eukaryotic RNA polymerases (Pols) II and III and archaeal Pol requires structurally related general transcription factors TFIIB, Brf1, and TFB, respectively, which are essential for polymerase recruitment and initiation events. A TFIIB-like protein was not evident in the Pol I basal transcription machinery. We report that TAF1B, a subunit of human Pol I basal transcription factor SL1, is structurally related to TFIIB/TFIIB-like proteins, through predicted amino-terminal zinc ribbon and cyclin-like fold domains. SL1, essential for Pol I recruitment to the ribosomal RNA gene promoter, also has an essential postpolymerase recruitment role, operating through TAF1B. Therefore, a TFIIB-related protein is implicated in preinitiation complex assembly and postpolymerase recruitment events in Pol I transcription, underscoring the parallels between eukaryotic Pol I, II, and III and archaeal transcription machineries. PMID:21921199

  2. Is it easy to stop RNA polymerase?

    Science.gov (United States)

    Artsimovitch, Irina; Vassylyev, Dmitry G

    2006-02-01

    Among transcription factors that bind to bacterial RNA polymerase (RNAP) and modulate its activity, a number of small molecules irreversibly inhibit RNAP thereby causing cell death. To be of clinical significance such inhibitors must (1) inhibit a broad range of bacterial RNAPs but not affect human cells, (2) penetrate bacterial cell walls and (3) circumvent bacterial resistance mechanisms. Rifamycins, the only class of RNAP inhibitors that have found their way into clinical practice, are widely used in the treatment of tuberculosis and leprosy. However, the practical value of this class of antibiotics is limited by a rapid rise in resistant bacterial isolates. In this review we focus on recent advances in studies of prokaryotic transcription that allow a detailed structural and functional characterization of a number of RNAP/rifamycins complexes, thereby opening new opportunities for the design of superior antibacterial agents. PMID:16479153

  3. The polymerase of negative-stranded RNA viruses

    OpenAIRE

    Morin, Benjamin; Kranzusch, Philip J.; Rahmeh, Amal A.; Whelan, Sean P. J.

    2013-01-01

    Negative-sense (NS) RNA viruses deliver into cells a mega-dalton RNA-protein complex competent for transcription. Within this complex, the RNA is protected in a nucleocapsid protein (NP) sheath which the viral polymerase negotiates during RNA synthesis. The NP-RNA templates come as nonsegemented (NNS) or segmented (SNS), necessitating distinct strategies for transcription by their polymerases. Atomic-level understanding of the NP-RNA of both NNS and SNS RNA viruses show that the RNA must be t...

  4. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.C.J.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.Previously, an RNA-dependent RNA polymerase produced upon infection of Vigna unguiculata

  5. Chemical fidelity of an RNA polymerase ribozyme

    DEFF Research Database (Denmark)

    Attwater, J.; Tagami, S.; Kimoto, M.;

    2013-01-01

    strands. Our results indicate specificity mechanisms that are found in functionally analogous forms in natural polymerases. They also reveal a level of chemical fidelity over multiple catalytic steps that is remarkable for a comparatively unoptimized enzyme developed de novo from a random sequence pool....... The convergent evolution of specificity mechanisms in phylogenetically unrelated proteinaceous polymerases and polymerase ribozymes suggests that chemical as well as informational fidelity are emergent properties of polymerase enzymes. © 2013 The Royal Society of Chemistry....

  6. Diversity of putative archaeal RNA viruses in metagenomic datasets of a yellowstone acidic hot spring.

    OpenAIRE

    Hongming WANG; Yu, Yongxin; Liu, Taigang; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2015-01-01

    Two genomic fragments (5,662 and 1,269 nt in size, GenBank accession no. JQ756122 and JQ756123, respectively) of novel, positive-strand RNA viruses that infect archaea were first discovered in an acidic hot spring in Yellowstone National Park (Bolduc et al., 2012). To investigate the diversity of these newly identified putative archaeal RNA viruses, global metagenomic datasets were searched for sequences that were significantly similar to those of the viruses. A total of 3,757 associated read...

  7. RNA polymerase III transcription in cancer: the BRF2 connection

    OpenAIRE

    Schramm Laura; Cabarcas Stephanie

    2011-01-01

    Abstract RNA polymerase (pol) III transcription is responsible for the transcription of small, untranslated RNAs involved in fundamental metabolic processes such mRNA processing (U6 snRNA) and translation (tRNAs). RNA pol III transcription contributes to the regulation of the biosynthetic capacity of a cell and a direct link exists between cancer cell proliferation and deregulation of RNA pol III transcription. Accurate transcription by RNA pol III requires TFIIIB, a known target of regulatio...

  8. Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase.

    OpenAIRE

    Van Der Werf, S.; Bradley, J; Wimmer, E; Studier, F W; Dunn, J J

    1986-01-01

    Plasmids containing the entire cDNA sequence of poliovirus type 1 (Mahoney strain) under control of a promoter for T7 RNA polymerase have been constructed. Purified T7 RNA polymerase efficiently transcribes the entire poliovirus cDNA in either direction to produce full-length poliovirus RNA [(+)RNA] or its complement [(-)RNA]. The (+)RNA produced initially had 60 nucleotides on the 5' side of the poliovirus RNA sequence, including a string of 18 consecutive guanine residues generated in the o...

  9. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity

    Science.gov (United States)

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  10. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

    Science.gov (United States)

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  11. RNA Silencing in Aspergillus nidulans Is Independent of RNA-Dependent RNA Polymerases

    OpenAIRE

    Hammond, T. M.; Keller, N P

    2005-01-01

    The versatility of RNA-dependent RNA polymerases (RDRPs) in eukaryotic gene silencing is perhaps best illustrated in the kingdom Fungi. Biochemical and genetic studies of Schizosaccharomyces pombe and Neurospora crassa show that these types of enzymes are involved in a number of fundamental gene-silencing processes, including heterochromatin regulation and RNA silencing in S. pombe and meiotic silencing and RNA silencing in N. crassa. Here we show that Aspergillus nidulans, another model fung...

  12. Conformational changes in E. coli RNA polymerase during promoter recognition.

    OpenAIRE

    Brodolin, K L; Studitsky, V M; Mirzabekov, A D

    1993-01-01

    We analysed complexes formed during recognition of the lacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solutio...

  13. Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

    International Nuclear Information System (INIS)

    mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in that the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template

  14. RNA aptamers selected against DNA polymerase β inhibit the polymerase activities of DNA polymerases β and κ

    OpenAIRE

    Gening, Leonid V.; Klincheva, Svetlana A.; Reshetnjak, Anastasia; Grollman, Arthur P; Miller, Holly

    2006-01-01

    DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 1012 individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift a...

  15. Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase.

    OpenAIRE

    Adkins, S; Stawicki, S S; Faurote, G; Siegel, R W; Kao, C. C.

    1998-01-01

    The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a hig...

  16. A closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

    OpenAIRE

    Bruenn, J A

    1993-01-01

    Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one o...

  17. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

    Directory of Open Access Journals (Sweden)

    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  18. The archaeal TFIIE homologue facilitates transcription initiation by enhancing TATA-box recognition

    NARCIS (Netherlands)

    Bell, S.D.; Brinkman, A.B.; Oost, van der J.; Jackson, S.P.

    2001-01-01

    Transcription from many archaeal promoters can be reconstituted in vitro using recombinant TATA-box binding protein (TBP) and transcription factor B (TFB)—homologues of eukaryal TBP and TFIIB—together with purified RNA polymerase (RNAP). However, all archaeal genomes sequenced to date reveal the pre

  19. File list: Pol.Bon.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bon.50.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Bone ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Bon.50.RNA_Polymerase_III.AllCell.bed ...

  20. File list: Pol.CDV.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.CDV.20.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Cardiovas...X320034,SRX346170,SRX346169,SRX373605,SRX680476 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.CDV.20.RNA_Polymerase_II.AllCell.bed ...

  1. File list: Pol.Emb.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.10.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Embryo... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.10.RNA_polymerase_III.AllCell.bed ...

  2. File list: Pol.Pup.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Gon.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Oth.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

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  5. File list: Pol.ALL.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Pol.Adl.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

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  7. File list: Pol.ALL.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.ALL.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Bld.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Kid.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Epd.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.Spl.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Unc.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.PSC.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.Unc.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Pol.Bld.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Pol.Pup.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Utr.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Adp.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.PSC.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Lar.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: Pol.Unc.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Unc.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Spl.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.Bon.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Pol.Dig.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.CDV.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.Adp.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Pan.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Gon.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Epd.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.YSt.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Plc.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Plc.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Placen...ta http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Plc.20.RNA_polymerase_III.AllCell.bed ...

  14. File list: Pol.Prs.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Prs.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Prostate...932,SRX020922,SRX022582 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Prs.50.RNA_polymerase_II.AllCell.bed ...

  15. File list: Pol.Neu.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Neu.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Neural ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Neu.05.RNA_Polymerase_III.AllCell.bed ...

  16. File list: Pol.CDV.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.CDV.20.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Cardiov...ascular http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.CDV.20.RNA_Polymerase_III.AllCell.bed ...

  17. File list: Pol.Oth.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Utr.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Emb.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Lng.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Bon.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bon.20.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Bone SRX1...035115,SRX731126 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Bon.20.RNA_Polymerase_II.AllCell.bed ...

  2. File list: Pol.Oth.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Pan.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Pancre...as http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Pan.05.RNA_polymerase_III.AllCell.bed ...

  4. File list: Pol.Adl.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.Neu.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Pol.Lng.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.Utr.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.ALL.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Brs.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Oth.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Gon.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.Oth.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Myo.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.Emb.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.Emb.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Pol.Bon.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Pol.Utr.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Myo.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Myo.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Unc.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Kid.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: Pol.Adl.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Myo.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Utr.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.Neu.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

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  6. File list: Pol.Emb.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.CDV.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.Unc.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Kid.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Unc.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Plc.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.Adp.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Lar.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.Spl.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.Prs.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Pol.Prs.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Pol.Pan.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Oth.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Emb.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Unc.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.ALL.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: Pol.Neu.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Myo.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Epd.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.ALL.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Pol.PSC.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.Plc.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.Emb.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Unc.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Unc.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Emb.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.PSC.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Dig.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.Emb.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.Gon.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Pol.Spl.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Spl.20.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Spleen ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Spl.20.RNA_Polymerase_III.AllCell.bed ...

  17. File list: Pol.Liv.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.PSC.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Epd.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Pan.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Plc.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: Pol.YSt.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Unc.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Lar.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.Unc.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Pol.Bon.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.Oth.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.Unc.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Pan.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.ALL.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Unc.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.Bon.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Oth.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.ALL.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.PSC.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Pol.Dig.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Pol.Unc.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Lar.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Adl.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Kid.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Unc.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: Pol.ALL.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Oth.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Kid.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Kid.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Kidney... SRX016996 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Kid.20.RNA_polymerase_III.AllCell.bed ...

  5. File list: Pol.Emb.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Embryo... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.05.RNA_polymerase_III.AllCell.bed ...

  6. File list: Pol.Neu.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.CDV.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.Myo.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Pol.Unc.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Neu.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Bld.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Pol.Plc.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Pol.Myo.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.Gon.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.Emb.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Pol.Pan.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Pol.ALL.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Emb.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.ALL.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Epd.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Lng.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Lng.20.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Lung SRX... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Lng.20.RNA_polymerase_II.AllCell.bed ...

  2. File list: Pol.Pan.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Pol.Utr.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Pol.Emb.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Embryo... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.20.RNA_polymerase_III.AllCell.bed ...

  5. File list: Pol.Gon.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Gonad ...http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Gon.50.RNA_polymerase_III.AllCell.bed ...

  6. File list: Pol.Epd.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.Pan.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Pancreas... SRX190244 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Pan.05.RNA_polymerase_II.AllCell.bed ...

  8. File list: Pol.YSt.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.YSt.20.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II Yeast... strain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.YSt.20.RNA_polymerase_II.AllCell.bed ...

  9. Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

    Science.gov (United States)

    Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

    2006-08-18

    DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes. PMID:16859707

  10. A cinematographic view of Escherichia coli RNA polymerase translocation.

    OpenAIRE

    Metzger, W.; Schickor, P; Heumann, H

    1989-01-01

    A series of RNA synthesizing transcription complexes, initiated at the T7 A1 promoter and halted at specific base positions ranging from +12 to +40, were analyzed by footprinting techniques; exonuclease III was used to determine the position of the bound RNA polymerase on the DNA and hydroxyl radicals were used to visualize the protein--DNA contact sites within the protected areas. In the binding (open) complex without RNA there are two DNA-domains, differing in their protection pattern. The ...

  11. NEW TARGET FOR INHIBITION OF BACTERIAL RNA POLYMERASE: "SWITCH REGION"

    OpenAIRE

    Srivastava, Aashish; Talaue, Meliza; Liu, Shuang; Degen, David; Ebright, Richard Y.; Sineva, Elena; Chakraborty, Anirban; Druzhinin, Sergey Y.; Chatterjee, Sujoy; Mukhopadhyay, Jayanta; Ebright, Yon W.; Zozula, Alex; Shen, Juan; Sengupta, Sonali; Niedfeldt, Rui Rong

    2011-01-01

    A new drug target-- the "switch region"--has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, c...

  12. The green tea component EGCG inhibits RNA polymerase III transcription

    OpenAIRE

    Jacob, Joby; Cabarcas, Stephanie; Veras, Ingrid; Zaveri, Nurulain; Schramm, Laura

    2007-01-01

    RNA polymerase III (RNA pol III) transcribes many small structural RNA molecules involved in RNA processing and translation, and thus regulates the growth rate of a cell. Accurate initiation by RNA pol III requires the initiation factor TFIIIB. TFIIIB has been demonstrated to be regulated by tumor suppressors, including ARF, p53, RB, and the RB-related pocket proteins, and is a target of the oncogene c-myc and the mitogen-activated protein kinase ERK. EGCG has been demonstrated to inhibit the...

  13. A Cross-chiral RNA Polymerase Ribozyme

    OpenAIRE

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer. 1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition 2 or chiral symmetry was broken through chemical pr...

  14. Stochastic resetting in backtrack recovery by RNA polymerases

    CERN Document Server

    Roldán, Édgar; Sánchez-Taltavull, Daniel; Grill, Stephan W

    2016-01-01

    Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally-inactive backtracked state is determined by a kinetic competition between 1D diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process, and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate o...

  15. Modeling RNA polymerase interaction in mitochondria of chordates

    Directory of Open Access Journals (Sweden)

    Lyubetsky Vassily A

    2012-08-01

    Full Text Available Abstract Background In previous work, we introduced a concept, a mathematical model and its computer realization that describe the interaction between bacterial and phage type RNA polymerases, protein factors, DNA and RNA secondary structures during transcription, including transcription initiation and termination. The model accurately reproduces changes of gene transcription level observed in polymerase sigma-subunit knockout and heat shock experiments in plant plastids. The corresponding computer program and a user guide are available at http://lab6.iitp.ru/en/rivals. Here we apply the model to the analysis of transcription and (partially translation processes in the mitochondria of frog, rat and human. Notably, mitochondria possess only phage-type polymerases. We consider the entire mitochondrial genome so that our model allows RNA polymerases to complete more than one circle on the DNA strand. Results Our model of RNA polymerase interaction during transcription initiation and elongation accurately reproduces experimental data obtained for plastids. Moreover, it also reproduces evidence on bulk RNA concentrations and RNA half-lives in the mitochondria of frog, human with or without the MELAS mutation, and rat with normal (euthyroid or hyposecretion of thyroid hormone (hypothyroid. The transcription characteristics predicted by the model include: (i the fraction of polymerases terminating at a protein-dependent terminator in both directions (the terminator polarization, (ii the binding intensities of the regulatory protein factor (mTERF with the termination site and, (iii the transcription initiation intensities (initiation frequencies of all promoters in all five conditions (frog, healthy human, human with MELAS syndrome, healthy rat, and hypothyroid rat with aberrant mtDNA methylation. Using the model, absolute levels of all gene transcription can be inferred from an arbitrary array of the three transcription characteristics, whereas, for

  16. Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro.

    Science.gov (United States)

    Reichert, Vienna L; Choi, Mehee; Petrillo, Jessica E; Gehrke, Lee

    2007-07-20

    Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals. PMID:17400272

  17. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis.

    Science.gov (United States)

    Te Velthuis, Aartjan J W; Fodor, Ervin

    2016-08-01

    The genomes of influenza viruses consist of multiple segments of single-stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, which form viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets. PMID:27396566

  18. Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

    OpenAIRE

    Černý, Jiří; Černá Bolfíková, Barbora; Valdés, James J.; Grubhoffer, Libor; Růžek, Daniel

    2014-01-01

    Viral RNA dependent polymerases (vRdPs) are present in all RNA viruses; unfortunately, their sequence similarity is too low for phylogenetic studies. Nevertheless, vRdP protein structures are remarkably conserved. In this study, we used the structural similarity of vRdPs to reconstruct their evolutionary history. The major strength of this work is in unifying sequence and structural data into a single quantitative phylogenetic analysis, using powerful a Bayesian approach. The resulting phylog...

  19. Active RNA polymerases: mobile or immobile molecular machines?

    Directory of Open Access Journals (Sweden)

    Argyris Papantonis

    Full Text Available It is widely assumed that active RNA polymerases track along their templates to produce a transcript. We test this using chromosome conformation capture and human genes switched on rapidly and synchronously by tumour necrosis factor alpha (TNFalpha; one is 221 kbp SAMD4A, which a polymerase takes more than 1 h to transcribe. Ten minutes after stimulation, the SAMD4A promoter comes together with other TNFalpha-responsive promoters. Subsequently, these contacts are lost as new downstream ones appear; contacts are invariably between sequences being transcribed. Super-resolution microscopy confirms that nascent transcripts (detected by RNA fluorescence in situ hybridization co-localize at relevant times. Results are consistent with an alternative view of transcription: polymerases fixed in factories reel in their respective templates, so different parts of the templates transiently lie together.

  20. The Functions of RNA-Dependent RNA Polymerases in Arabidopsis

    OpenAIRE

    Willmann, Matthew R.; Endres, Matthew W.; Cook, Rebecca T.; Gregory, Brian D.

    2011-01-01

    One recently identified mechanism that regulates mRNA abundance is RNA silencing, and pioneering work in Arabidopsis thaliana and other genetic model organisms helped define this process. RNA silencing pathways are triggered by either self-complementary fold-back structures or the production of double-stranded RNA (dsRNA) that gives rise to small RNAs (smRNAs) known as microRNAs (miRNAs) or small-interfering RNAs (siRNAs). These smRNAs direct sequence-specific regulation of various gene trans...

  1. Transcriptional sequencing: A method for DNA sequencing using RNA polymerase

    OpenAIRE

    Sasaki, Nobuya; Izawa, Masaki; Watahiki, Masanori; Ozawa, Kaori; Tanaka, Takumi; Yoneda, Yuko; Matsuura, Shuji; Carninci, Piero; Muramatsu, Masami; Okazaki, Yasushi; Hayashizaki, Yoshihide

    1998-01-01

    We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3′-deoxynucleoside triphosphate (3′-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in

  2. The mechanism of pRNA-mediated release of RNA polymerase from Bacillus subtilis 6S-1 RNA

    OpenAIRE

    Beckmann, Benedikt

    2010-01-01

    Adaptation of the transcriptome to nutrient limitation and resupply is a fundamental process in bacteria, particularly in natural habitats. Bacterial 6S RNA, an ubiquitous and growth phasedependent regulator of transcription, binds to RNA polymerase (RNAP) and inhibits transcription during stationary growth. Upon nutrient resupply, RNAP acts as an RNA-dependent RNA polymerase by transcribing large amounts of short RNAs (pRNAs) fro...

  3. RNA Polymerase V transcription guides ARGONAUTE4 to chromatin

    OpenAIRE

    Wierzbicki, Andrzej T.; Ream, Thomas; Haag, Jeremy R.; Pikaard, Craig S.

    2009-01-01

    Summary Retrotransposons and repetitive DNA elements in eukaryotes are silenced by small RNA-directed heterochromatin formation. In Arabidopsis, this process involves 24 nt siRNAs that bind to ARGONAUTE4 (AGO4) and facilitate the targeting of complementary loci1,2 via unknown mechanisms. Nuclear RNA Polymerase V is an RNA silencing enzyme recently shown to generate noncoding transcripts at loci silenced by 24nt siRNAs3. We show that AGO4 physically interacts with these Pol V transcripts and i...

  4. RNA polymerase of the killer virus of yeast

    International Nuclear Information System (INIS)

    The L/sub A/ and M double-stranded (ds) RNA segments of the cytoplasmically inherited killer virus of Saccharomyces cerevisiae are encapsidated in virions that contain a DNA-independent transcriptase activity. This enzyme catalyzes the synthesis of full-length (+) stranded copies of the genomic dsRNA segments, denoted l/sub A/ and m. The L/sub A/ dsRNA segment appears to encode the major capsid protein in which both dsRNA molecules are encapsidated, while M dsRNA encodes products responsible for the two killer phenotypes of toxin production and resistance to toxin. Proteins extracted from transcriptionally active virions fail to cross-react with antibody to yeast DNA-dependent RNA polymerases, suggesting that none of the subunits of the host cell polymerases are active in viral transcription. Sequence analysis of the in vitro transcripts reveals neither to be 3'-terminally polyadenylated, although m contains an apparent internal polyA-like tract. In the presence of any three ribonucleoside triphosphates (0.5 mM), the fourth ribonucleoside triphosphate shows an optimal rate of incorporation into transcript at a concentration of 20 μM. However, in a 3-hour reaction, the yield of a product RNA increases with the concentration of the limiting ribonucleotide up to 0.5 mM. Gel electrophoresis of the reaction products reveals that increasing the substrate concentration accelerates the appearance of radioactivity in full-length l/sub A/ and m transcripts

  5. In vitro RNA synthesis by infectious pancreatic necrosis virus-associated RNA polymerase.

    Science.gov (United States)

    Mertens, P P; Jamieson, P B; Dobos, P

    1982-03-01

    The presence of an RNA-dependent RNA polymerase was demonstrated in purified infectious pancreatic necrosis virus (IPNV). The enzyme was active in vitro without any pretreatment of the virus. Optimum activity was shown at 30 degrees C, pH 8 and in the presence of 6 mM-magnesium ions. Approx. 50% of the polymerase product remained associated with the dsRNA template of the virions. The remainder was found as extravirion ssRNA broken down to 5S to 7S fragments by virus-associated RNase(s). Although the addition of bentonite considerably reduced the amount of RNA synthesized, it protected the ssRNA product from degradation. This, in turn, permitted the synthesis of small amounts of ssRNA, which when analysed by sucrose gradient centrifugation or polyacrylamide gel electrophoresis behaved identically to the 24S single-stranded virus mRNA produced in infected cells. The virion polymerase was not stimulated by S-adenosyl-L-methionine or the addition of cellular or capped reovirus ssRNA. Several other modifications of the assay system were tried in an attempt to increase 24S RNA synthesis, but with little success. When [3H]uridine-labelled virus was used in the polymerase reaction, some labelled 24S ssRNA was obtained, indicating that in vitro transcription may proceed by a semi-conservative (displacement) mechanism. PMID:6175731

  6. RNA-Dependent RNA Polymerase 6 Is Required for Efficient hpRNA-Induced Gene Silencing in Plants

    OpenAIRE

    Harmoko, Rikno; Fanata, Wahyu Indra Duwi; Yoo, Jae Yong; Ko, Ki Seong; Rim, Yeong Gil; Uddin, Mohammad Nazim; Siswoyo, Tri Agus; Lee, Seung Sik; Kim, Dool Yi; Lee, Sang Yeol; Lee, Kyun Oh

    2013-01-01

    In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back o...

  7. Coupling of RNA Polymerase II Transcription Elongation with Pre-mRNA Splicing.

    Science.gov (United States)

    Saldi, Tassa; Cortazar, Michael A; Sheridan, Ryan M; Bentley, David L

    2016-06-19

    Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II. The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes the current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes. PMID:27107644

  8. Characterization of an RNA-dependent RNA polymerase activity associated with La France isometric virus.

    OpenAIRE

    Goodin, M M; Schlagnhaufer, B; Weir, T; Romaine, C. P.

    1997-01-01

    Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent ...

  9. Nascent transcription affected by RNA polymerase IV in Zea mays.

    Science.gov (United States)

    Erhard, Karl F; Talbot, Joy-El R B; Deans, Natalie C; McClish, Allison E; Hollick, Jay B

    2015-04-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

  10. Gender Specific Differences in RNA Polymerase III Transcription

    Science.gov (United States)

    Diette, N; Koo, J; Cabarcas-Petroski, S; Schramm, L

    2016-01-01

    Background RNA polymerase (pol) III transcribes a variety of untranslated RNAs responsible for regulating cellular growth and is deregulated in a variety of cancers. In this study, we examined gender differences in RNA pol III transcription in vitro and in vivo. Methods Expression levels of U6 snRNA, tMet, and known modulators of RNA pol III transcription were assayed in male and female derived adenocarcinoma (AC) lung cancer cell lines and male and female C57BL/6J mice using real time quantitative PCR. Methylation status of the U6 snRNA promoter was determined for lung and liver tissue isolated from male and female C57BL/6J mice by digesting genomic DNA with methylation sensitive restriction enzymes and digestion profiles were analyzed by qPCR using primers spanning the U6 promoter. Results Here, we demonstrate that RNA pol III transcription is differentially regulated by EGCG in male and female derived AC lung cancer cell lines. Basal RNA pol III transcript levels are significantly different in male and female derived AC lung cancer cell lines. These data prompted an investigation of gender specific differences in RNA pol III transcription in vivo in lung and liver tissue. Herein, we report that U6 snRNA RNA pol III transcription is significantly stimulated in the liver tissue of male C57BL/6J mice. Further, the increase in U6 transcription correlates with a significant inhibition in the expression of p53, a negative regulator of RNA pol III transcription, and demethylation of the U6 promoter in the liver tissue of male C57BL/6J mice. Conclusions To the best of our knowledge, this is the first study demonstrating gender specific differences in RNA pol III transcription both in vivo and in vitro and further highlights the need to include both male and female cell lines and animals in experimental design.

  11. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

    OpenAIRE

    Li, W B; Bzik, D J; Gu, H M; Tanaka, M.; Fox, B.A.; Inselburg, J

    1989-01-01

    We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distin...

  12. Maize RNA polymerase IV defines trans-generational epigenetic variation.

    Science.gov (United States)

    Erhard, Karl F; Parkinson, Susan E; Gross, Stephen M; Barbour, Joy-El R; Lim, Jana P; Hollick, Jay B

    2013-03-01

    The maize (Zea mays) RNA Polymerase IV (Pol IV) largest subunit, RNA Polymerase D1 (RPD1 or NRPD1), is required for facilitating paramutations, restricting expression patterns of genes required for normal development, and generating small interfering RNA (siRNAs). Despite this expanded role for maize Pol IV relative to Arabidopsis thaliana, neither the general characteristics of Pol IV-regulated haplotypes, nor their prevalence, are known. Here, we show that specific haplotypes of the purple plant1 locus, encoding an anthocyanin pigment regulator, acquire and retain an expanded expression domain following transmission from siRNA biogenesis mutants. This conditioned expression pattern is progressively enhanced over generations in Pol IV mutants and then remains heritable after restoration of Pol IV function. This unusual genetic behavior is associated with promoter-proximal transposon fragments but is independent of sequences required for paramutation. These results indicate that trans-generational Pol IV action defines the expression patterns of haplotypes using co-opted transposon-derived sequences as regulatory elements. Our results provide a molecular framework for the concept that induced changes to the heterochromatic component of the genome are coincident with heritable changes in gene regulation. Alterations of this Pol IV-based regulatory system can generate potentially desirable and adaptive traits for selection to act upon. PMID:23512852

  13. RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage

    International Nuclear Information System (INIS)

    The authors have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a hammerhead) to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's). When the transcript can form the hammerhead structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [α-thio]ATP in place of ATP. They have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 ± 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the hammerhead structure does not disrupt the hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed

  14. RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.; Kahn, J.D.; Hearst, J.E. (Univ. of California, Berkeley (USA) Lawrence Berkeley Lab., CA (USA))

    1990-08-28

    The authors have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a hammerhead) to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3{prime}-deoxyNTP's or 3{prime}-O-methylNTP's). When the transcript can form the hammerhead structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using ({alpha}-thio)ATP in place of ATP. They have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 {plus minus} 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the hammerhead structure does not disrupt the hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.

  15. Unusual properties of adenovirus E2E transcription by RNA polymerase III.

    Science.gov (United States)

    Huang, Wenlin; Flint, S J

    2003-04-01

    In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual. PMID:12634361

  16. A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases

    OpenAIRE

    Bruenn, Jeremy A.

    2003-01-01

    A systematic bioinformatic approach to identifying the evolutionarily conserved regions of proteins has verified the universality of a newly described conserved motif in RNA-dependent RNA polymerases (motif F). In combination with structural comparisons, this approach has defined two regions that may be involved in unwinding double-stranded RNA (dsRNA) for transcription. One of these is the N-terminal portion of motif F and the second is a large insertion in motif F present in the RNA-depende...

  17. Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases.

    OpenAIRE

    Bruenn, J A

    1991-01-01

    The sequences of 50 RNA-dependent RNA polymerases (RDRPs) from 43 positive strand and 7 double strand RNA (dsRNA) viruses have been compared. The alignment permitted calculation of distances among the 50 viruses and a resultant dendrogram based on every amino acid, rather than just those amino acids in the conserved motifs. Remarkably, a large subgroup of these viruses, including vertebrate, plant, and insect viruses, forms a single cluster whose only common characteristic is exploitation of ...

  18. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

    Directory of Open Access Journals (Sweden)

    Isabel X. Wang

    2014-03-01

    Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

  19. Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing.

    Science.gov (United States)

    Bremer, H; Ehrenberg, M

    1995-05-17

    A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp-synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis. PMID:7539631

  20. RNA polymerase motors: dwell time distribution, velocity and dynamical phases

    International Nuclear Information System (INIS)

    Polymerization of RNA from a template DNA is carried out by a molecular machine called RNA polymerase (RNAP). It also uses the template as a track on which it moves as a motor, utilizing chemical energy input. The time it spends at each successive monomer of DNA is random; we derive the exact distribution of these 'dwell times' in our model. The inverse of the mean dwell time satisfies a Michaelis–Menten-like equation and is also consistent with a general formula derived earlier by Fisher and Kolomeisky for molecular motors with unbranched mechano-chemical cycles. Often many RNAP motors move simultaneously on the same track. Incorporating the steric interactions among the RNAPs in our model, we also plot the three-dimensional phase diagram of our model for RNAP traffic using an extremum current hypothesis

  1. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination

  2. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Jabafi, Ilham; Selisko, Barbara; Coutard, Bruno; De Palma, Armando M.; Neyts, Johan; Egloff, Marie-Pierre; Grisel, Sacha; Dalle, Karen; Campanacci, Valerie; Spinelli, Silvia; Cambillau, Christian; Canard, Bruno; Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

    2007-06-01

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination.

  3. File list: Pol.NoD.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.05.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.NoD.05.RNA_polymerase_II.AllCell.bed ...

  4. File list: Pol.NoD.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.05.RNA_Polymerase_II.AllCell sacCer3 RNA polymerase RNA Polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.05.RNA_Polymerase_II.AllCell.bed ...

  5. File list: Pol.NoD.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.10.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.10.RNA_polymerase_II.AllCell.bed ...

  6. File list: Pol.NoD.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.50.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III No desc...ription http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.NoD.50.RNA_Polymerase_III.AllCell.bed ...

  7. File list: Pol.NoD.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.10.RNA_Polymerase_II.AllCell sacCer3 RNA polymerase RNA Polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.10.RNA_Polymerase_II.AllCell.bed ...

  8. File list: Pol.NoD.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III No des...cription http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.50.RNA_polymerase_III.AllCell.bed ...

  9. File list: Pol.CeL.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.CeL.50.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Cell line...70,SRX749072,SRX749071,SRX749073,SRX017852,SRX529168 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.CeL.50.RNA_polymerase_II.AllCell.bed ...

  10. File list: Pol.NoD.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III No des...cription http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.20.RNA_polymerase_III.AllCell.bed ...

  11. File list: Pol.NoD.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.20.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.20.RNA_polymerase_II.AllCell.bed ...

  12. File list: Pol.NoD.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.20.RNA_Polymerase_II.AllCell sacCer3 RNA polymerase RNA Polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.20.RNA_Polymerase_II.AllCell.bed ...

  13. File list: Pol.NoD.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II No descr...iption http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.10.RNA_polymerase_II.AllCell.bed ...

  14. File list: Pol.NoD.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III No des...cription http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.05.RNA_polymerase_III.AllCell.bed ...

  15. File list: Pol.NoD.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.50.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.50.RNA_polymerase_II.AllCell.bed ...

  16. File list: Pol.NoD.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.NoD.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III No des...cription http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.10.RNA_polymerase_III.AllCell.bed ...

  17. Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I.

    OpenAIRE

    Bateman, E.; Iida, C T; Kownin, P; Paule, M R

    1985-01-01

    The binding of a species-specific transcription initiation factor (TIF) and purified RNA polymerase I to the promoter region of the 39S ribosomal RNA gene from Acanthamoeba were studied by using DNase I "footprinting." Conditions were chosen such that the footprints obtained could be correlated with the transcriptional activity of the TIF-containing fractions used and that the labeled DNA present would itself serve as a template for transcription. The transcription factor binds upstream from ...

  18. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

    Science.gov (United States)

    Li, W B; Bzik, D J; Gu, H M; Tanaka, M; Fox, B A; Inselburg, J

    1989-12-11

    We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension. PMID:2690004

  19. Functional insights from molecular modeling, docking, and dynamics study of a cypoviral RNA dependent RNA polymerase.

    Science.gov (United States)

    Kundu, Anirban; Dutta, Anirudha; Biswas, Poulomi; Das, Amit Kumar; Ghosh, Ananta Kumar

    2015-09-01

    Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) contains 11 double stranded RNA genome segments and infects tasar silkworm A. mylitta. RNA-dependent RNA polymerase (RdRp) is reported as a key enzyme responsible for propagation of the virus in the host cell but its structure function relationship still remains elusive. Here a computational approach has been taken to compare sequence and secondary structure of AmCPV RdRp with other viral RdRps to identify consensus motifs. Then a reliable pairwise sequence alignment of AmCPV RdRp with its closest sequence structure homologue λ3 RdRp is done to predict three dimensional structure of AmCPV RdRp. After comparing with other structurally known viral RdRps, important sequence and/or structural features involved in substrate entry or binding, polymerase reaction and the product release events have been identified. A conserved RNA pentanucleotide (5'-AGAGC-3') at the 3'-end of virus genome is predicted as cis-acting signal for RNA synthesis and its docking and simulation study along with the model of AmCPV RdRp has allowed to predict mode of template binding by the viral polymerase. It is found that template RNA enters into the catalytic center through nine sequence-independent and two sequence-dependent interactions with the specific amino acid residues. However, number of sequence dependent interactions remains almost same during 10 nano-second simulation time while total number of interactions decreases. Further, docking of N(7)-methyl-GpppG (mRNA cap) on the model as well as prediction of RNA secondary structure has shown the template entry process in the active site. These findings have led to postulate the mechanism of RNA-dependent RNA polymerization process by AmCPV RdRp. To our knowledge, this is the first report to evaluate structure function relationship of a cypoviral RdRp. PMID:26264734

  20. UvrD facilitates DNA repair by pulling RNA polymerase backwards

    OpenAIRE

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2014-01-01

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD a...

  1. Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

    Science.gov (United States)

    Minaker, Sean W.; Filiatrault, Megan C.; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C.

    2013-01-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport. PMID:23267056

  2. Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNAIle2 and ATP

    International Nuclear Information System (INIS)

    A. fulgidus TiaS was cocrystallized with tRNAIle2 and ATP and X-ray diffraction data were collected to 2.9 Å resolution using a synchrotron-radiation source. The cytidine at the first anticodon position of archaeal tRNAIle2, which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm2C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNAIle-agm2C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNAIle2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNAIle2 and ATP by the vapour-diffusion method. The crystals of the TiaS–tRNAIle2–ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3221, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS–tRNAIle2–ATP complex, with a Matthews coefficient of 2.8 Å3 Da−1 and a solvent content of 61%

  3. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

    Directory of Open Access Journals (Sweden)

    Aaron M Collier

    2016-04-01

    Full Text Available During the replication cycle of double-stranded (ds RNA viruses, the viral RNA-dependent RNA polymerase (RdRP replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV. In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1 the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2 the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3 RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4 the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

  4. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

    Science.gov (United States)

    Collier, Aaron M; Lyytinen, Outi L; Guo, Yusong R; Toh, Yukimatsu; Poranen, Minna M; Tao, Yizhi J

    2016-04-01

    During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly. PMID:27078841

  5. Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases.

    Science.gov (United States)

    Qian, Xinlei; Hamid, Fursham M; El Sahili, Abbas; Darwis, Dina Amallia; Wong, Yee Hwa; Bhushan, Shashi; Makeyev, Eugene V; Lescar, Julien

    2016-04-22

    Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr) Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure. PMID:26907693

  6. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts.

    Science.gov (United States)

    Turowski, Tomasz W; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-07-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5' peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3'-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3'-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5'-exonuclease Rat1. PMID:27206856

  7. Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B

    Directory of Open Access Journals (Sweden)

    Megan H. Powdrill

    2010-09-01

    Full Text Available More than 20 years after the identification of the hepatitis C virus (HCV as a novel human pathogen, the only approved treatment remains a combination of pegylated interferon-α and ribavirin. This rather non-specific therapy is associated with severe side effects and by far not everyone benefits from treatment. Recently, progress has been made in the development of specifically targeted antiviral therapy for HCV (STAT-C. A major target for such direct acting antivirals (DAAs is the HCV RNA-dependent RNA polymerase or non-structural protein 5B (NS5B, which is essential for viral replication. This review will examine the current state of development of inhibitors targeting the polymerase and issues such as the emergence of antiviral resistance during treatment, as well as strategies to address this problem.

  8. Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation.

    Science.gov (United States)

    Shu, Bo; Gong, Peng

    2016-07-12

    Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTP-induced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the template-product duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases. PMID:27339134

  9. Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

    OpenAIRE

    Mejia, Yara X.; Nudler, Evgeny; Bustamante, Carlos,

    2014-01-01

    RNA polymerase is a vital enzyme responsible for the first step in gene expression. Despite extensive studies, fundamental questions about its kinetic and mechanistic properties still remain unanswered. The trigger loop is a conserved domain within RNA polymerase that has been linked to the enzyme’s average elongation velocity and pausing behavior. In this study, we use optical tweezers, a single molecule technique, to analyze the behavior of two mutant polymerases with a single point mutatio...

  10. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    Science.gov (United States)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  11. Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

    Science.gov (United States)

    Pinkney, M; Hoggett, J G

    1988-03-15

    Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase. PMID:2839152

  12. Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors.

    Science.gov (United States)

    Zhang, Pinghu; Liu, Fei; Guo, Fang; Zhao, Qiong; Chang, Jinhong; Guo, Ju-Tao

    2016-07-01

    Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted. PMID:27083116

  13. RNA polymerase pausing regulates translation initiation by providing additional time for TRAP-RNA interaction.

    Science.gov (United States)

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2006-11-17

    RNA polymerase (RNAP) pause sites have been identified in several prokaryotic genes. Although the presumed biological function of RNAP pausing is to allow synchronization of RNAP position with regulatory factor binding and/or RNA folding, a direct causal link between pausing and changes in gene expression has been difficult to establish. RNAP pauses at two sites in the Bacillus subtilis trpEDCFBA operon leader. Pausing at U107 and U144 participates in transcription attenuation and trpE translation control mechanisms, respectively. Substitution of U144 caused a substantial pausing defect in vitro and in vivo. These mutations led to increased trp operon expression that was suppressed by overproduction of TRAP, indicating that pausing at U144 provides additional time for TRAP to bind to the nascent transcript and promote formation of an RNA structure that blocks translation of trpE. These results establish that pausing is capable of playing a role in regulating translation in bacteria. PMID:17114058

  14. Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

    OpenAIRE

    Schultz, M C; Choe, S Y; Reeder, R H

    1991-01-01

    A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, II, and III. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast.

  15. An interaction between gramicidin and the sigma subunit of RNA polymerase.

    OpenAIRE

    Fisher, R.; Blumenthal, T

    1982-01-01

    Gramicidin, a peptide antibiotic produced by Bacillus brevis, inhibits initiation of transcription by RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). We show here that the presence of gramicidin causes an increase in the rate of cleavage of the sigma subunit of Escherichia coli RNA polymerase by trypsin, although it does not alter the cleavage rate of any of the core subunits. Furthermore, whereas isolated sigma is cleaved much faster than is sigma in holoenzym...

  16. Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds.

    OpenAIRE

    Mustaev, A; Zaychikov, E; Severinov, K.; Kashlev, M; Polyakov, A.; Nikiforov, V.; Goldfarb, A

    1994-01-01

    Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent...

  17. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

    Directory of Open Access Journals (Sweden)

    Rodrigo Jácome

    Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

  18. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  19. Bacterial and archaeal communities in Lake Nyos (Cameroon, Central Africa)

    OpenAIRE

    Tiodjio, Rosine E.; Sakatoku, Akihiro; Nakamura, Akihiro; Tanaka, Daisuke; Fantong, Wilson Y.; Tchakam, Kamtchueng B.; Tanyileke, Gregory; Ohba, Takeshi; Hell, Victor J.; Kusakabe, Minoru; Nakamura, Shogo; Ueda, Akira

    2014-01-01

    The aim of this study was to assess the microbial diversity associated with Lake Nyos, a lake with an unusual chemistry in Cameroon. Water samples were collected during the dry season on March 2013. Bacterial and archaeal communities were profiled using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) approach of the 16S rRNA gene. The results indicate a stratification of both communities along the water column. Altogether, the physico-chemical data and microbial s...

  20. Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B.

    Science.gov (United States)

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups. PMID:26485449

  1. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

    Directory of Open Access Journals (Sweden)

    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  2. Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

    Science.gov (United States)

    Kidd, G H; Link, G; Bogorad, L

    1979-10-01

    Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID:16661032

  3. RNA polymerase II/III transcription specificity determined by TATA box orientation.

    OpenAIRE

    Wang, Y.; Stumph, W E

    1995-01-01

    The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and...

  4. Staf, a promiscuous activator for enhanced transcription by RNA polymerases II and III.

    OpenAIRE

    Schaub, M; Myslinski, E; Schuster, C.; Krol, A.; Carbon, P

    1997-01-01

    Staf is a zinc finger protein that we recently identified as the transcriptional activator of the RNA polymerase III-transcribed selenocysteine tRNA gene. In this work we demonstrate that enhanced transcription of the majority of vertebrate snRNA and snRNA-type genes, transcribed by RNA polymerases II and III, also requires Staf. DNA binding assays and microinjection of mutant genes into Xenopus oocytes showed the presence of Staf-responsive elements in the genes for human U4C, U6, Y4 and 7SK...

  5. RNA polymerase motors on DNA track: effects of traffic congestion on RNA synthesis

    CERN Document Server

    Tripathi, Tripti

    2007-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a ssDNA. In some circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track. We refer to such collective movements of the RNAPs as RNAP traffic because of the similarities between the collective dynamics of the RNAPs on ssDNA track and that of vehicles in highway traffic. In this paper we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the ssDNA track. We also suggest novel experiments for testing o...

  6. Site-directed mutagenesis of the foot-and-mouth disease virus RNA-polymerase gene

    International Nuclear Information System (INIS)

    The foot-and-mouth disease virus RNA-polymerase gene was mutagenised in its active site. Pst I digestion of the polymerase gene (cDNA) generated a 790 bp fragment containing the critical sequence. This fragment was subcloned in M13mp8 for mutagenesis method. The polymerase gene was then reconstructed and subcloned in pUC19. These mutants will be used to study the enzyme structure and activity and to develop intracellular immunization assays in eukaryotic cells. (author)

  7. Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions

    OpenAIRE

    Hausner, Winfried; Thomm, Michael

    2001-01-01

    Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex s...

  8. relA-dependent RNA polymerase activity in Escherichia coli.

    OpenAIRE

    Ryals, J; Bremer, H

    1982-01-01

    Parameters relating to RNA synthesis were measured after a temperature shift from 30 to 42 degrees C, in a relA+ and relA- isogenic pair of Escherichia coli strains containing a temperature-sensitive valyl tRNA synthetase. The following results were obtained: (i) the rRNA chain growth rate increased 2-fold in both strains; (ii) newly synthesized rRNA became unstable in both strains; (iii) the stable RNA gene activity (rRNA and tRNA, measured as stable RNA synthesis rate relative to the total ...

  9. RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification.

    Directory of Open Access Journals (Sweden)

    Aneeshkumar G Arimbasseri

    2015-12-01

    Full Text Available Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(22G26 modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(22G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(22G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(22G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(22G26 modification and that this response is conserved among highly divergent yeasts and human cells.

  10. Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn2+ concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp

  11. Foot-and-mouth disease virus-induced RNA polymerase is associated with Golgi apparatus.

    OpenAIRE

    Polatnick, J; Wool, S H

    1985-01-01

    Electrophoretic analysis of the Golgi apparatus isolated by differential centrifugation from radiolabeled cells infected with foot-and-mouth disease virus showed about 10 protein bands. The virus-induced RNA polymerase was identified by immunoprecipitation and electron microscope staining procedures. Pulse-chase experiments indicated that the polymerase passed through the Golgi apparatus in less than 1 h.

  12. RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II

    Science.gov (United States)

    Martínez-Alonso, Mónica; Hengrung, Narin

    2016-01-01

    ABSTRACT Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation. IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for

  13. Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity

    OpenAIRE

    Charpentier, Emmanuelle; Richter, Hagen; van der Oost, John; White, Malcolm F

    2015-01-01

    CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-as...

  14. The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases.

    OpenAIRE

    Ferrero, Diego S.; Mònica Buxaderas; Rodríguez, José F.; Núria Verdaguer

    2015-01-01

    Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded...

  15. Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.

    OpenAIRE

    Learned, R M; Cordes, S; Tjian, R

    1985-01-01

    A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have...

  16. Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate.

    Science.gov (United States)

    Dreyer, C; Hausen, P

    1978-01-01

    DRB triphosphate inhibits activity of isolated RNA polymerase B, and, to a lesser extent, that of polymerase A. The same holds true for transcription in isolated nuclei. It does not act as an initiation inhibitor. In all cases, high concentrations of DRB triphosphate are required. Cells do not phosphorylate DRB to a measurable extent. hn RNA resistant to DRB is initiated with both ATP and GTP in the presence of the drug. These experiments render the hypothesis unlikely that DRB triphosphate in the cell specifically interferes with the initiation reaction of polymerase B. PMID:704359

  17. A reevaluation of the higher taxonomy of viruses based on RNA polymerases.

    OpenAIRE

    Zanotto, P M; Gibbs, M. J.; Gould, E. A.; Holmes, E.C.

    1996-01-01

    In order to assess the validity of classifications of RNA viruses, published alignments and phylogenies of RNA-dependent RNA and DNA polymerase sequences were reevaluated by a Monte Carlo randomization procedure, bootstrap resampling, and phylogenetic signal analysis. Although clear relationships between some viral taxa were identified, overall the sequence similarities and phylogenetic signals were insufficient to support many of the proposed evolutionary groupings of RNA viruses. Likewise, ...

  18. Structure-function studies of the influenza virus RNA polymerase PA subunit

    Institute of Scientific and Technical Information of China (English)

    Mark; BARTLAM

    2009-01-01

    The influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex (PA, PB1 and PB2) with multiple enzymatic activities for catalyzing viral RNA transcription and replication. The roles of PB1 and PB2 have been clearly defined, but PA is less well understood. The critical role of the polymerase complex in the influenza virus life cycle and high sequence conservation suggest it should be a major target for therapeutic intervention. However, until very recently, functional studies and drug discovery targeting the influenza polymerase have been hampered by the lack of three-dimensional structural information. We will review the recent progress in the structure and function of the PA subunit of influenza polymerase, and discuss prospects for the development of anti-influenza therapeutics based on available structures.

  19. Structure-function studies of the influenza virus RNA polymerase PA subunit

    Institute of Scientific and Technical Information of China (English)

    LIU YingFang; LOU ZhiYong; Mark BARTLAM; RAO ZiHe

    2009-01-01

    The influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex (PA, PB1 and PB2) with multiple enzymatic activities for catalyzing viral RNA transcription and replication. The roles of PB1 and PB2 have been clearly defined, but PA is less well understood. The critical role of the poly-merase complex in the influenza virus life cycle and high sequence conservation suggest it should be a major target for therapeutic intervention. However, until very recently, functional studies and drug discovery targeting the influenza polymerase have been hampered by the lack of three-dimensional structural information. We will review the recent progress in the structure and function of the PA sub-unit of influenza polymerase, and discuss prospects for the development of anti-influenza therapeutics based on available structures.

  20. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

    Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

  1. Transcription of the major neurospora crassa microRNA-like small RNAs relies on RNA polymerase III.

    Directory of Open Access Journals (Sweden)

    Qiuying Yang

    Full Text Available Most plant and animal microRNAs (miRNAs are transcribed by RNA polymerase II. We previously discovered miRNA-like small RNAs (milRNAs in the filamentous fungus Neurospora crassa and uncovered at least four different pathways for milRNA production. To understand the evolutionary origin of milRNAs, we determined the roles of polymerases II and III (Pol II and Pol III in milRNA transcription. Our results show that Pol III is responsible for the transcription of the major milRNAs produced in this organism. The inhibition of Pol III activity by an inhibitor or by gene silencing abolishes the production of most abundant milRNAs and pri-milRNAs. In addition, Pol III associates with these milRNA producing loci. Even though silencing of Pol II does not affect the synthesis of the most abundant milRNAs, Pol II or both Pol II and Pol III are associated with some milRNA-producing loci, suggesting a regulatory interaction between the two polymerases for some milRNA transcription. Furthermore, we show that one of the Pol III-transcribed milRNAs is derived from a tRNA precursor, and its biogenesis requires RNase Z, which cleaves the tRNA moiety to generate pre-milRNA. Our study identifies the transcriptional machinery responsible for the synthesis of fungal milRNAs and sheds light on the evolutionary origin of eukaryotic small RNAs.

  2. The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases.

    Science.gov (United States)

    Ferrero, Diego S; Buxaderas, Mònica; Rodríguez, José F; Verdaguer, Núria

    2015-12-01

    Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5'-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups. PMID:26625123

  3. The influence of different land uses on the structure of archaeal communities in Amazonian anthrosols based on 16S rRNA and amoA genes.

    Science.gov (United States)

    Taketani, Rodrigo Gouvêa; Tsai, Siu Mui

    2010-05-01

    Soil from the Amazonian region is usually regarded as unsuitable for agriculture because of its low organic matter content and low pH; however, this region also contains extremely rich soil, the Terra Preta Anthrosol. A diverse archaeal community usually inhabits acidic soils, such as those found in the Amazon. Therefore, we hypothesized that this community should be sensitive to changes in the environment. Here, the archaeal community composition of Terra Preta and adjacent soil was examined in four different sites in the Brazilian Amazon under different anthropic activities. The canonical correspondence analysis of terminal restriction fragment length polymorphisms has shown that the archaeal community structure was mostly influenced by soil attributes that differentiate the Terra Preta from the adjacent soil (i.e., pH, sulfur, and organic matter). Archaeal 16S rRNA gene clone libraries indicated that the two most abundant genera in both soils were Candidatus nitrosphaera and Canditatus nitrosocaldus. An ammonia monoxygenase gene (amoA) clone library analysis indicated that, within each site, there was no significant difference between the clone libraries of Terra Preta and adjacent soils. However, these clone libraries indicated there were significant differences between sites. Quantitative PCR has shown that Terra Preta soils subjected to agriculture displayed a higher number of amoA gene copy numbers than in adjacent soils. On the other hand, soils that were not subjected to agriculture did not display significant differences on amoA gene copy numbers between Terra Preta and adjacent soils. Taken together, our findings indicate that the overall archaeal community structure in these Amazonian soils is determined by the soil type and the current land use. PMID:20204349

  4. Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity

    NARCIS (Netherlands)

    Charpentier, Emmanuelle; Richter, Hagen; Oost, van der John; White, Malcolm F.

    2015-01-01

    CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences

  5. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    OpenAIRE

    Ferrero, Diego; Buxaderas, Mònica; Rodríguez, José F.; Verdaguer, Núria

    2012-01-01

    Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a...

  6. Evolution of plant phage-type RNA polymerases: the genome of the basal angiosperm Nuphar advena encodes two mitochondrial and one plastid phage-type RNA polymerases

    Directory of Open Access Journals (Sweden)

    Börner Thomas

    2010-12-01

    Full Text Available Abstract Background In mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP available, we searched for the respective genes in the basal angiosperm Nuphar advena. Results By screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp gene, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage

  7. The delta subunit of RNA polymerase from Bacillus subtilis

    Czech Academy of Sciences Publication Activity Database

    Švenková, Alžběta; Šanderová, Hana; Motáčková, V.; Žídek, L.; Krásný, Libor

    Brno, 2009. Roč. 7, - (2009), s. 9-9. ISSN 1214-8598. [RNA Club, 7. ročník. 30.10.2009-30.10.2009, Brno] R&D Projects: GA ČR GA204/09/0583 Institutional research plan: CEZ:AV0Z50200510 Keywords : rna Subject RIV: EE - Microbiology, Virology

  8. New pseudodimeric aurones as palm pocket inhibitors of Hepatitis C virus RNA-dependent RNA polymerase.

    Science.gov (United States)

    Meguellati, Amel; Ahmed-Belkacem, Abdelhakim; Nurisso, Alessandra; Yi, Wei; Brillet, Rozenn; Berqouch, Nawel; Chavoutier, Laura; Fortuné, Antoine; Pawlotsky, Jean-Michel; Boumendjel, Ahcène; Peuchmaur, Marine

    2016-06-10

    The NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme for Hepatitis C Virus (HCV) replication. In addition to the catalytic site, this enzyme is characterized by the presence of at least four allosteric pockets making it an interesting target for development of inhibitors as potential anti-HCV drugs. Based on a previous study showing the potential of the naturally occurring aurones as inhibitors of NS5B, we pursued our efforts to focus on pseudodimeric aurones that have never been investigated so far. Hence, 14 original compounds characterized by the presence of a spacer between the benzofuranone moieties were synthesized and investigated as HCV RdRp inhibitors by means of an in vitro assay. The most active inhibitor, pseudodimeric aurone 4, induced high inhibition activity (IC50 = 1.3 μM). Mutagenic and molecular modeling studies reveal that the binding site for the most active derivatives probably is the palm pocket I instead of the thumb pocket I as for the monomeric derivatives. PMID:27017550

  9. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

    Directory of Open Access Journals (Sweden)

    Xinran Liu

    2015-10-01

    Full Text Available The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I in the RNA-dependent RNA polymerase (RdRp. We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

  10. An RNA-dependent RNA polymerase gene in bat genomes derived from an ancient negative-strand RNA virus.

    Science.gov (United States)

    Horie, Masayuki; Kobayashi, Yuki; Honda, Tomoyuki; Fujino, Kan; Akasaka, Takumi; Kohl, Claudia; Wibbelt, Gudrun; Mühldorfer, Kristin; Kurth, Andreas; Müller, Marcel A; Corman, Victor M; Gillich, Nadine; Suzuki, Yoshiyuki; Schwemmle, Martin; Tomonaga, Keizo

    2016-01-01

    Endogenous bornavirus-like L (EBLL) elements are inheritable sequences derived from ancient bornavirus L genes that encode a viral RNA-dependent RNA polymerase (RdRp) in many eukaryotic genomes. Here, we demonstrate that bats of the genus Eptesicus have preserved for more than 11.8 million years an EBLL element named eEBLL-1, which has an intact open reading frame of 1,718 codons. The eEBLL-1 coding sequence revealed that functional motifs essential for mononegaviral RdRp activity are well conserved in the EBLL-1 genes. Genetic analyses showed that natural selection operated on eEBLL-1 during the evolution of Eptesicus. Notably, we detected efficient transcription of eEBLL-1 in tissues from Eptesicus bats. To the best of our knowledge, this study is the first report showing that the eukaryotic genome has gained a riboviral polymerase gene from an ancient virus that has the potential to encode a functional RdRp. PMID:27174689

  11. A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II

    OpenAIRE

    Pellizzoni, Livio; Charroux, Bernard; Rappsilber, Juri; Mann, Matthias; Dreyfuss, Gideon

    2001-01-01

    The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex–associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interacti...

  12. Close association of RNA polymerase II and many transcription factors with Pol III genes.

    Science.gov (United States)

    Raha, Debasish; Wang, Zhong; Moqtaderi, Zarmik; Wu, Linfeng; Zhong, Guoneng; Gerstein, Mark; Struhl, Kevin; Snyder, Michael

    2010-02-23

    Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using alpha-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression. PMID:20139302

  13. UvrD facilitates DNA repair by pulling RNA polymerase backwards.

    Science.gov (United States)

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2014-01-16

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes. PMID:24402227

  14. Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

    Directory of Open Access Journals (Sweden)

    Stefanie A Krumm

    Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

  15. NusG Is a Sequence-specific RNA Polymerase Pause Factor That Binds to the Non-template DNA within the Paused Transcription Bubble.

    Science.gov (United States)

    Yakhnin, Alexander V; Murakami, Katsuhiko S; Babitzke, Paul

    2016-03-01

    NusG, referred to as Spt5 in archaeal and eukaryotic organisms, is the only transcription factor conserved in all three domains of life. This general transcription elongation factor binds to RNA polymerase (RNAP) soon after transcription initiation and dissociation of the RNA polymerase σ factor. Escherichia coli NusG increases transcription processivity by suppressing RNAP pausing, whereas Bacillus subtilis NusG dramatically stimulates pausing at two sites in the untranslated leader of the trpEDCFBA operon. These two regulatory pause sites participate in transcription attenuation and translational control mechanisms, respectively. Here we report that B. subtilis NusG makes sequence-specific contacts with a T-rich sequence in the non-template DNA (ntDNA) strand within the paused transcription bubble. NusG protects T residues of the recognition sequence from permanganate oxidation, and these T residues increase the affinity of NusG to the elongation complex. Binding of NusG to RNAP does not require interaction with RNA. These results indicate that bound NusG prevents forward movement of RNA polymerase by simultaneously contacting RNAP and the ntDNA strand. Mutational studies indicate that amino acid residues of two short regions within the NusG N-terminal domain are primarily responsible for recognition of the trp operon pause signals. Structural modeling indicates that these two regions are adjacent to each another in the protein. We propose that recognition of specific sequences in the ntDNA and stimulation of RNAP pausing is a conserved function of NusG-like transcription factors. PMID:26742846

  16. Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase

    OpenAIRE

    Teng, Chang Ying; Dang, Yunkun; Danne, Jillian C.; Waller, Ross F.; Green, Beverley R.

    2013-01-01

    Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) ...

  17. Evidence that Transcript Cleavage Is Essential for RNA Polymerase II Transcription and Cell Viability

    OpenAIRE

    Sigurdsson, Stefan; Dirac-Svejstrup, A. Barbara; Svejstrup, Jesper Q.

    2010-01-01

    Summary During transcript elongation in vitro, backtracking of RNA polymerase II (RNAPII) is a frequent occurrence that can lead to transcriptional arrest. The polymerase active site can cleave the transcript during such backtracking, allowing transcription to resume. Transcript cleavage is either stimulated by elongation factor TFIIS or occurs much more slowly in its absence. However, whether backtracking actually occurs in vivo, and whether transcript cleavage is important to escape it, has...

  18. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference

    DEFF Research Database (Denmark)

    Peng, Wenfang; Feng, Mingxia; Feng, Xu;

    2015-01-01

    CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids...... carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis....... islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA....

  19. Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide beta,gamma hydrolysis nor protein phosphorylation.

    OpenAIRE

    Lofquist, A K; Li, H; Imboden, M A; Paule, M. R.

    1993-01-01

    With some bacterial RNA polymerases and in eukaryotic RNA polymerase II, DNA melting during initiation requires the coupling of energy derived from beta,gamma hydrolysis of ATP. A detailed analysis of this possible requirement for eukaryotic RNA polymerase I reveals no such requirement. However, in some cases, beta,gamma non-hydrolyzable derivatives (beta,gamma imido or methylene) of nucleotide substrates have been found to significantly inhibit transcription initiation because of their ineff...

  20. Different human TFIIIB activities direct RNA polymerase III transcription from TATA-containing and TATA-less promoters

    OpenAIRE

    Schramm, Laura; Pendergrast, P. Shannon; Sun, Yuling; Hernandez, Nouria

    2000-01-01

    Transcription initiation at RNA polymerase III promoters requires transcription factor IIIB (TFIIIB), an activity that binds to RNA polymerase III promoters, generally through protein–protein contacts with DNA binding factors, and directly recruits RNA polymerase III. Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide β″. Although human homologs for two of the TFIIIB subunits, the TATA box–binding prot...

  1. A Model of Sequence Dependent Rna-Polymerase Diffusion Along Dna

    CERN Document Server

    Barbi, M; Popkov, V; Salerno, M; Barbi, Maria; Place, Christophe; Popkov, Vladislav; Salerno, Mario

    2001-01-01

    We introduce a probabilistic model for the RNA-polymerase sliding motion along DNA during the promoter search. The model accounts for possible effects due to sequence-dependent interactions between the nonspecific DNA and the enzyme. We focus on T7 RNA-polymerase and exploit the available information about its interaction at the promoter site in order to investigate the influence of bacteriophage T7 DNA sequence on the dynamics of the sliding process. Hydrogen bonds in the major groove are used as the main sequence-dependent interaction between the RNA-polymerase and the DNA. The resulting dynamical properties and the possibility of an experimental validation are discussed in details. We show that, while at large times the process reaches a pure diffusive regime, it initially displays a sub-diffusive behavior. The crossover from anomalous to normal diffusion may occur at times large enough to be of biological interest.

  2. Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.

    Directory of Open Access Journals (Sweden)

    Shilpa Aggarwal

    Full Text Available BACKGROUND: It is widely accepted that the highly error prone replication process of influenza A virus (IAV, together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol, is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized. PRINCIPAL FINDINGS: Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT of human immunodeficiency virus (HIV-1 and murine leukemia virus (MuLV, which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg(++ with Mn(++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn(++. Finally, when the IAV nucleoprotein (NP was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity. SIGNIFICANCE: Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be

  3. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  4. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Tan, B H; Fu, J; Sugrue, R J; Yap, E H; Chan, Y C; Tan, Y H

    1996-02-15

    The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA. PMID:8607261

  5. The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome

    International Nuclear Information System (INIS)

    The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem-loop domains of both polarities of HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA

  6. Promoter and nonspecific DNA binding by the T7 RNA polymerase.

    OpenAIRE

    Smeekens, S.P.; Romano, L J

    1986-01-01

    T7 RNA polymerase plays an important role in both the transcription and replication of bacteriophage T7. In this study we have used a nitrocellulose filter binding assay to examine the binding properties of the T7 RNA polymerase with T7 promoters cloned into plasmid DNAs. Promoter-specific binding was shown to be relatively insensitive to variations in the ionic strength of the incubation solution but dependent on the helical structure of the DNA. On the other hand, nonpromoter interior-site ...

  7. The MTE, a new core promoter element for transcription by RNA polymerase II

    OpenAIRE

    LIM, CHIN YAN; Santoso, Buyung; Boulay, Thomas; Dong, Emily; Ohler, Uwe; Kadonaga, James T.

    2004-01-01

    The core promoter is the ultimate target of the vast network of regulatory factors that contribute to the initiation of transcription by RNA polymerase II. Here we describe the MTE (motif ten element), a new core promoter element that appears to be conserved from Drosophila to humans. The MTE promotes transcription by RNA polymerase II when it is located precisely at positions +18 to +27 relative to A+1 in the initiator (Inr) element. MTE sequences from +18 to +22 relative to A+1 are importan...

  8. Transmissible Gastroenteritis Coronavirus RNA-Dependent RNA Polymerase and Nonstructural Proteins 2, 3, and 8 Are Incorporated into Viral Particles

    OpenAIRE

    Nogales, Aitor; Márquez-Jurado, Silvia; Galán, Carmen; Enjuanes, Luis; Almazán, Fernando

    2012-01-01

    Coronavirus replication and transcription are processes mediated by a protein complex, with the RNA-dependent RNA polymerase (RdRp) as a main component. Proteomic analysis of highly purified transmissible gastroenteritis virus showed the RdRp to be a component of the viral particles. This finding was confirmed by Western blotting, immunofluorescence, and immunoelectron microscopy analyses. Interestingly, the replicase nonstructural proteins 2, 3, and 8 colocalized with the RdRp in the viral f...

  9. The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases.

    Directory of Open Access Journals (Sweden)

    Diego S Ferrero

    2015-12-01

    Full Text Available Thosea asigna virus (TaV, an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5'-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups.

  10. The juxtamembrane sequence of the Hepatitis C virus polymerase can affect RNA synthesis and inhibition by allosteric polymerase inhibitors.

    Science.gov (United States)

    Wen, Y; Lin, X; Fan, B; Ranjith-Kumar, C T; Kao, C C

    2015-08-01

    The Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), nonstructural protein 5B (NS5B), is anchored in the membrane through a C-terminal helix. A sequence of ca. 12 residues that connects the catalytically competent portion of the RdRp and the C-terminal helix, the juxtamembrane sequence (JMS), has a poorly defined role in RdRp function in a large part since it is translated from a cis-acting RNA element (CRE) that is essential for HCV replication. Using a HCV replicon that transposed a second copy of CRE to the 3' UTR of the HCV replicon, we demonstrate that amino acid substitutions in the JMS were detrimental for HCV replicon replication. Substitutions in the JMS also resulted in a defect in de novo-initiated RNAs synthesis in vitro and in a cell-based reporter assay. A nonnucleoside inhibitor of the NS5B that binds to the catalytic pocket was less potent in inhibiting NS5B in the presence of JMS mutations. The JMS mutants exhibit reduced stability in thermodenaturation assays, suggesting that the JMS helps confer a more stable conformation to NS5B that could impact RNA synthesis. PMID:25895103

  11. Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

    Czech Academy of Sciences Publication Activity Database

    Černý, Jiří; Černá, B.; Valdés, James J.; Grubhoffer, Libor; Růžek, Daniel

    2014-01-01

    Roč. 9, č. 5 (2014), e96070. E-ISSN 1932-6203 R&D Projects: GA ČR GAP502/11/2116; GA ČR GAP302/12/2490; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Q-BETA replicase * C virus RNA * crystal structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  12. The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

    NARCIS (Netherlands)

    van der Linden, Lonneke; Vives-Adrián, Laia; Selisko, Barbara; Ferrer-Orta, Cristina; Liu, Xinran; Lanke, Kjerstin; Ulferts, Rachel; De Palma, Armando M; Tanchis, Federica; Goris, Nesya; Lefebvre, David; De Clercq, Kris; Leyssen, Pieter; Lacroix, Céline; Pürstinger, Gerhard; Coutard, Bruno; Canard, Bruno; Boehr, David D; Arnold, Jamie J; Cameron, Craig E; Verdaguer, Nuria; Neyts, Johan; van Kuppeveld, Frank J M

    2015-01-01

    The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-b

  13. Purification of RNA Polymerase II General Transcription Factors from Rat Liver

    OpenAIRE

    Conaway, Ronald C.; Reines, Daniel; Garrett, Karla Pfeil; Powell, Wade; Conaway, Joan Weliky

    1996-01-01

    Eukaryotic messenger RNA synthesis is a complex biochemical process requiring the concerted action of multiple “general” transcription factors (TFs) that control the activity of RNA polymerase II at both the initiation1 and elongation2,3 stages of transcription. Because the general transcription factors are present at low levels in mammalian cells, their purification is a formidable undertaking. For this reason we explored the feasibility of using rat liver as a source for purification of the...

  14. RNA Polymerase III in Cajal Bodies and Lampbrush Chromosomes of the Xenopus Oocyte Nucleus

    OpenAIRE

    Murphy, Christine; Wang, Zhengxin; Roeder, Robert G.; Gall, Joseph G.

    2002-01-01

    We used immunofluorescence to study the distribution and targeting of RNA polymerase (pol) III subunits and pol III transcription factors in the Xenopus laevis oocyte nucleus. Antibodies against several of these proteins stained Cajal bodies and ∼90 specific sites on the lampbrush chromosomes. Some of the chromosomal sites had been identified previously by in situ hybridization as the genes for 5S rRNA. The remaining sites presumably encode tRNAs and other pol III transcripts. Pol III sites w...

  15. Enhanced RNA Polymerase III-dependent Transcription Is Required for Oncogenic Transformation*♦

    OpenAIRE

    Johnson, Sandra A. S.; Dubeau, Louis; Johnson, Deborah L.

    2008-01-01

    RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformatio...

  16. BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies.

    OpenAIRE

    Borck, G; Hög, F.; Dentici, M.; Tan, P; Sowada, N.; Medeira, A.; Gueneau, L.; Thiele, H; Kousi, M.; Lepri, F.; Wenzeck, L.; Blumenthal, I; Radicioni, A.; Schwarzenberg, T.; Mandriani, B.

    2015-01-01

    RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well...

  17. Identification of dengue viral RNA-dependent RNA polymerase inhibitor using computational fragment-based approaches and molecular dynamics study.

    Science.gov (United States)

    Anusuya, Shanmugam; Velmurugan, Devadasan; Gromiha, M Michael

    2016-07-01

    Dengue is a major public health concern in tropical and subtropical countries of the world. There are no specific drugs available to treat dengue. Even though several candidates targeted both viral and host proteins to overcome dengue infection, they have not yet entered into the later stages of clinical trials. In order to design a drug for dengue fever, newly emerged fragment-based drug designing technique was applied. RNA-dependent RNA polymerase, which is essential for dengue viral replication is chosen as a drug target for dengue drug discovery. A cascade of methods, fragment screening, fragment growing, and fragment linking revealed the compound [2-(4-carbamoylpiperidin-1-yl)-2-oxoethyl]8-(1,3-benzothiazol-2-yl)naphthalene-1-carboxylate as a potent dengue viral polymerase inhibitor. Both strain energy and binding free energy calculations predicted that this could be a better inhibitor than the existing ones. Molecular dynamics simulation studies showed that the dengue polymerase-lead complex is stable and their interactions are consistent throughout the simulation. The hydrogen-bonded interactions formed by the residues Arg792, Thr794, Ser796, and Asn405 are the primary contributors for the stability and the rigidity of the polymerase-lead complex. This might keep the polymerase in closed conformation and thus inhibits viral replication. Hence, this might be a promising lead molecule for dengue drug designing. Further optimization of this lead molecule would result in a potent drug for dengue. PMID:26262439

  18. Interaction of sigma 70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance.

    Science.gov (United States)

    Ferguson, A L; Hughes, A D; Tufail, U; Baumann, C G; Scott, D J; Hoggett, J G

    2000-09-22

    The interaction between the core form of bacterial RNA polymerases and sigma factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and sigma 70 has been investigated by surface plasmon resonance. The His-tagged form of sigma 70 factor was immobilised on a Ni2+-NTA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9x10(-7) M, and the dissociation rate constant for release of sigma from core, in the absence of DNA or transcription, was 4x10(-3) s(-1), corresponding to a half-life of about 200 s. PMID:11007979

  19. GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein

    DEFF Research Database (Denmark)

    Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara; Mitter, Richard; Svejstrup, Jesper Q

    2011-01-01

    We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent......We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron...... in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support...

  20. epsilon, a New Subunit of RNA Polymerase Found in Gram-Positive Bacteria

    Czech Academy of Sciences Publication Activity Database

    Keller, A. N.; Yang, X.; Wiedermannová, Jana; Delumeau, O.; Krásný, Libor; Lewis, P. J.

    2014-01-01

    Roč. 196, č. 20 (2014), s. 3622-3632. ISSN 0021-9193 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:61388971 Keywords : RNA polymerase * subunit * X-ray crystallography Subject RIV: EE - Microbiology, Virology Impact factor: 2.808, year: 2014

  1. Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lydia J R Hunter

    Full Text Available BACKGROUND: RNA-dependent RNA polymerases (RDRs function in anti-viral silencing in Arabidopsis thaliana and other plants. Salicylic acid (SA, an important defensive signal, increases RDR1 gene expression, suggesting that RDR1 contributes to SA-induced virus resistance. In Nicotiana attenuata RDR1 also regulates plant-insect interactions and is induced by another important signal, jasmonic acid (JA. Despite its importance in defense RDR1 regulation has not been investigated in detail. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis, SA-induced RDR1 expression was dependent on 'NON-EXPRESSER OF PATHOGENESIS-RELATED GENES 1', indicating regulation involves the same mechanism controlling many other SA- defense-related genes, including pathogenesis-related 1 (PR1. Isochorismate synthase 1 (ICS1 is required for SA biosynthesis. In defensive signal transduction RDR1 lies downstream of ICS1. However, supplying exogenous SA to ics1-mutant plants did not induce RDR1 or PR1 expression to the same extent as seen in wild type plants. Analysing ICS1 gene expression using transgenic plants expressing ICS1 promoter:reporter gene (β-glucuronidase constructs and by measuring steady-state ICS1 transcript levels showed that SA positively regulates ICS1. In contrast, ICS2, which is expressed at lower levels than ICS1, is unaffected by SA. The wound-response hormone JA affects expression of Arabidopsis RDR1 but jasmonate-induced expression is independent of CORONATINE-INSENSITIVE 1, which conditions expression of many other JA-responsive genes. Transiently increased RDR1 expression following tobacco mosaic virus inoculation was due to wounding and was not a direct effect of infection. RDR1 gene expression was induced by ethylene and by abscisic acid (an important regulator of drought resistance. However, rdr1-mutant plants showed normal responses to drought. CONCLUSIONS/SIGNIFICANCE: RDR1 is regulated by a much broader range of phytohormones than previously thought

  2. The RNA silencing enzyme RNA polymerase v is required for plant immunity.

    Directory of Open Access Journals (Sweden)

    Ana López

    2011-12-01

    Full Text Available RNA-directed DNA methylation (RdDM is an epigenetic control mechanism driven by small interfering RNAs (siRNAs that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1. NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V, which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA-mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA-mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA-related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence

  3. Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C. (Harvard-Med); (NIH); (CH-Boston)

    2009-04-08

    Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

  4. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    Energy Technology Data Exchange (ETDEWEB)

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  5. Repression of RNA Polymerase I Transcription by the Tumor Suppressor p53

    OpenAIRE

    Zhai, Weiguo; Comai, Lucio

    2000-01-01

    The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. Moreover, it appears that p53 also suppresses RNA Pol I transcription. In this study, we examined the molecular mechanism of Pol I transcriptional inhibition by p53. We show that wild-type, but not mutant, p53 can repress Pol I transcription from a huma...

  6. Promoter Clearance by RNA Polymerase II Is an Extended, Multistep Process Strongly Affected by Sequence

    OpenAIRE

    Pal, Mahadeb; McKean, David; Luse, Donal S.

    2001-01-01

    We have characterized RNA polymerase II complexes halted from +16 to +49 on two templates which differ in the initial 20 nucleotides (nt) of the transcribed region. On a template with a purine-rich initial transcript, most complexes halted between +20 and +32 become arrested and cannot resume RNA synthesis without the SII elongation factor. These arrested complexes all translocate upstream to the same location, such that about 12 to 13 bases of RNA remain in each of the complexes after SII-me...

  7. Phosphorylation of histone H3 serine 28 modulates RNA polymerase III-dependent transcription

    OpenAIRE

    Zhang, Qingsong; Zhong, Qian; Evans, Austin G.; Levy, Daniel; Zhong, Shuping

    2011-01-01

    Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell proliferation, transformation and tumor formation. Phosphorylation of histone H3 (H3ph) is induced by tumor promoters (EGF, UV and TPA) and immediate early genes, such as c-myc, c-jun and c-fos. However, it remains to be determined whether H3ph is involved in RNA Pol III transcription. Here, we report that EGF strongly indu...

  8. Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    OpenAIRE

    Gouge, Jerome; Satia, Karishma; Guthertz, Nicolas; Widya, Marcella; Thompson, Andrew James; Cousin, Pascal; Dergai, Oleksandr; Hernandez, Nouria; Vannini, Alessandro

    2015-01-01

    Summary TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular intera...

  9. Constructing kinetic models to elucidate structural dynamics of a complete RNA polymerase II elongation cycle

    International Nuclear Information System (INIS)

    The RNA polymerase II elongation is central in eukaryotic transcription. Although multiple intermediates of the elongation complex have been identified, the dynamical mechanisms remain elusive or controversial. Here we build a structure-based kinetic model of a full elongation cycle of polymerase II, taking into account transition rates and conformational changes characterized from both single molecule experimental studies and computational simulations at atomistic scale. Our model suggests a force-dependent slow transition detected in the single molecule experiments corresponds to an essential conformational change of a trigger loop (TL) opening prior to the polymerase translocation. The analyses on mutant study of E1103G and on potential sequence effects of the translocation substantiate this proposal. Our model also investigates another slow transition detected in the transcription elongation cycle which is independent of mechanical force. If this force-independent slow transition happens as the TL gradually closes upon NTP binding, the analyses indicate that the binding affinity of NTP to the polymerase has to be sufficiently high. Otherwise, one infers that the slow transition happens pre-catalytically but after the TL closing. Accordingly, accurate determination of intrinsic properties of NTP binding is demanded for an improved characterization of the polymerase elongation. Overall, the study provides a working model of the polymerase II elongation under a generic Brownian ratchet mechanism, with most essential structural transition and functional kinetics elucidated. (paper)

  10. A new yeast poly(A polymerase complex involved in RNA quality control.

    Directory of Open Access Journals (Sweden)

    Stepánka Vanácová

    2005-06-01

    Full Text Available Eukaryotic cells contain several unconventional poly(A polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAiMet. Here we show that Trf4p is the catalytic subunit of a new poly(A polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAiMet with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAiMet by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.

  11. The σ enigma: Bacterial σ factors, archaeal TFB and eukaryotic TFIIB are homologs

    OpenAIRE

    Burton, Samuel P; Burton, Zachary F.

    2014-01-01

    Structural comparisons of initiating RNA polymerase complexes and structure-based amino acid sequence alignments of general transcription initiation factors (eukaryotic TFIIB, archaeal TFB and bacterial σ factors) show that these proteins are homologs. TFIIB and TFB each have two-five-helix cyclin-like repeats (CLRs) that include a C-terminal helix-turn-helix (HTH) motif (CLR/HTH domains). Four homologous HTH motifs are present in bacterial σ factors that are relics of CLR/HTH domains. Sequen...

  12. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    International Nuclear Information System (INIS)

    The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P21212 and C2221 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P21212 and diffracts up to 2.1 Å and the RdRp-Lu3+ derivative co-crystals belong to the C2221 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

  13. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.

    OpenAIRE

    Dvir, A; Peterson, S R; Knuth, M W; Lu, H.; Dynan, W S

    1992-01-01

    The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription ...

  14. “Silencing the ribosomal locus of Saccharomyces cerevisiae: role of RNA polymerase I transcription and chromatin acetylation”

    OpenAIRE

    Cesarini, Elisa

    2011-01-01

    During my PhD I investigated the transcriptional silencing occurring at the ribosomal DNA of Saccharomyces cerevisiae. In yeast the ribosomal locus (rDNA) is transcribed with high efficiency by RNA polymerase I (Pol I) and III to synthetize ribosomal RNAs. It has been discovered that RNA polymerase Pol II (Pol II) can also transcribe the ribosomal locus, at low level, starting from cryptic promoters and generating non coding RNAs (ncRNAs). ncRNA transcription leads to genome...

  15. Quantitation of RNA Polymerase II and Its Transcription Factors in an HeLa Cell: Little Soluble Holoenzyme but Significant Amounts of Polymerases Attached to the Nuclear Substructure

    OpenAIRE

    Kimura, Hiroshi; Tao, Yong; Roeder, Robert G.; Cook, Peter R.

    1999-01-01

    Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; ∼140,000 unengaged polymerases (mainly of form IIA) were released. Only ∼4,000 of these solub...

  16. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    Science.gov (United States)

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  17. Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction.

    Science.gov (United States)

    Tanriverdi, Kahraman; Kucukural, Alper; Mikhalev, Ekaterina; Tanriverdi, Selim E; Lee, Rosalind; Ambros, Victor R; Freedman, Jane E

    2016-05-15

    MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)-qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids. PMID:26969789

  18. Divergent Contributions of Conserved Active Site Residues to Transcription by Eukaryotic RNA Polymerases I and II

    Directory of Open Access Journals (Sweden)

    Olga V. Viktorovskaya

    2013-09-01

    Full Text Available Multisubunit RNA polymerases (msRNAPs exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL in the largest subunit of RNA polymerase I (Pol I yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G. Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.

  19. Purification of Crystallization-Grade RNA Polymerase I from S. cerevisiae.

    Science.gov (United States)

    Engel, Christoph

    2016-01-01

    Purification of RNA polymerase (Pol) I is essential for functional as well as for structural studies. The product needs to be extremely pure in order to exclude secondary effects, e.g., caused by copurified nucleic acids in subsequent experiments. For this purpose, the method presented here was originally introduced nearly a decade ago but underwent constant optimization [1]. The polymerase is extracted from its endogenous source, since no overexpression system for the entire 590 kDa, 14-subunit complex is available thus far. Following yeast cultivation, a number of standard protein purification techniques are applied and combined to a robust but elaborate procedure that takes 3 days. In brief, a yeast strain with histidine-tagged RNA polymerase I is fermented, cells are broken by bead beating, and cell debris is removed by a two-step centrifugation. The lysate is then dialyzed, the Pol-I-containing pellet resuspended, and polymerase I enriched by a His-trap affinity step, followed by sequential purification via anion and cation exchange and a final size exclusion chromatography. PMID:27576712

  20. Effects of substitutions at position 180 in the Escherichia coli RNA polymerase 70 subunit

    Indian Academy of Sciences (India)

    Olga N Koroleva; Stephen Jw Busby; Valeriy L Drutsa

    2011-03-01

    In order to investigate the role of His180 residue, located in the non-conserved region of the 70 subunit of Escherichia coli RNA polymerase, two mutant variants of the protein with substitutions for either alanine or glutamic acid were constructed and purified using the IMPACT system. The ability of mutant 70 subunits to interact with core RNA polymerase was investigated using native gel-electrophoresis. The properties of the corresponding reconstituted holoenzymes, as provided by gel shift analysis of their complexes with single- and double-stranded promoter-like DNA and by in vitro transcription experiments, allowed one to deduce that His180 influences several steps of transcription initiation, including core binding, promoter DNA recognition and open complex formation.

  1. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    OpenAIRE

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-01-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate p...

  2. The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells

    OpenAIRE

    Gaspari, Martina; Falkenberg, Maria; Larsson, Nils-Göran; Gustafsson, Claes M.

    2004-01-01

    Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specifi...

  3. TFB2 is a transient component of the catalytic site of the human mitochondrial RNA polymerase

    OpenAIRE

    Sologub, Marina; Litonin, Dmitry; Anikin, Michael; Mustaev, Arkady; Temiakov, Dmitry

    2009-01-01

    Transcription in human mitochondria is carried out by a single-subunit, T7-like RNA polymerase assisted by several auxiliary factors. We demonstrate that an essential initiation factor, TFB2, forms a network of interactions with DNA near the transcription start site and facilitates promoter melting but may not be essential for promoter recognition. Unexpectedly, catalytic autolabeling reveals that TFB2 interacts with the priming substrate, suggesting that TFB2 acts as a transient component of...

  4. Cyanobacterial community structure as seen from RNA polymerase gene sequence analysis.

    OpenAIRE

    Palenik, B

    1994-01-01

    PCR was used to amplify DNA-dependent RNA polymerase gene sequences specifically from the cyanobacterial population in a seawater sample from the Sargasso Sea. Sequencing and analysis of the cloned fragments suggest that the population in the sample consisted of two distinct clusters of Prochlorococcus-like cyanobacteria and four clusters of Synechococcus-like cyanobacteria. The diversity within these clusters was significantly different, however. Clones within each Synechococcus-like cluster...

  5. Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system

    OpenAIRE

    Polosa, Paola Loguercio; Deceglie, Stefania; Falkenberg, Maria; Roberti, Marina; Di Ponzio, Barbara; Gadaleta, Maria Nicola; Cantatore, Palmiro

    2007-01-01

    Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. T...

  6. Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

    OpenAIRE

    Revyakin, Andrey; Zhang, Zhengjian; Coleman, Robert A.; Li, Yan; Inouye, Carla; Lucas, Julian K.; Park, Sang-Ryul; Chu, Steven; Tjian, Robert

    2012-01-01

    RNA polymerase II (Pol II) transcription is an immensely complex process that involves a myriad of regulatory factors and elements. In a technical tour de force, Tjian and colleagues now define an in vitro reconstituted Pol II system to detect and quantify Pol II transcription at single-molecule resolution using fluorescence video-microscopy. The study provides valuable insight into transcription reinitiation and, significantly, paves the way for a new era of opportunities in investigating th...

  7. Identifizierung des zentralen Polypeptids des generellen RNA-Polymerase III Transkriptionsfaktors TFIIIC1

    OpenAIRE

    Gruber, Christin

    2004-01-01

    Der Transkriptionsfaktor TFIIIC1 wird für die RNA-Synthese aller humanen Polymerase III-abhängigen Gene unabhängig vom jeweiligen Promotortyp essentiell benötigt. Innerhalb des humanen Pol III Systems ist TFIIIC1 der einzige Transkriptionsfaktor dessen Untereinheiten-Zusammensetzung bisher noch immer ungeklärt blieb. Die Hauptaufgabe der vorliegenden Arbeit lag daher in der chromatographischen Reinigung von TFIIIC1-aktiven ...

  8. In vitro analysis of a transcription termination site for RNA polymerase II.

    OpenAIRE

    Wiest, D K; Hawley, D K

    1990-01-01

    Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction cond...

  9. Tailing and 3'-end labeling of RNA with yeast poly(A) polymerase and various nucleotides.

    OpenAIRE

    Martin, G; Keller, W

    1998-01-01

    We have tested conditions for the labeling and tailing the 3'-end of RNAs with yeast poly(A) polymerase. Conditions were optimized for addition of NTP, dNTP, or ddNTP nucleotides to RNA. ATP, GTP, and UTP were useful for adding homopolymer tracts of various lengths. The nonradioactive nucleotides biotin-N6-ATP and digoxigenin-11-UTP also were used efficiently.

  10. Determinants for Dephosphorylation of the RNA Polymerase II C-Terminal Domain by Scp1

    OpenAIRE

    Yan ZHANG; Kim, Youngjun; Genoud, Nicolas; Gao, Jianmin; Kelly, Jeffery W.; Pfaff, Samuel L.; Gill, Gordon N.; Dixon, Jack E.; Noel, Joseph P.

    2006-01-01

    Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+-dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray cry...

  11. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    OpenAIRE

    Noireaux Vincent; Shin Jonghyeon

    2010-01-01

    Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been opt...

  12. CONSERVED FUNCTIONAL DOMAINS OF THE RNA-POLYMERASE-III GENERAL TRANSCRIPTION FACTOR BRF

    OpenAIRE

    Khoo, B; Brophy, B; Jackson, S P

    1994-01-01

    In Saccharomyces cerevisiae, two components of the RNA polymerase III (Pol III) general transcription factor TFIIIB are the TATA-binding protein (TBP) and the B-related factor (BRF), so called because its amino-terminal half is homologous to the Pol II transcription factor IIB (TFIIB). We have cloned BRF genes from the yeasts Kluyveromyces lactis and Candida albicans, Despite the large evolutionary distance between these species and S. cerevisiae, the BRF proteins are conserved highly. Althou...

  13. Transcription-independent role for human mitochondrial RNA polymerase in mitochondrial ribosome biogenesis

    OpenAIRE

    Surovtseva, Yulia V; Shadel, Gerald S.

    2013-01-01

    Human mitochondrial RNA polymerase, POLRMT, is required for mitochondrial DNA (mtDNA) transcription and forms initiation complexes with human mitochondrial transcription factor B2 (h-mtTFB2). However, POLRMT also interacts with the paralogue of h-mtTFB2, h-mtTFB1, which is a 12S ribosomal RNA methyltransferase required for small (28S) mitochondrial ribosome subunit assembly. Herein, we show that POLRMT associates with h-mtTFB1 in 28S mitochondrial ribosome complexes that are stable in the abs...

  14. The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

    OpenAIRE

    Hempel, W M; Cavanaugh, A H; Hannan, R D; Taylor, L.; Rothblum, L I

    1996-01-01

    Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UB...

  15. PTEN Represses RNA Polymerase I Transcription by Disrupting the SL1 Complex†

    OpenAIRE

    Zhang, Cheng; Comai, Lucio; Johnson, Deborah L.

    2005-01-01

    PTEN is a tumor suppressor whose function is frequently lost in human cancer. It possesses a lipid phosphatase activity that represses the activation of PI3 kinase/Akt signaling, leading to decreased cell growth, proliferation, and survival. The potential for PTEN to regulate transcription of the large rRNAs by RNA polymerase I (RNA Pol I) was investigated. As increased synthesis of rRNAs is a hallmark of neoplastic transformation, the ability of PTEN to control the transcription of rRNAs mig...

  16. A novel TBP-associated factor of SL1 functions in RNA polymerase I transcription

    OpenAIRE

    Gorski, Julia J; Pathak, Shalini; Panov, Kostya; Kasciukovic, Taciana; Panova, Tanya; Russell, Jackie; Zomerdijk, Joost C. B. M.

    2007-01-01

    In mammalian RNA polymerase I transcription, SL1, an assembly of TBP and associated factors (TAFs), is essential for preinitiation complex formation at ribosomal RNA gene promoters in vitro. We provide evidence for a novel component of SL1, TAFI41 (MGC5306), which functions in Pol I transcription. TAFI41 resides at the rDNA promoter in the nucleolus and co-purifies and co-immunoprecipitates with SL1. TAFI41 immunodepletion from nuclear extracts dramatically reduces Pol I transcription; additi...

  17. Relationship between RNA polymerase II and efficiency of vaccinia virus replication

    International Nuclear Information System (INIS)

    It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study the authors examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L6H9 rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing cured enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with [35S]methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rate of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, the authors suggest that mobilization of pol II depends on the efficiency of vaccinia virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types

  18. Real-time dynamics of RNA Polymerase II clustering in live human cells

    Science.gov (United States)

    Cisse, Ibrahim

    2014-03-01

    Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

  19. Interacting RNA polymerase motors on DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis

    CERN Document Server

    Tripathi, Tripti

    2007-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two new measures of {\\it fluctuations} in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis dep...

  20. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus.

    Science.gov (United States)

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F; Verdaguer, Núria

    2012-10-01

    Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2(1)2(1)2 and diffracts up to 2.1 Å and the RdRp-Lu(3+) derivative co-crystals belong to the C222(1) space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses. PMID:23027763

  1. Fidelity of a viral RNA polymerase in an intact host: template structure and host factors affect rates of deletion mutations

    Science.gov (United States)

    Plant RNA viruses often have very diverse populations. One of the commonly proposed mechanisms for generating these diverse populations is the error prone RNA-dependent RNA polymerase, based on examples from a number of human and bacterial viruses. This paper describes work to determine the insertio...

  2. Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents

  3. Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

    Directory of Open Access Journals (Sweden)

    Butler Margaret I

    2006-10-01

    Full Text Available Abstract Background Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi. Results We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2 from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2 of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2, one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins. Conclusion The identification of these new inteins

  4. Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter.

    OpenAIRE

    Kassavetis, G A; Bardeleben, C; Kumar, A; Ramirez, E.; Geiduschek, E P

    1997-01-01

    Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-binding protein, the TFIIB-related protein Brf, and B". TFIIIB, which is brought to RNA polymerase III-transcribed genes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by the TATA-binding protein, in turn recruits RNA polymerase III to the promoter. N-terminally deleted derivatives of Brf have been examined for their ability to interact with DNA-bound T...

  5. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding

    Science.gov (United States)

    Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G

    2015-01-01

    Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1. PMID:25654332

  6. Maize RNA Polymerase IV Defines trans-Generational Epigenetic Variation[W

    Science.gov (United States)

    Erhard, Karl F.; Parkinson, Susan E.; Gross, Stephen M.; Barbour, Joy-El R.; Lim, Jana P.; Hollick, Jay B.

    2013-01-01

    The maize (Zea mays) RNA Polymerase IV (Pol IV) largest subunit, RNA Polymerase D1 (RPD1 or NRPD1), is required for facilitating paramutations, restricting expression patterns of genes required for normal development, and generating small interfering RNA (siRNAs). Despite this expanded role for maize Pol IV relative to Arabidopsis thaliana, neither the general characteristics of Pol IV–regulated haplotypes, nor their prevalence, are known. Here, we show that specific haplotypes of the purple plant1 locus, encoding an anthocyanin pigment regulator, acquire and retain an expanded expression domain following transmission from siRNA biogenesis mutants. This conditioned expression pattern is progressively enhanced over generations in Pol IV mutants and then remains heritable after restoration of Pol IV function. This unusual genetic behavior is associated with promoter-proximal transposon fragments but is independent of sequences required for paramutation. These results indicate that trans-generational Pol IV action defines the expression patterns of haplotypes using co-opted transposon-derived sequences as regulatory elements. Our results provide a molecular framework for the concept that induced changes to the heterochromatic component of the genome are coincident with heritable changes in gene regulation. Alterations of this Pol IV–based regulatory system can generate potentially desirable and adaptive traits for selection to act upon. PMID:23512852

  7. Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

    Science.gov (United States)

    Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

    2011-06-01

    A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

  8. [Factors influencing the pulse character of RNA elongation in vitro by E. coli RNA polymerase].

    Science.gov (United States)

    Aivazashvili, V A; Bibilashvili, R Sh; Vartikian, R M; Kutateladze, T V

    1981-01-01

    Pause location along primary structure of two RNA fragments each 200 nucleotide residues in the length synthesized from A1 promoters of T7 phage DNA and delta D111 T7 phage DNA was analyzed. No correlation between the location of pauses and GC-rich or self complementary regions of RNA were found. The location of pauses does not change upon the variation of the temperature or ionic strength. Concurrent variation of all four NTP concentrations also did not influence pausing pattern. However the distribution of pauses depends highly on the ratio of the individual substrate concentrations. Substitution of GTP by ITP changes the pausing pattern completely. Inorganic pyrophosphate (PPi) of inhibits RNA elongation preferentially in the regions: NAUN, CGUAG. The study of PPi action on RNA terminated with 3' OCH3-NMP suggest that the sequence-specific inhibition of RNA elongation may be a result of pyrophosphorolysis of terminal nucleotide residues of RNA. It was proposed that the pulse character of RNA elongation stems rather from differences in the kinetic constants of nucleotides attachment and pyrophosphorolysis from the 3'-termini of RNA than by termination signals encoded in the primary structure of DNA. The stable location of pauses in certain short oligonucleotides: AUG, AUU, AAU and some others is in favour of the hypothesis. PMID:6265762

  9. The structural basis for an essential subunit interaction in influenza virus RNA polymerase

    International Nuclear Information System (INIS)

    Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2, and PA are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The C terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the N-terminal residues of PB1 can fit by forming a 310 helix. (author)

  10. DNA - dependent RNA-polymerase of irradiated animal organs Communication 5. Physico-chemical changes in the enzyme molecules

    International Nuclear Information System (INIS)

    The authors investigate the changes noted in the properties of macromolecules of a DNA-dependent RNA-polymerase from rat liver and spleen 120 hours after irradiation (800 rad). In particular, they find changes in the sedimentation constant, in the electrophoretic mobility of sub-units and in their concentration and also suppression of the fermentative activity of RNA-polymerases, both in DNA isolated after irradiation and in DNA from normal organs. (V.A.P.)

  11. Functional and Structural Organization of Brf, the TFIIB-Related Component of the RNA Polymerase III Transcription Initiation Complex

    OpenAIRE

    Kassavetis, George A.; Kumar, Ashok; Ramirez, Enrique; Geiduschek, E.Peter

    1998-01-01

    Brf is the TFIIB-related component of Saccharomyces cerevisiae RNA polymerase III transcription initiation factor IIIB (TFIIIB). An extensive set of Brf fragments has been examined for the abilities to assemble the TFIIIB-DNA complex and recruit RNA polymerase III to accurately initiate transcription. The principal TFIIIB-assembly function of Brf was found to be contributed by a C-proximal segment spanning amino acids 435 to 545, while the principal transcription-directing function was contri...

  12. Electrostatic map of T7 DNA. Comparative analysis of functional and electrostatic properties of T7 RNA polymerase specific promoters

    OpenAIRE

    Kamzolova, S. G.; Beskaravainy, P. M.; Osypov, A. A.; Dzhelyadin, T. R.; Temlyakova, E. A.; Sorokin, A. A.

    2013-01-01

    The entire T7 bacteriophage genome contains 39937 base pairs (Database NCBI RefSeq N1001604). Here, electrostatic potential distribution around double helical T7 DNA was calculated by Coulomb method using the computer program of Sorokin A.A. Electrostatic profiles of 17 promoters recognized by T7 phage specific RNA polymerase were analyzed. It was shown that electrostatic profiles of all T7 RNA polymerase specific promoters can be characterized by distinctive motifs which are specific for eac...

  13. RNA polymerase motor on DNA track: effects of interactions, external force and torque

    CERN Document Server

    Tripathi, Tripti

    2008-01-01

    RNA polymerase (RNAP) is like a mobile molecular workshop that polymerizes a RNA molecule by adding monomeric subunits one by one, while moving step by step on the DNA template itself. Here we develop a theoretical model by incorporating their steric interactions and mechanochemical cycles which explicitly captures the cyclical shape changes of each motor. Using this model, we explain not only the dependence of the average velocity of a RNAP on the externally applied load force, but also predict a {\\it nonmotonic} variation of the average velocity on external torque. We also show the effect of steric interactions of the motors on the total rate of RNA synthesis. In principle, our predictions can be tested by carrying out {\\it in-vitro} experiments.

  14. Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

    KAUST Repository

    Da, Lin-Tai

    2016-04-19

    The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

  15. Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

    OpenAIRE

    Rodriguez, P L; Carrasco, L.

    1992-01-01

    The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in...

  16. Interplay of positive and negative effectors in function of the C-terminal repeat domain of RNA polymerase II.

    OpenAIRE

    Li, Y.; Kornberg, R D

    1994-01-01

    RNA polymerase II lacking a C-terminal domain (CTD) was active in transcription with purified proteins from yeast but failed to support transcription in a yeast extract. CTD dependence could be reconstituted in the purified system by addition of two fractions from the extract. An inhibitory fraction abolished transcription by both wild-type and CTD-less RNA polymerases; a stimulatory fraction restored activity of the wild-type polymerase but had a much lesser effect on the CTD-less enzyme. Pa...

  17. Structure–function studies of the RNA polymerase II elongation complex

    International Nuclear Information System (INIS)

    X-ray crystallographic and complementary functional studies have contributed significantly to the current understanding of gene transcription. Here, recent structure–function studies on various aspects of the elongation phase of transcription are summarized. RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin α-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template

  18. The JNKs differentially regulate RNA polymerase III transcription by coordinately modulating the expression of all TFIIIB subunits

    OpenAIRE

    Zhong and, Shuping; Johnson, Deborah L.

    2009-01-01

    RNA polymerase (pol) III-dependent transcription is subject to stringent regulation by tumor suppressors and oncogenic proteins and enhanced RNA pol III transcription is essential for cellular transformation and tumorigenesis. Since the c-Jun N-terminal kinases (JNKs) display both oncogenic and tumor suppressor properties, the roles of these proteins in regulating RNA pol III transcription were examined. In both mouse and human cells, loss or reduction in JNK1 expression represses RNA pol III...

  19. Functional Diversification of Maize RNA Polymerase IV and V Subtypes via Alternative Catalytic Subunits

    Directory of Open Access Journals (Sweden)

    Jeremy R. Haag

    2014-10-01

    Full Text Available Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two subtypes of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  20. The N-terminus of the RNA polymerase from infectious pancreatic necrosis virus is the determinant of genome attachment.

    Directory of Open Access Journals (Sweden)

    Stephen C Graham

    2011-06-01

    Full Text Available The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxynucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.

  1. Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II

    International Nuclear Information System (INIS)

    Eukaryotic RNA polymerase II consists of three subspecies, designated II0, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with γ[32P]-ATP in the presence of casein kinase I and 32P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of 32P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription

  2. Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II

    Energy Technology Data Exchange (ETDEWEB)

    Cadena, D.; Dahmus, M.E.

    1986-05-01

    Eukaryotic RNA polymerase II consists of three subspecies, designated II/sub 0/, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with ..gamma..(/sup 32/P)-ATP in the presence of casein kinase I and /sup 32/P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of /sup 32/P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription.

  3. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  4. Ancient Origin and Recent Innovations of RNA Polymerase IV and V.

    Science.gov (United States)

    Huang, Yi; Kendall, Timmy; Forsythe, Evan S; Dorantes-Acosta, Ana; Li, Shaofang; Caballero-Pérez, Juan; Chen, Xuemei; Arteaga-Vázquez, Mario; Beilstein, Mark A; Mosher, Rebecca A

    2015-07-01

    Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highly in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence. PMID:25767205

  5. Mechanism of Action and Antiviral Activity of Benzimidazole-Based Allosteric Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase

    OpenAIRE

    Tomei, Licia; Altamura, Sergio; Bartholomew, Linda; Biroccio, Antonino; Ceccacci, Alessandra; Pacini, Laura; Narjes, Frank; Gennari, Nadia; Bisbocci, Monica; Incitti, Ilario; Orsatti, Laura; Harper, Steven; Stansfield, Ian; Rowley, Michael; De Francesco, Raffaele

    2003-01-01

    The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis...

  6. Avian reovirus L2 genome segment sequences and predicted structure/function of the encoded RNA-dependent RNA polymerase protein

    OpenAIRE

    Xu Wanhong; Coombs Kevin M

    2008-01-01

    Abstract Background The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp) enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreovir...

  7. Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    Directory of Open Access Journals (Sweden)

    Nadya Urakova

    2016-04-01

    Full Text Available Rabbit haemorrhagic disease virus (RHDV is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus. The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV.

  8. Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    Science.gov (United States)

    Urakova, Nadya; Netzler, Natalie; Kelly, Andrew G.; Frese, Michael; White, Peter A.; Strive, Tanja

    2016-01-01

    Rabbit haemorrhagic disease virus (RHDV) is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus). The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp) is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C) for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV. PMID:27089358

  9. Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle.

    Science.gov (United States)

    Pfannschmidt, Thomas; Blanvillain, Robert; Merendino, Livia; Courtois, Florence; Chevalier, Fabien; Liebers, Monique; Grübler, Björn; Hommel, Elisabeth; Lerbs-Mache, Silva

    2015-12-01

    Chloroplasts are the sunlight-collecting organelles of photosynthetic eukaryotes that energetically drive the biosphere of our planet. They are the base for all major food webs by providing essential photosynthates to all heterotrophic organisms including humans. Recent research has focused largely on an understanding of the function of these organelles, but knowledge about the biogenesis of chloroplasts is rather limited. It is known that chloroplasts develop from undifferentiated precursor plastids, the proplastids, in meristematic cells. This review focuses on the activation and action of plastid RNA polymerases, which play a key role in the development of new chloroplasts from proplastids. Evolutionarily, plastids emerged from the endosymbiosis of a cyanobacterium-like ancestor into a heterotrophic eukaryote. As an evolutionary remnant of this process, they possess their own genome, which is expressed by two types of plastid RNA polymerase, phage-type and prokaryotic-type RNA polymerase. The protein subunits of these polymerases are encoded in both the nuclear and plastid genomes. Their activation and action therefore require a highly sophisticated regulation that controls and coordinates the expression of the components encoded in the plastid and nucleus. Stoichiometric expression and correct assembly of RNA polymerase complexes is achieved by a combination of developmental and environmentally induced programmes. This review highlights the current knowledge about the functional coordination between the different types of plastid RNA polymerases and provides working models of their sequential expression and function for future investigations. PMID:26355147

  10. DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation

    DEFF Research Database (Denmark)

    Close, Pierre; East, Philip; Dirac-Svejstrup, A Barbara;

    2012-01-01

    Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation. As the nascent pre...... and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1...... (ZIRD)) as subunits of a novel protein complex--named DBIRD--that binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in (A + T)-rich DNA, and is present at the affected exons. RNA-interference-mediated DBIRD depletion results in region-specific decreases in...

  11. On the promoter complex formation rate of E. coli RNA polymerases with T7 phage DNA.

    OpenAIRE

    Belintsev, B N; Zavriev, S.K.; Shemyakin, M.F.

    1980-01-01

    Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is disc...

  12. Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli.

    OpenAIRE

    Muffler, A; Traulsen, D D; Lange, R. de; Hengge-Aronis, R

    1996-01-01

    The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth. Osmotic induction of rpoS can be t...

  13. Catching RNA Polymerase in the act of Binding: Intermediates in Transcription Illuminated by Synchrotron Footprinting

    International Nuclear Information System (INIS)

    The article by Sclavi et al. in this issue of PNAS addresses 'initiation, ' the first step in transcription. Gene transcription is catalyzed in cells by large multisubunit proteins called RNA polymerases (RNAP). The eubacteria holoenzyme of RNAP is composed of five core subunits (α, α2, β, β', and ω) that contain the amino acid residues required for the enzyme's catalytic activity. A sixth subunit (σ) guides RNAP to specific sequences on the genomic DNA (promoters) that mark the beginning of a gene or group of genes

  14. Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea

    Czech Academy of Sciences Publication Activity Database

    Loza-Muller, L.; Rodriguez-Corona, U.; Sobol, Margaryta; Rodriguez-Zapata, L.C.; Hozák, Pavel; Castano, E.

    2015-01-01

    Roč. 6, Nov 6 (2015). ISSN 1664-462X R&D Projects: GA ČR GAP305/11/2232; GA ČR GA15-08738S; GA MPO FR-TI3/588; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : histones * methylation * RNA polymerase I * Brassica * phosphoinositide Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.948, year: 2014

  15. Alternatively spliced hBRF variants function at different RNA polymerase III promoters

    OpenAIRE

    McCulloch, Vicki; Hardin, Peter; Peng, Wenchen; Ruppert, J. Michael; Lobo-Ruppert, Susan M.

    2000-01-01

    In yeast, a single form of TFIIIB is required for transcription of all RNA polymerase III (pol III) genes. It consists of three subunits: the TATA box-binding protein (TBP), a TFIIB-related factor, BRF, and B′′. Human TFIIIB is not as well defined and human pol III promoters differ in their requirements for this activity. A human homolog of yeast BRF was shown to be required for transcription at the gene-internal 5S and VA1 promoters. Whether or not it was also involved in transcription from ...

  16. A common site on TBP for transcription by RNA polymerases II and III

    OpenAIRE

    Schröder, Oliver; Bryant, Gene O.; Geiduschek, E.Peter; Berk, Arnold J.; Kassavetis, George A.

    2003-01-01

    The TATA-binding protein (TBP) is involved in all nuclear transcription. We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III. TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB. A photochemical cross-linking approach was used to survey a collection of human TBP surface residue mutants for their ability to form TFIIIB–DNA complexes reliant on only the TFII...

  17. A post-recruitment function for the RNA polymerase III transcription–initiation factor IIIB

    OpenAIRE

    Kassavetis, George A.; Kumar, Ashok; Letts, Garth A.; Geiduschek, E.Peter

    1998-01-01

    Transcription factor (TF) IIIB, which directs RNA polymerase (pol) III to its promoters, is made up of three components: the TATA box-binding protein, the TFIIB-related Brf, and the pol III-specific B′′. Certain mutations in Saccharomyces cerevisiae Brf and B′′ retain TFIIIB transcription factor activity with supercoiled DNA but are inactive with linear duplex DNA. Further analysis shows that these inactive TFIIIB–DNA complexes bind pol III and position it appropriately over the transcription...

  18. Cloning and expression of the gene for bacteriophage T7 RNA polymerase.

    OpenAIRE

    Davanloo, P; Rosenberg, A H; Dunn, J J; Studier, F W

    1984-01-01

    The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitabl...

  19. Determination of mRNA fate by different RNA polymerase II promoters.

    OpenAIRE

    Enssle, J; Kugler, W; Hentze, M.W.; Kulozik, A E

    1993-01-01

    Translational stop mutations of the human beta-globin gene cause a reduction of cytoplasmic mRNA accumulation in thalassemia patients and in transfection models. The exact mechanism underlying this phenomenon has remained enigmatic but is known to be post-transcriptional. We have used transfected HeLa cells to study the expression of beta-globin mRNAs with nonsense or frameshift mutations within the three exons of this gene. Mutations in exons 1 or 2 reduce cytoplasmic mRNA accumulation where...

  20. Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria

    Czech Academy of Sciences Publication Activity Database

    Hnilicová, Jarmila; Jirát-Matějčková, Jitka; Šiková, Michaela; Pospíšil, Jiří; Halada, Petr; Pánek, Josef; Krásný, Libor

    2014-01-01

    Roč. 42, č. 18 (2014), s. 11763-11776. ISSN 0305-1048 R&D Projects: GA ČR(CZ) GBP305/12/G034; GA ČR GP13-27150P Grant ostatní: Magistrát hl. m. P.(CZ) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : ESCHERICHIA-COLI * 6S RNA * NONCODING RNA Subject RIV: EE - Microbiology, Virology Impact factor: 9.112, year: 2014

  1. An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

    KAUST Repository

    Gao, Zhihuan

    2010-04-21

    DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm. © 2010 Macmillan Publishers Limited. All rights reserved.

  2. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    Science.gov (United States)

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-07-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

  3. Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

    KAUST Repository

    Ariel, Federico D.

    2014-08-01

    The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

  4. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    Science.gov (United States)

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-01-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. PMID:27418187

  5. Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

    Directory of Open Access Journals (Sweden)

    Chang Ying Teng

    Full Text Available Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

  6. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    Science.gov (United States)

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-01-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted. Images PMID:1696290

  7. The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses

    International Nuclear Information System (INIS)

    Short interfering double-stranded RNAs (siRNAs) expressed under the control of an RNA polymerase I promoter system were used to target gene expression of influenza A and West Nile virus. Decreased RNA and protein expression was induced in a sequence-specific manner--reducing sequence complementarity from 21 to 17 nucleotides abrogated the siRNA effect. Reduced M2 expression resulted in a decrease in total and infectious influenza A virus production. WNV protein expression, genomic RNA, and infectious virus production were all dramatically reduced by siRNAs targeting two distinct viral sequences. The data demonstrate the utility of plasmid-driven siRNAs in regulating the expression of single viral genes, global viral gene expression, as a potential antiviral treatment, and as a genetic tool for viruses whose genomes are difficult to manipulate

  8. Nucleoproteins of Negative Strand RNA Viruses; RNA Binding, Oligomerisation and Binding to Polymerase Co-Factor

    OpenAIRE

    Thibaut Crépin; Ruigrok, Rob W H

    2010-01-01

    Commentary on Tawar, R.G.; Duquerroy, S.; Vonrhein, C.; Varela, P.F.; Damier-Piolle, L.; Castagné, N.; MacLellan, K.; Bedouelle, H.; Bricogne, G.; Bhella, D.; Eléouët, J.-F.; Rey, F.A. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 2009, 326, 1279-1283.

  9. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

    Science.gov (United States)

    Thiffault, Isabelle; Wolf, Nicole I.; Forget, Diane; Guerrero, Kether; Tran, Luan T.; Choquet, Karine; Lavallée-Adam, Mathieu; Poitras, Christian; Brais, Bernard; Yoon, Grace; Sztriha, Laszlo; Webster, Richard I.; Timmann, Dagmar; van de Warrenburg, Bart P.; Seeger, Jürgen; Zimmermann, Alíz; Máté, Adrienn; Goizet, Cyril; Fung, Eva; van der Knaap, Marjo S.; Fribourg, Sébastien; Vanderver, Adeline; Simons, Cas; Taft, Ryan J.; Yates III, John R.; Coulombe, Benoit; Bernard, Geneviève

    2015-01-01

    A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy. PMID:26151409

  10. Phenotypic Traits of Arabidopsis Plants Deficient in RpoTmp RNA Polymerase

    Directory of Open Access Journals (Sweden)

    V.I. Tarasenko

    2015-12-01

    Full Text Available In Arabidopsis, three nuclear-encoded RNA polymerases participate in the transcription of organellar genes. RpoTmp is a RNA polymerase that localizes both in mitochondria and chloroplasts, but is involved predominantly in the control of gene expression in mitochondria. Insertion mutant rpotmp is characterized by a number of phenotypic and molecular-biological peculiarities including decreased activities of the mitochondrial respiratory complexes I and IV. In the present study we compared growth characteristics of the rpotmp mutant and fro1 mutant which is characterized by the absence of functional complex I. We showed that in spite of the similar molecular defects and phenotypic appearance, the investigated mutants can be distinguished by the growth rate under different photoperiod as well as by the age of leaf senescence onset. Moreover, the growth rate of suspension cell culture of the rpotmp line is extremely retarded which clearly distinguished it from the fro1 suspension cell culture. We propose that unique properties of the rpotmp mutant are associated with the decreased level of respiratory complex IV activity.

  11. hRRN3 is essential in the SL1-mediated recruitment of RNA Polymerase I to rRNA gene promoters

    OpenAIRE

    Miller, Gail; Panov, Kostya I.; Friedrich, J. Karsten; TRINKLE-MULCAHY, LAURA; Lamond, Angus I; Zomerdijk, Joost C. B. M.

    2001-01-01

    A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (Iα) are tra...

  12. Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

    Science.gov (United States)

    Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin; Hernandez, Nouria; Willis, Ian M

    2015-05-01

    MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. PMID:25934505

  13. Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain reaction detection of enterovirus RNA in clinical specimens.

    OpenAIRE

    Muir, P; Nicholson, F; Jhetam, M; Neogi, S; Banatvala, J E

    1993-01-01

    We describe a rapid method for extraction and detection of enterovirus RNA in clinical samples. By using magnetic bead technology, enterovirus RNA was efficiently and rapidly extracted from cerebrospinal fluid, stool, saliva, blood, pericardial fluid, urine, and cryopreserved or formalin-fixed solid tissue. Enterovirus RNA was then detected by reverse transcription followed by polymerase chain reaction amplification with primers designed to allow detection of most enterovirus serotypes. For d...

  14. Human Maf1 negatively regulates RNA Polymerase III transcription via the TFIIB family members Brf1 and Brf2

    OpenAIRE

    Rollins, Janet; Veras, Ingrid; Cabarcas, Stephanie; Willis, Ian; Schramm, Laura

    2007-01-01

    RNA polymerase III (RNA pol III) transcribes many of the small structural RNA molecules involved in processing and translation, thereby regulating the growth rate of a cell. Initiation of pol III transcription requires the evolutionarily conserved pol III initiation factor TFIIIB. TFIIIB is the molecular target of regulation by tumor suppressors, including p53, RB and the RB-related pocket proteins. However, our understanding of negative regulation of human TFIIIB-mediated transcription by ot...

  15. A conformational switch is responsible for the reversal of the 6S RNA-dependent RNA polymerase inhibition in Escherichia coli.

    Science.gov (United States)

    Steuten, Benedikt; Wagner, Rolf

    2012-12-01

    6S RNA is a bacterial transcriptional regulator,which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ 70 -containing RNA polymerase. This inhibitory RNA polymerase ∼ 6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs.Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6SRNA ∼ pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem(conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented. PMID:23667906

  16. The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

    Directory of Open Access Journals (Sweden)

    Lonneke van der Linden

    2015-03-01

    Full Text Available The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71 for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylenebis(oxy]bis(5-nitro-benzonitrile with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family. Surprisingly, coxsackievirus B3 (CVB3 and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight

  17. Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

    Directory of Open Access Journals (Sweden)

    Jerome Deval

    2015-06-01

    Full Text Available Respiratory syncytial virus (RSV causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV, whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

  18. Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp

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    Devendra K. Rai

    2012-07-01

    Full Text Available Bovine Rhinitis B Virus (BRBV is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp, which is also known as 3D polymerase (3Dpol. In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P, nucleotide tri-phosphate (NTP and pyrophosphate (PPi bound FMDV 3Dpol (PDB, 1U09 and 2E9Z. The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16 and rabbit hemorrhagic disease virus (RHDV 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the

  19. Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high-throughput lead-finding campaign.

    Science.gov (United States)

    Smith, Thomas M; Lim, Siew Pheng; Yue, Kimberley; Busby, Scott A; Arora, Rishi; Seh, Cheah Chen; Wright, S Kirk; Nutiu, Razvan; Niyomrattanakit, Pornwaratt; Wan, Kah Fei; Beer, David; Shi, Pei-Yong; Benson, Timothy E

    2015-01-01

    Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase. PMID:25252731

  20. Transposable B2 SINE elements can provide mobile RNA polymerase II promoters.

    Science.gov (United States)

    Ferrigno, O; Virolle, T; Djabari, Z; Ortonne, J P; White, R J; Aberdam, D

    2001-05-01

    Short interspersed elements (SINEs) are highly abundant components of mammalian genomes that are propagated by retrotransposition. SINEs are recognized as a causal agent of human disease and must also have had a profound influence in shaping eukaryotic genomes. The B2 SINE family constitutes approximately 0.7% of total mouse genomic DNA (ref. 2) and is also found at low abundance in humans. It resembles the Alu family in several respects, such as its mechanism of propagation. B2 SINEs are derived from tRNA and are transcribed by RNA polymerase (pol) III to generate short transcripts that are not translated. We find here, however, that one B2 SINE also carries an active pol II promoter located outside the tRNA region. Indeed, a B2 element is responsible for the production of a mouse Lama3 transcript. The B2 pol II promoters can be bound and stimulated by the transcription factor USF (for upstream stimulatory factor), as shown by transient transfection experiments. Moreover, this pol II activity does not preclude the pol III transcription necessary for retrotransposition. Dispersal of B2 SINEs by retrotransposition may therefore have provided numerous opportunities for creating regulated pol II transcription at novel genomic sites. This mechanism may have allowed the evolution of new transcription units and new genes. PMID:11326281

  1. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  2. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    OpenAIRE

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-01-01

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstr...

  3. Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes.

    OpenAIRE

    Colland, F; Fujita, N; Kotlarz, D; Bown, J A; Meares, C F; Ishihama, A; Kolb, A

    1999-01-01

    The sigma(S) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of Esigma(S) RNA polymerase, using sigma(S) derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 ...

  4. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB

    Directory of Open Access Journals (Sweden)

    Lenice Roteia Cardoso Jung

    2015-06-01

    Full Text Available Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR. The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

  5. Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system

    Science.gov (United States)

    Polosa, Paola Loguercio; Deceglie, Stefania; Falkenberg, Maria; Roberti, Marina; Di Ponzio, Barbara; Gadaleta, Maria Nicola; Cantatore, Palmiro

    2007-01-01

    Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms. PMID:17392338

  6. UV damage-induced RNA polymerase II stalling stimulates H2B deubiquitylation.

    Science.gov (United States)

    Mao, Peng; Meas, Rithy; Dorgan, Kathleen M; Smerdon, Michael J

    2014-09-01

    Histone H2B monoubiquitylation plays an important role in RNA polymerase II (RNAPII) elongation. Whether this modification responds to RNAPII stalling is not yet known. We report that both yeast and human cells undergo a rapid and significant H2B deubiquitylation after exposure to UV irradiation. This deubiquitylation occurs concurrently with UV-induced transcription arrest and is significantly reduced in a DNA damage-bypassing RNAPII yeast mutant. Consistent with these results, yeast deubiquitylases Ubp8 and Ubp10 are associated with the RNAPII complex. Moreover, simultaneous deletion of Ubp8 and Ubp10 leads to a lack of H2B deubiquitylation after UV exposure. Consequently, nucleotide excision repair at an actively transcribed gene locus is decreased, whereas UV-induced RNAPII degradation is increased in ubp8Δubp10Δ mutant cells. These results indicate that eukaryotic cells respond to RNAPII arrest by deubiquitylating H2B to coordinate DNA repair and RNAPII degradation. PMID:25136098

  7. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    DEFF Research Database (Denmark)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C;

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the...... methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA...... and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator...

  8. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    International Nuclear Information System (INIS)

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

  9. Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase.

    Science.gov (United States)

    Ma, Ming-Yue; Lan, Xin-Ran; Niu, Deng-Ke

    2016-01-01

    The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In an RNA-dependent RNA polymerase (RdRp) gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among other Solanum plants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3' splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals. PMID:27547574

  10. Metabolism and expression of RNA polymerase II transcripts in Influenza virus-infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Katze, M.G.; Krug, R.M.

    1984-10-01

    Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for BETA-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleas cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplamic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in biro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.

  11. Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Hashimi, Hassan; Číčová, Zdeňka; Novotná, Lucie; Wen, Y.-Z.; Lukeš, Julius

    2009-01-01

    Roč. 15, č. 4 (2009), s. 588-599. ISSN 1355-8382 R&D Projects: GA ČR GA204/09/1667; GA AV ČR IAA500960705; GA MŠk LC07032; GA MŠk 2B06129; GA ČR GD524/03/H133 Institutional research plan: CEZ:AV0Z60220518 Keywords : RNA editing * guide RNA * mitochondrion * trypanosome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.198, year: 2009

  12. Optimization and Validation of a Real Time Reverse Transcriptase Polymerase Chain Reaction with RNA Internal Control to Detect Rubella RNA

    Directory of Open Access Journals (Sweden)

    Winny Xie

    2013-12-01

    Full Text Available BACKGROUND: According to a report from WHO, cases of rubella infection in Indonesia has increased up to 10-fold from 2007 to 2011. Despite no data of congenital rubella syndrome in the report, there are approximately 45,000 cases of babies born with heart failure and 0.1-0.3% live births with congenital deafness in Indonesia. Allegedly, rubella infection during pregnancy may play a role in this condition. This study aimed to optimize and validate a real-time reverse transcriptase polymerase chain reaction (RT-qPCR method to detect rubella virus RNA as an aid for the diagnosis of congenital rubella infection. METHODS: Method optimization was conducted using nucleic acids extracted from Trimovax Merieux vaccine with the High Pure Viral Nucleic Acid Kit. One step RT-qPCR was performed with Quantifast Multiplex RTPCR+R Kit. Target synthetic DNA was designed and used to determine the sensitivity of the method. RNA internal control was synthesized to control the process of extraction and amplification. RESULTS: The analytical sensitivity of this method was as low as 5 copies target synthetic DNA/μl. The mean Coefficient of Variation (CV % of the critical threshold (Ct obtained were 2.71%, 1.20%, 1.62%, and 1.59% for within run, between run, between kit lots, and between operators, respectively. Recovery of the target synthetic DNA from amniotic fluid was 100.51% (by the log copies/μl at the concentration of 1,000,000 copies/μl. CONCLUSIONS: RT-qPCR is successfully used for the detection of rubella virus RNA in vaccine and synthetic nucleic acid. With its high sensitivity, good precision and recovery, this method offers a means to improve the diagnosis of congenital rubella infection in developing countries like Indonesia. KEYWORDS: congenital rubella, RT-qPCR, prenatal diagnosis, amniotic fluid.

  13. Molecular characterization of the largest subunit of Plasmodium falciparum RNA polymerase I.

    Science.gov (United States)

    Fox, B A; Li, W B; Tanaka, M; Inselburg, J; Bzik, D J

    1993-09-01

    Plasmodium species possess developmentally regulated ribosomal RNA (rRNA) genes. This report describes the expression and gene structure of the largest subunit of P. falciparum RNA polymerase I (RNAPI), which is responsible for the synthesis of rRNA. The RNAPI largest subunit gene was present as a single copy gene on chromosome 9. Three exons encode the 2910-amino acid RNAPI polypeptide (340 140 Da). A comparison of Plasmodium, Trypanosoma, and Saccharomyces cerevisiae nuclear RNAP largest subunits identified conserved amino acid positions and class-specific amino acid positions. Novel amino acid insertions were found between RNAPI conserved regions A and B (region A'), D and DE1 (region D'), DE2 and E (region DE2'), and F and G (region F'). Leucine zipper domains were found within regions D', DE2, and DE2'. A novel serine-rich repeat domain, a domain with homology to the C-terminal domain of eukaryotic upstream binding factor (UBF), and 4 highly conserved casein kinase II (CKII) Ser/Thr phosphorylation motifs were found within a 127-amino acid sub-region of enlarged region F'. The novel RNAPI serine-rich repeat contained a conserved motif, Ser-X3-Ser, which was also identified in the serine-rich repeat domains of the P. falciparum RNAPII and RNAPIII largest subunits, as well as within a highly homologous serine-rich repeat from trophozoite antigen R45. The results of this molecular analysis indicate that phosphorylation and dephosphorylation mechanisms regulate the activity of P. falciparum RNAPI. PMID:8259131

  14. Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

    Science.gov (United States)

    Xie, Lili; Wartchow, Charles; Shia, Steven; Uehara, Kyoko; Steffek, Micah; Warne, Robert; Sutton, James; Muiru, Gladys T; Leonard, Vincent H J; Bussiere, Dirksen E; Ma, Xiaolei

    2016-01-01

    Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2. PMID:26559973

  15. Modeling RNA polymerase competition: the effect of σ-subunit knockout and heat shock on gene transcription level

    Directory of Open Access Journals (Sweden)

    Seliverstov Alexandr V

    2011-01-01

    Full Text Available Abstract Background Modeling of a complex biological process can explain the results of experimental studies and help predict its characteristics. Among such processes is transcription in the presence of competing RNA polymerases. This process involves RNA polymerases collision followed by transcription termination. Results A mathematical and computer simulation model is developed to describe the competition of RNA polymerases during genes transcription on complementary DNA strands. E.g., in the barley Hordeum vulgare the polymerase competition occurs in the locus containing plastome genes psbA, rpl23, rpl2 and four bacterial type promoters. In heat shock experiments on isolated chloroplasts, a twofold decrease of psbA transcripts and even larger increase of rpl23-rpl2 transcripts were observed, which is well reproduced in the model. The model predictions are in good agreement with virtually all relevant experimental data (knockout, heat shock, chromatogram data, etc.. The model allows to hypothesize a mechanism of cell response to knockout and heat shock, as well as a mechanism of gene expression regulation in presence of RNA polymerase competition. The model is implemented for multiprocessor platforms with MPI and supported on Linux and MS Windows. The source code written in C++ is available under the GNU General Public License from the laboratory website. A user-friendly GUI version is also provided at http://lab6.iitp.ru/en/rivals. Conclusions The developed model is in good agreement with virtually all relevant experimental data. The model can be applied to estimate intensities of binding of the holoenzyme and phage type RNA polymerase to their promoters using data on gene transcription levels, as well as to predict characteristics of RNA polymerases and the transcription process that are difficult to measure directly, e.g., the intensity (frequency of holoenzyme binding to the promoter in correlation to its nucleotide composition and the

  16. Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

    OpenAIRE

    Firzlaff, J M; Diggelmann, H

    1984-01-01

    In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the tra...

  17. Tamoxifen represses alcohol-induced transcription of RNA polymerase III-dependent genes in breast cancer cells

    OpenAIRE

    Zhong, Qian; Shi, Ganggang; Zhang, Qingsong; Lu, Lei; Levy, Daniel; Zhong, Shuping

    2014-01-01

    Alcohol consumption in women has been associated with an increased risk of breast cancer, particular in estrogen receptor positive (ER+) cases. Deregulation of RNA polymerase III-dependent (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell transformation and tumor formation. Our recent studies demonstrated that alcohol induces Brf1 expression and Pol III gene transcription via ER. Here, we report that Tamoxi...

  18. Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III.

    Science.gov (United States)

    Li, W B; Bzik, D J; Tanaka, M; Gu, H M; Fox, B A; Inselburg, J

    1991-06-01

    We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum. The P. falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13. The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions. Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits. Variable region C' represented nearly one-third of the P. falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II. Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed. PMID:1656254

  19. The Structure of Fcp1, an Essential RNA Polymerase II CTD Phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, A.; Shuman, S.; Lima, C.D. (SKI)

    2009-03-27

    Kinases and phosphatases regulate mRNA synthesis and processing by phosphorylating and dephosphorylating the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Fcp1 is an essential CTD phosphatase that preferentially hydrolyzes Ser2-PO{sub 4} of the tandem YSPTSPS CTD heptad array. Fcp1 crystal structures were captured at two stages of the reaction pathway: a Mg-BeF{sub 3} complex that mimics the aspartylphosphate intermediate and a Mg-AlF{sub 4}{sup -} complex that mimics the transition state of the hydrolysis step. Fcp1 is a Y-shaped protein composed of an acylphosphatase domain located at the base of a deep canyon formed by flanking modules that are missing from the small CTD phosphatase (SCP) clade: an Fcp1-specific helical domain and a C-terminal BRCA1 C-terminal (BRCT) domain. The structure and mutational analysis reveals that Fcp1 and Scp1 (a Ser5-selective phosphatase) adopt different CTD-binding modes; we surmise the CTD threads through the Fcp1 canyon to access the active site.

  20. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.

    Science.gov (United States)

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

    2016-04-20

    DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in anin vitrotranscription assay using RNA polymerase fromBacillus subtilisandEscherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by theE. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. PMID:27001521

  1. Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.

    Science.gov (United States)

    Fishburn, James; Galburt, Eric; Hahn, Steven

    2016-06-17

    Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction. PMID:27129284

  2. Repair of UV induced DNA lesions in ribosomal gene chromatin and the role of "Odd" RNA polymerases (I and III).

    Science.gov (United States)

    Charton, Romain; Guintini, Laetitia; Peyresaubes, François; Conconi, Antonio

    2015-12-01

    In fast growing eukaryotic cells, a subset of rRNA genes are transcribed at very high rates by RNA polymerase I (RNAPI). Nuclease digestion-assays and psoralen crosslinking have shown that they are open; that is, largely devoid of nucleosomes. In the yeast Saccharomyces cerevisae, nucleotide excision repair (NER) and photolyase remove UV photoproducts faster from open rRNA genes than from closed and nucleosome-loaded inactive rRNA genes. After UV irradiation, rRNA transcription declines because RNAPI halt at UV photoproducts and are then displaced from the transcribed strand. When the DNA lesion is quickly recognized by NER, it is the sub-pathway transcription-coupled TC-NER that removes the UV photoproduct. If dislodged RNAPI are replaced by nucleosomes before NER recognizes the lesion, then it is the sub-pathway global genome GG-NER that removes the UV photoproducts from the transcribed strand. Also, GG-NER maneuvers in the non-transcribed strand of open genes and in both strands of closed rRNA genes. After repair, transcription resumes and elongating RNAPI reopen the rRNA gene. In higher eukaryotes, NER in rRNA genes is inefficient and there is no evidence for TC-NER. Moreover, TC-NER does not occur in RNA polymerase III transcribed genes of both, yeast and human fibroblast. PMID:26411875

  3. Complete Structural Model of Escherichia coli RNA Polymerase from a Hybrid Approach

    Energy Technology Data Exchange (ETDEWEB)

    Opalka, N.; Brown, J; Lane, W; Twist, K; Landick, R; Asturias, F; Darst, S

    2010-01-01

    The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program. Transcription, or the synthesis of RNA from DNA, is one of the most important processes in the cell. The central enzyme of transcription is the DNA-dependent RNA polymerase (RNAP), a large, macromolecular assembly consisting of at least five subunits. Historically, much of our fundamental information on the process of transcription has come from genetic and biochemical studies of RNAP from the model bacterium Escherichia coli. More recently, major breakthroughs in our understanding of the mechanism of action of RNAP have come from high resolution crystal structures of various bacterial, archaebacterial, and eukaryotic enzymes. However, all of our high-resolution bacterial RNAP structures are of enzymes from the thermophiles Thermus aquaticus or T. thermophilus, organisms with poorly characterized transcription systems. It has thus far proven impossible to obtain a high-resolution structure of E. coli RNAP, which has made it difficult to relate the large collection

  4. Characterization of the RNA polymerase II and III complexes in Leishmania major.

    Science.gov (United States)

    Martínez-Calvillo, Santiago; Saxena, Alka; Green, Amanda; Leland, Aaron; Myler, Peter J

    2007-04-01

    Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function. PMID:17275824

  5. CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Brian [Rockefeller Univ., New York, NY (United States); Nayak, Dhananjaya [Univ. of Wisconsin, Madison, WI (United States); Ray, Ananya [Univ. of Wisconsin, Madison, WI (United States); Mustaev, Arkady [New Jersey Medical School, Newark, NJ (United States); Landick, Robert [Univ. of Wisconsin, Madison, WI (United States); Darst, Seth A. [Rockefeller Univ., New York, NY (United States)

    2015-07-20

    RNA polymerase inhibitors like the CBR class that target the enzyme’s complex catalytic center are attractive leads for new antimicrobials. The catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. Here, we report crystal structures of CBR inhibitor/Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3'-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.

  6. The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, F.; Petrovicova, I.;

    2008-01-01

    The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition...

  7. E3-ubiquitin ligase Nedd4 determines the fate of AID-associated RNA polymerase II in B cells.

    Science.gov (United States)

    Sun, Jianbo; Keim, Celia D; Wang, Jiguang; Kazadi, David; Oliver, Paula M; Rabadan, Raul; Basu, Uttiya

    2013-08-15

    Programmed mutagenesis of the immunoglobulin locus of B lymphocytes during class switch recombination (CSR) and somatic hypermutation requires RNA polymerase II (polII) transcription complex-dependent targeting of the DNA mutator activation-induced cytidine deaminase (AID). AID deaminates cytidine residues on substrate sequences in the immunoglobulin (Ig) locus via a transcription-dependent mechanism, and this activity is stimulated by the RNA polII stalling cofactor Spt5 and the 11-subunit cellular noncoding RNA 3'-5' exonucleolytic processing complex RNA exosome. The mechanism by which the RNA exosome recognizes immunoglobulin locus RNA substrates to stimulate AID DNA deamination activity on its in vivo substrate sequences is an important question. Here we report that E3-ubiquitin ligase Nedd4 destabilizes AID-associated RNA polII by a ubiquitination event, leading to generation of 3' end free RNA exosome RNA substrates at the Ig locus and other AID target sequences genome-wide. We found that lack of Nedd4 activity in B cells leads to accumulation of RNA exosome substrates at AID target genes and defective CSR. Taken together, our study links noncoding RNA processing following RNA polII pausing with regulation of the mutator AID protein. Our study also identifies Nedd4 as a regulator of noncoding RNAs that are generated by stalled RNA polII genome-wide. PMID:23964096

  8. A Step Subsequent to Preinitiation Complex Assembly at the Ribosomal RNA Gene Promoter Is Rate Limiting for Human RNA Polymerase I-Dependent Transcription

    OpenAIRE

    Panov, Kostya I.; Friedrich, J. Karsten; Zomerdijk, Joost C. B. M.

    2001-01-01

    The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contain...

  9. Cloning of RNA-dependent RNA Polymerase (RdRp Gene from Genotype Dengue Type-2 (New Guinea-C Strain

    Directory of Open Access Journals (Sweden)

    Samian, R.

    2005-01-01

    Full Text Available Dengue virus causes febrile disease in human. Dengue infection causes dengue fever that is not life threatening. However, a severe form of the disease called dengue hemorrhagic fever (DH or dengue shock syndrome (DSS, proven to be fatal. A positive single stranded RNA virus genome encodes for a single polyprotein precursor and is arranged in the order of NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. The purpose of this study was to clone NS5 gene that encodes for the RNA-dependent RNA polymerase (RdRp. This enzyme plays an important role in viral RNA replication. The RdRp associated by cofactors produce minus-strand single stranded RNA, which in turn, serves as a template for the production of new plus-strand single stranded genome. The virus RNA was extracted from Aedes albopictus cell line C6/36 that was infected with dengue virus type 2. Then, the extracted virus RNA was used as the template for RT-PCR. A 2.7 kb DNA fragment, representing the RNA-dependent RNA polymerase gene, wassuccessfully amplified using specific primers. The PCR product was then cloned into cloning vector (pGEM-T and transformed into E. coli JM109.

  10. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

    2016-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. PMID:26574011

  11. Structural relationships among the multiple forms of DNA-dependent RNA polymerase II from cultured parsley cells

    International Nuclear Information System (INIS)

    DNA-dependent RNA polymerase II (or B) was purified from cultured parsley cells, and its molecular structure was examined in detail. Upon centrifugation through glycerol gradients, RNA polymerase II sediments as a single band with an apparent sedimentation constant of 15S. No contamination with RNA polymerases I or III could be detected when the activity of purified RNA polymerase II was assayed in the presence of high concentrations of α-amanitin. Analysis of purified RNA polymerase II be nondenaturing and denaturing polyacrylamide gel electrophoresis revealed that this enzyme exists in multiple forms. They were designated II(O), II(A), and II(B). It is suggested that each form has a subunit of Mr = 140000 as well as smaller polypeptides in common. They differ, however, in the molecular weights of their largest subunits which is 220000 in form II(O), 200000 in form II(A), and 180000 in form II(B). These large subunits were labelled with 125I, digested with trypsin, and tryptic digests were compared by two-dimensional analysis on thin-layer plates (Elder et al. (1977) J. Biol. Chem. 252, 6510-6515). Fingerprints of tryptic digests from the polypeptides with Mr = 220000, Mr = 200000, and Mr = 180000 were similar. It is, therefore, suggested that these subunits are stucturally related. A tryptic digest was also produced from the subunit with Mr = 140000. Its fingerprint was found to yield a considerably different distribution of peptides as compared to those from the three large subunits. (orig.)

  12. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    Science.gov (United States)

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-10-25

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1. PMID:7971267

  13. An integrated one-chip-sensor system for microRNA quantitative analysis based on digital droplet polymerase chain reaction

    Science.gov (United States)

    Tsukuda, Masahiko; Wiederkehr, Rodrigo Sergio; Cai, Qing; Majeed, Bivragh; Fiorini, Paolo; Stakenborg, Tim; Matsuno, Toshinobu

    2016-04-01

    A silicon microfluidic chip was developed for microRNA (miRNA) quantitative analysis. It performs sequentially reverse transcription and polymerase chain reaction in a digital droplet format. Individual processes take place on different cavities, and reagent and sample mixing is carried out on a chip, prior to entering each compartment. The droplets are generated on a T-junction channel before the polymerase chain reaction step. Also, a miniaturized fluorescence detector was developed, based on an optical pick-up head of digital versatile disc (DVD) and a micro-photomultiplier tube. The chip integrated in the detection system was tested using synthetic miRNA with known concentrations, ranging from 300 to 3,000 templates/µL. Results proved the functionality of the system.

  14. Factors related to RNA polymerase II transcription are localized in interchromatin granule clusters of Panorpa communis oocytes.

    Directory of Open Access Journals (Sweden)

    Vladimir Parfenov

    2009-05-01

    Full Text Available Diplotene oocyte nucleus of the scorpionfly Panorpa communis is transcriptionally silent and contains numerous nuclear bodies including interchromatin granule clusters (IGCs. The latter consist of the granules of 30-50 nm in diameter and contain IGC marker protein SC35 as well as RNA polymerase II. In this study, we also localized in P. communis oocyte IGCs the transcription coactivators CBP/p300, TATA-binding protein (TBP which is a component of the basal transcription factor TFIID and the basal transcription factor TFIIH. We belive that IGCs in transcriptionally inert P. communis oocytes are storage sites for the components of RNA polymerase II holoenzyme and other factors of RNA pol II transcription.

  15. The fission yeast TFIIB-related factor limits RNA polymerase III to a TATA-dependent pathway of TBP recruitment

    OpenAIRE

    Ying HUANG; McGillicuddy, Edward; Weindel, Michael; Dong, Steven; Maraia, Richard J.

    2003-01-01

    The RNA polymerase (pol) III-transcribed (e.g. tRNA and 5S rRNA) genes of traditionally studied organisms rely on gene-internal promoters that precisely position the initiation factor, TFIIIB, on the upstream promoter-less DNA. This is accomplished by the ability of the TFIIIB subunit, TFIIB-related factor (Brf1), to make stable protein–protein interactions with TATA-binding protein (TBP) and place it on the promoter-less upstream DNA. Unlike traditional model organisms, Schizosaccharomyces p...

  16. Phylogenetic analysis of DNA and RNA polymerases from a Moniliophthora perniciosa mitochondrial plasmid reveals probable lateral gene transfer.

    Science.gov (United States)

    Andrade, B S; Góes-Neto, A

    2015-01-01

    The filamentous fungus Moniliophthora perniciosa is a hemibiotrophic basidiomycete that causes witches' broom disease of cacao (Theobroma cacao L.). Many fungal mitochondrial plasmids are DNA and RNA polymerase-encoding invertrons with terminal inverted repeats and 5'-linked proteins. The aim of this study was to carry out comparative and phylogenetic analyses of DNA and RNA polymerases for all known linear mitochondrial plasmids in fungi. We performed these analyses at both gene and protein levels and assessed differences between fungal and viral polymerases in order to test the lateral gene transfer (LGT) hypothesis. We analyzed all mitochondrial plasmids of the invertron type within the fungal clade, including five from Ascomycota, seven from Basidiomycota, and one from Chytridiomycota. All phylogenetic analyses generated similar tree topologies regardless of the methods and datasets used. It is likely that DNA and RNA polymerase genes were inserted into the mitochondrial genomes of the 13 fungal species examined in our study as a result of different LGT events. These findings are important for a better understanding of the evolutionary relationships between fungal mitochondrial plasmids. PMID:26535725

  17. Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2015-07-01

    Full Text Available The RNA polymerase II (Pol II is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.

  18. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    International Nuclear Information System (INIS)

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

  19. Inhomogeneities and nonlinear dynamics of a helical DNA interacting with a RNA-polymerase

    International Nuclear Information System (INIS)

    We have numerically investigated the effects of helicity and inhomogeneities on DNA base pairs opening. The inhomogeneities are due to the site-dependent stacking and hydrogen bonding energies in DNA and protein molecules. We have considered a situation in which the active site of the RNA-polymerase molecule binds onto the promoter site of the spin-like model of the DNA molecule at the physiological temperature and creates a bubble. During the study, we have found that the helical coupling has to be very weak compared to intra-strand coupling in the real DNA molecule. Results show that inhomogeneities do not affect the general pattern of base pair opening, even as the periodic inhomogeneity introduces a train of periodic oscillations on the tail of the bubble; while the height of the bubble is an increasing function of the helical coupling parameter. The basic properties of breather-like modes, obtained here by taking into account helical structure and inhomogeneities, are essential for DNA functioning since such breathing-like modes are considered to be much better candidates for the nonlinear modes responsible for a locally open state where biological functioning takes place. (paper)

  20. Characterization of a novel RNA polymerase mutant that alters DksA activity.

    Science.gov (United States)

    Satory, Dominik; Halliday, Jennifer A; Sivaramakrishnan, Priya; Lua, Rhonald C; Herman, Christophe

    2013-09-01

    The auxiliary factor DksA is a global transcription regulator and, with the help of ppGpp, controls the nutritional stress response in Escherichia coli. Although the consequences of its modulation of RNA polymerase (RNAP) are becoming better explained, it is still not fully understood how the two proteins interact. We employed a series of genetic suppressor selections to find residues in RNAP that alter its sensitivity to DksA. Our approach allowed us to identify and genetically characterize in vivo three single amino acid substitutions: β' E677G, β V146F, and β G534D. We demonstrate that the mutation β' E677G affects the activity of both DksA and its homolog, TraR, but does not affect the action of other secondary interactors, such as GreA or GreB. Our mutants provide insight into how different auxiliary transcription factors interact with RNAP and contribute to our understanding of how different stages of transcription are regulated through the secondary channel of RNAP in vivo. PMID:23852871

  1. Divergence of RNA polymerase α subunits in angiosperm plastid genomes is mediated by genomic rearrangement

    Science.gov (United States)

    Blazier, J. Chris; Ruhlman, Tracey A.; Weng, Mao-Lun; Rehman, Sumaiyah K.; Sabir, Jamal S. M.; Jansen, Robert K.

    2016-01-01

    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA. PMID:27087667

  2. Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

    Directory of Open Access Journals (Sweden)

    Kevin M. Harlen

    2016-06-01

    Full Text Available Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II C-terminal domain (CTD and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7, we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

  3. The molecular basis of selective promoter activation by the sigmaS subunit of RNA polymerase.

    Science.gov (United States)

    Typas, Athanasios; Becker, Gisela; Hengge, Regine

    2007-03-01

    Different environmental stimuli cause bacteria to exchange the sigma subunit in the RNA polymerase (RNAP) and, thereby, tune their gene expression according to the newly emerging needs. Sigma factors are usually thought to recognize clearly distinguishable promoter DNA determinants, and thereby activate distinct gene sets, known as their regulons. In this review, we illustrate how the principle sigma factor in stationary phase and in stressful conditions in Escherichia coli, sigmaS (RpoS), can specifically target its large regulon in vivo, although it is known to recognize the same core promoter elements in vitro as the housekeeping sigma factor, sigma70 (RpoD). Variable combinations of cis-acting promoter features and trans-acting protein factors determine whether a promoter is recognized by RNAP containing sigmaS or sigma70, or by both holoenzymes. How these promoter features impose sigmaS selectivity is further discussed. Moreover, additional pathways allow sigmaS to compete more efficiently than sigma70 for limiting amounts of core RNAP (E) and thereby enhance EsigmaS formation and effectiveness. Finally, these topics are discussed in the context of sigma factor evolution and the benefits a cell gains from retaining competing and closely related sigma factors with overlapping sets of target genes. PMID:17302812

  4. The interaction of Bacillus subtilis sigmaA with RNA polymerase.

    Science.gov (United States)

    Johnston, Elecia B; Lewis, Peter J; Griffith, Renate

    2009-11-01

    RNA polymerase (RNAP) is an essential and highly conserved enzyme in all organisms. The process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. In prokaryotes, initiation is regulated by sigma factors, making the essential interaction between sigma factors and RNAP an attractive target for antimicrobial agents. Our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the interaction between a bacterial RNAP and an essential sigma factor can be disrupted by introducing carefully designed mutations into sigma(A) of Bacillus subtilis. This disruption was demonstrated qualitatively by Far-Western blotting. Design of mutant sigmas was achieved by computer-aided visualization of the RNAP-sigma interface of the B. subtilis holoenzyme (RNAP + sigma) constructed using a homology modeling approach with published crystal structures of bacterial RNAPs. Models of the holoenzyme and the core RNAP were rigorously built, evaluated, and validated. To allow a high-quality RNAP-sigma interface model to be constructed for the design of mutations, a crucial error in the B. subtilis sigma(A) sequence in published databases at amino acid 165 had to be corrected first. The new model was validated through determination of RNAP-sigma interactions using targeted mutations. PMID:19735077

  5. Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop

    International Nuclear Information System (INIS)

    Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta

  6. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    Science.gov (United States)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  7. The genome of AR9, a giant transducing Bacillus phage encoding two multisubunit RNA polymerases.

    Science.gov (United States)

    Lavysh, Daria; Sokolova, Maria; Minakhin, Leonid; Yakunina, Maria; Artamonova, Tatjana; Kozyavkin, Sergei; Makarova, Kira S; Koonin, Eugene V; Severinov, Konstantin

    2016-08-01

    Bacteriophage AR9 and its close relative PBS1 have been extensively used to construct early Bacillus subtilis genetic maps. Here, we present the 251,042bp AR9 genome, a linear, terminally redundant double-stranded DNA containing deoxyuridine instead of thymine. Multiple AR9 genes are interrupted by non-coding sequences or sequences encoding putative endonucleases. We show that these sequences are group I and group II self-splicing introns. Eight AR9 proteins are homologous to fragments of bacterial RNA polymerase (RNAP) subunits β/β'. These proteins comprise two sets of paralogs of RNAP largest subunits, with each paralog encoded by two disjoint phage genes. Thus, AR9 is a phiKZ-related giant phage that relies on two multisubunit viral RNAPs to transcribe its genome independently of host transcription apparatus. Purification of one of PBS1/AR9 RNAPs has been reported previously, which makes AR9 a promising object for further studies of RNAP evolution, assembly and mechanism. PMID:27236306

  8. Maf1, a new player in the regulation of human RNA polymerase III transcription.

    Directory of Open Access Journals (Sweden)

    Jaime H Reina

    Full Text Available BACKGROUND: Human RNA polymerase III (pol III transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the alpha-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells.

  9. Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila.

    Science.gov (United States)

    Marshall, Lynne; Rideout, Elizabeth J; Grewal, Savraj S

    2012-04-18

    The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth. PMID:22367393

  10. Bacteriophage T7 RNA polymerase-based expression in Pichia pastoris.

    Science.gov (United States)

    Hobl, Birgit; Hock, Björn; Schneck, Sandra; Fischer, Reinhard; Mack, Matthias

    2013-11-01

    A novel Pichia pastoris expression vector (pEZT7) for the production of recombinant proteins employing prokaryotic bacteriophage T7 RNA polymerase (T7 RNAP) (EC 2.7.7.6) and the corresponding promoter pT7 was constructed. The gene for T7 RNAP was stably introduced into the P. pastoris chromosome 2 under control of the (endogenous) constitutive P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (pGAP). The gene product T7 RNAP was engineered to contain a nuclear localization signal, which directed recombinant T7 RNAP to the P. pastoris nucleus. To promote translation of uncapped T7 RNAP derived transcripts, the internal ribosomal entry site from hepatitis C virus (HCV-IRES) was inserted directly upstream of the multiple cloning site of pEZT7. A P. pastoris autonomous replicating sequence (PARS1) was integrated into pEZT7 enabling propagation and recovery of plasmids from P. pastoris. Rapid amplification of 5' complementary DNA ends (5' RACE) experiments employing the test plasmid pEZT7-EGFP revealed that transcripts indeed initiated at pT7. HCV-IRES mediated translation of the latter mRNAs, however, was not observed. Surprisingly, HCV-IRES and the reverse complement of PARS1 (PARS1rc) were both found to display significant promoter activity as shown by 5' RACE. PMID:24056257

  11. [Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].

    Science.gov (United States)

    Zheng, Hai-Xue; Jin, Ye; Yin, Shuang-Hui; Guo, Hui-Chen; Shang, You-Jun; Bai, Xing-Wen; Liu, Xiang-Tao; Xie, Qing-Ge

    2007-09-01

    To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system. PMID:18051880

  12. Mechanisms and Developmental Roles of Promoter-proximal Pausing of RNA Polymerase II

    Science.gov (United States)

    Robinson, Christine; Lowe, Matthew; Schwartz, Amanda; Kikyo, Nobuaki

    2016-01-01

    RNA polymerase II (Pol II) temporarily stops transcription after synthesizing 30–50 bases, and resumes elongation only after stimulations by various signaling molecules and developmental cues. This phenomenon, called promoter-proximal pausing, is observed in 10–50% of the entire genes from Drosophila embryos to human cells. Release of paused Pol II is primarily mediated by the activated form of positive transcription elongation factor b (P-TEFb) initially sequestered in the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Many proteins and RNAs have been discovered and studied in detail to explain the process of the pausing and release of Pol II in relation to P-TEFb. At the functional level, promoter-proximal pausing regulates genes involved in stimulus-response and development in Drosophila. In mammalian stem cell biology, pausing is important for proliferation and signaling in embryonic stem cells and the formation of induced pluripotent stem cells. Other than this, however, little is known about the biological significance of pausing in mammalian cell differentiation. Further study on pausing mechanisms as well as its functions will contribute to the development of stem cell biology and its clinical applications.

  13. Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter.

    Science.gov (United States)

    Tomescu, Alexandra I; Robb, Nicole C; Hengrung, Narin; Fodor, Ervin; Kapanidis, Achillefs N

    2014-08-12

    The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction. PMID:25071209

  14. Ups and Downs of Poised RNA Polymerase II in B-Cells.

    Science.gov (United States)

    Dao, Phuong; Wojtowicz, Damian; Nelson, Steevenson; Levens, David; Przytycka, Teresa M

    2016-04-01

    Recent genome-wide analyses have uncovered a high accumulation of RNA polymerase II (Pol II) at the 5' end of genes. This elevated Pol II presence at promoters, referred to here as Poll II poising, is mainly (but not exclusively) attributed to temporal pausing of transcription during early elongation which, in turn, has been proposed to be a regulatory step for processes that need to be activated "on demand". Yet, the full genome-wide regulatory role of Pol II poising is yet to be delineated. To elucidate the role of Pol II poising in B cell activation, we compared Pol II profiles in resting and activated B cells. We found that while Pol II poised genes generally overlap functionally among different B cell states and correspond to the functional groups previously identified for other cell types, non-poised genes are B cell state specific. Focusing on the changes in transcription activity upon B cell activation, we found that the majority of such changes were from poised to non-poised state. The genes showing this type of transition were functionally enriched in translation, RNA processing and mRNA metabolic process. Interestingly, we also observed a transition from non-poised to poised state. Within this set of genes we identified several Immediate Early Genes (IEG), which were highly expressed in resting B cell and shifted from non-poised to poised state after B cell activation. Thus Pol II poising does not only mark genes for rapid expression in the future, but it is also associated with genes that are silenced after a burst of their expression. Finally, we performed comparative analysis of the presence of G4 motifs in the context of poised versus non-poised but active genes. Interestingly we observed a differential enrichment of these motifs upstream versus downstream of TSS depending on poising status. The enrichment of G4 sequence motifs upstream of TSS of non-poised active genes suggests a potential role of quadruplexes in expression regulation. PMID:27078128

  15. Ups and Downs of Poised RNA Polymerase II in B-Cells.

    Directory of Open Access Journals (Sweden)

    Phuong Dao

    2016-04-01

    Full Text Available Recent genome-wide analyses have uncovered a high accumulation of RNA polymerase II (Pol II at the 5' end of genes. This elevated Pol II presence at promoters, referred to here as Poll II poising, is mainly (but not exclusively attributed to temporal pausing of transcription during early elongation which, in turn, has been proposed to be a regulatory step for processes that need to be activated "on demand". Yet, the full genome-wide regulatory role of Pol II poising is yet to be delineated. To elucidate the role of Pol II poising in B cell activation, we compared Pol II profiles in resting and activated B cells. We found that while Pol II poised genes generally overlap functionally among different B cell states and correspond to the functional groups previously identified for other cell types, non-poised genes are B cell state specific. Focusing on the changes in transcription activity upon B cell activation, we found that the majority of such changes were from poised to non-poised state. The genes showing this type of transition were functionally enriched in translation, RNA processing and mRNA metabolic process. Interestingly, we also observed a transition from non-poised to poised state. Within this set of genes we identified several Immediate Early Genes (IEG, which were highly expressed in resting B cell and shifted from non-poised to poised state after B cell activation. Thus Pol II poising does not only mark genes for rapid expression in the future, but it is also associated with genes that are silenced after a burst of their expression. Finally, we performed comparative analysis of the presence of G4 motifs in the context of poised versus non-poised but active genes. Interestingly we observed a differential enrichment of these motifs upstream versus downstream of TSS depending on poising status. The enrichment of G4 sequence motifs upstream of TSS of non-poised active genes suggests a potential role of quadruplexes in expression

  16. Electrochemical biosensor for microRNA detection based on poly(U) polymerase mediated isothermal signal amplification.

    Science.gov (United States)

    Zhou, Yunlei; Yin, Huanshun; Li, Jie; Li, Bingchen; Li, Xue; Ai, Shiyun; Zhang, Xiansheng

    2016-05-15

    MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3' end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3'-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3'-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p-nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7 fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000 fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones. PMID:26700579

  17. Archaeal extrachromosomal genetic elements

    DEFF Research Database (Denmark)

    Wang, Haina; Peng, Nan; Shah, Shiraz Ali;

    2015-01-01

    viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered...

  18. Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase.

    Science.gov (United States)

    Deredge, Daniel; Li, Jiawen; Johnson, Kenneth A; Wintrode, Patrick L

    2016-05-01

    New nonnucleoside analogs are being developed as part of a multi-drug regimen to treat hepatitis C viral infections. Particularly promising are inhibitors that bind to the surface of the thumb domain of the viral RNA-dependent RNA polymerase (NS5B). Numerous crystal structures have been solved showing small molecule non-nucleoside inhibitors bound to the hepatitis C viral polymerase, but these structures alone do not define the mechanism of inhibition. Our prior kinetic analysis showed that nonnucleoside inhibitors binding to thumb site-2 (NNI2) do not block initiation or elongation of RNA synthesis; rather, they block the transition from the initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex. Here we have mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange kinetics. All three inhibitors rigidify an extensive allosteric network extending >40 Å from the binding site, thus providing a structural rationale for the observed disruption of the transition from distributive initiation to processive elongation. The two more potent inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip, which may contribute their stronger inhibition. These results establish that NNI2 inhibitors act through long range allosteric effects, reveal important conformational changes underlying normal polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:27006396

  19. Differential response of archaeal groups to land use change in an acidic red soil.

    Science.gov (United States)

    Shen, Ju-Pei; Cao, Peng; Hu, Hang-Wei; He, Ji-Zheng

    2013-09-01

    Land use management, one of the most important aspects of anthropogenic disturbance to terrestrial ecosystems, has exerted overriding impacts on soil biogeochemical cycling and inhabitant microorganisms. However, the knowledge concerning response of different archaeal groups to long-term land use changes is still limited in terrestrial environments. Here we used quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE) approaches to investigate the response of archaeal communities to four different land use practices, i.e. cropland, pine forest, restoration land and degradation land. qPCR analyses showed that expression of the archaeal amoA gene responds more sensitively to changes of land use. In particular, we observed, occurring at significantly lower numbers of archaeal amoA genes in degradation land samples, while the abundance of total archaea and Group 1.1c based on 16S rRNA gene copy numbers remained constant among the different treatments examined. Soil nitrate content is significantly correlated with archaeal amoA gene abundance, but not their bacterial counterparts. The percentage of archaea among total prokaryote communities increases with increasing depth, but has no significant relationship with total carbon, total nitrogen or pH. Soil pH was significantly correlated with total bacterial abundance. Based on results from PCR-DGGE, three land use practices (i.e. cropland, pine forest, restoration land) showed distinct dominant bands, which were mostly affiliated with Group 1.1a. Degradation land, however, was dominated by sequences belonging to Group 1.1c. Results from this study suggest that community structure of ammonia oxidizing archaea were significantly impacted by land use practices. PMID:23774250

  20. A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II.

    Science.gov (United States)

    Wang, Ying; Qu, Jie; Ji, Shaoyi; Wallace, Andrew J; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

    2016-05-01

    Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP PSTVd replication in the nucleoplasm generates (-)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (-)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

  1. A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II[OPEN

    Science.gov (United States)

    Qu, Jie; Ji, Shaoyi; Wallace, Andrew J.; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

    2016-01-01

    Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (−)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (−)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

  2. Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter

    OpenAIRE

    Tomescu, Alexandra I.; Robb, Nicole C.; Hengrung, Narin; Fodor, Ervin; Kapanidis, Achillefs N.

    2014-01-01

    The genome of the influenza virus consists of eight single-stranded segments of RNA with highly conserved 5′ and 3′ termini. These termini associate to form double-stranded structures that act as promoters for viral transcription and replication. Structural information on the polymerase-bound promoter currently does not exist, so to address this we developed a sensitive single-molecule FRET assay that allowed us to measure distances between fluorescent dyes located on the promoter and map its...

  3. Structural Characterization of RNA Polymerase II Complexes Arrested by a Cyclobutane Pyrimidine Dimer in the Transcribed Strand of Template DNA*

    OpenAIRE

    Tornaletti, Silvia; Reines, Daniel; Hanawalt, Philip C.

    1999-01-01

    We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested...

  4. Casein Kinase 2 Associates with Initiation-Competent RNA Polymerase I and Has Multiple Roles in Ribosomal DNA Transcription

    OpenAIRE

    Panova, Tatiana B; Panov, Kostya I.; Russell, Jackie; Zomerdijk, Joost C. B. M.

    2006-01-01

    Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Iβ isoform and not with Pol Iα. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Iβ-associated CK2 can phosphorylate topoisomerase IIα in Pol Iβ, activator upstream binding factor (UB...

  5. A Region of Bdp1 Necessary for Transcription Initiation That Is Located within the RNA Polymerase III Active Site Cleft

    OpenAIRE

    Hu, Hui-Lan; Wu, Chih-Chien; Lee, Jin-Cheng; Chen, Hung-Ta

    2015-01-01

    The RNA polymerase III (Pol III)-specific transcription factor Bdp1 is crucial to Pol III recruitment and promoter opening in transcription initiation, yet structural information is sparse. To examine its protein-binding targets within the preinitiation complex at the residue level, photoreactive amino acids were introduced into Saccharomyces cerevisiae Bdp1. Mutations within the highly conserved SANT domain cross-linked to the transcription factor IIB (TFIIB)-related transcription factor Brf...

  6. UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity

    Czech Academy of Sciences Publication Activity Database

    Sobol, Margaryta; Yildirim, Sukriye; Philimonenko, Vlada; Marášek, Pavel; Castano, Enrique; Hozák, Pavel

    2013-01-01

    Roč. 4, č. 6 (2013), 478–486. ISSN 1949-1034 R&D Projects: GA ČR GAP305/11/2232; GA MŠk LD12063; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : PIP2 * mitosis * transcription * nucleolus * RNA polymerase I * UBF * fibrillarin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.148, year: 2013

  7. Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

    Directory of Open Access Journals (Sweden)

    Shrividhya Srinivasan

    2008-10-01

    Full Text Available Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II. Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

  8. Allele-specific distribution of RNA polymerase II on female X chromosomes.

    Science.gov (United States)

    Kucera, Katerina S; Reddy, Timothy E; Pauli, Florencia; Gertz, Jason; Logan, Jenae E; Myers, Richard M; Willard, Huntington F

    2011-10-15

    While the distribution of RNA polymerase II (PolII) in a variety of complex genomes is correlated with gene expression, the presence of PolII at a gene does not necessarily indicate active expression. Various patterns of PolII binding have been described genome wide; however, whether or not PolII binds at transcriptionally inactive sites remains uncertain. The two X chromosomes in female cells in mammals present an opportunity to examine each of the two alleles of a given locus in both active and inactive states, depending on which X chromosome is silenced by X chromosome inactivation. Here, we investigated PolII occupancy and expression of the associated genes across the active (Xa) and inactive (Xi) X chromosomes in human female cells to elucidate the relationship of gene expression and PolII binding. We find that, while PolII in the pseudoautosomal region occupies both chromosomes at similar levels, it is significantly biased toward the Xa throughout the rest of the chromosome. The general paucity of PolII on the Xi notwithstanding, detectable (albeit significantly reduced) binding can be observed, especially on the evolutionarily younger short arm of the X. PolII levels at genes that escape inactivation correlate with the levels of their expression; however, additional PolII sites can be found at apparently silenced regions, suggesting the possibility of a subset of genes on the Xi that are poised for expression. Consistent with this hypothesis, we show that a high proportion of genes associated with PolII-accessible sites, while silenced in GM12878, are expressed in other female cell lines. PMID:21791549

  9. Application of X-ray absorption spectroscopy and anomalous small angle scattering to RNA polymerase

    International Nuclear Information System (INIS)

    X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (≤∝5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the EXAFS region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near-physiological concentrations (as low as ∝100 μM). With careful choice of model compounds, judicious use of fitting procedures, and consideration of the results of biochemical and other spectrOScopic results, this data has provided pivotal information about the structures of these active sites which store energy in their conformation changes or ligand exchanges. Although the application of anomalous small angle scattering to biomolecules has occurred more recently, it clearly provides a method of determining distances between active sites that are outside the range of X-ray absorption spectroscopy. The wavelength dependence of the X-ray scattering power varies rapidly near the edge of the absorbing atom in both amplitude and phase. This behavior selectively alters the contribution of the absorbing atom to the scattering pattern. The structure-function relationship of the intermediate states provide the key to understanding the mechanisms of these complex molecules. It is this precise structural information about the active sites that is not obtainable by other spectroscopic techniques. Combination of these techniques offers a unique approach to the determination of the organization of active sites in biomolecules, especially metalloenzymes. Application of these methods to the substrate and template binding sites of RNA polymerase which contain zinc atoms demonstrates the versatility of this approach. (orig.)

  10. Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

    Science.gov (United States)

    Scott, D J; Ferguson, A L; Gallegos, M T; Pitt, M; Buck, M; Hoggett, J G

    2000-12-01

    The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed. PMID:11085949

  11. A human RNA polymerase II subunit is encoded by a recently generated multigene family

    Directory of Open Access Journals (Sweden)

    Mattei Marie-Geneviève

    2001-11-01

    Full Text Available Abstract Background The sequences encoding the yeast RNA polymerase II (RPB subunits are single copy genes. Results While those characterized so far for the human (h RPB are also unique, we show that hRPB subunit 11 (hRPB11 is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex ; the second generates polypeptides hRPB11bα and hRPB11bβ through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR. Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11bα is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G, and encodes a single polypeptide which is identical to subunit hRPB11a. Conclusions The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

  12. A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Ayan Banerjee

    Full Text Available BACKGROUND: Making the correct choice between transcription elongation and transcription termination is essential to the function of RNA polymerase II, and fundamental to gene expression. This choice can be influenced by factors modifying the transcription complex, factors modifying chromatin, or signals mediated by the template or transcript. To aid in the study of transcription elongation and termination we have developed a transcription elongation reporter system that consists of tandem luciferase reporters flanking a test sequence of interest. The ratio of expression from the reporters provides a measure of the relative rates of successful elongation through the intervening sequence. METHODOLOGY/PRINCIPAL FINDINGS: Size matched fragments containing the polyadenylation signal of the human beta-actin gene (ACTB and the human beta-globin gene (HBB were evaluated for transcription termination using this new ratiometric tandem reporter assay. Constructs bearing just 200 base pairs on either side of the consensus poly(A addition site terminated 98% and 86% of transcription for ACTB and HBB sequences, respectively. The nearly 10-fold difference in read-through transcription between the two short poly(A regions was eclipsed when additional downstream poly(A sequence was included for each gene. Both poly(A regions proved very effective at termination when 1100 base pairs were included, stopping 99.6% of transcription. To determine if part of the increased termination was simply due to the increased template length, we inserted several kilobases of heterologous coding sequence downstream of each poly(A region test fragment. Unexpectedly, the additional length reduced the effectiveness of termination of HBB sequences 2-fold and of ACTB sequences 3- to 5-fold. CONCLUSIONS/SIGNIFICANCE: The tandem construct provides a sensitive measure of transcription termination in human cells. Decreased Xrn2 or Senataxin levels produced only a modest release from

  13. Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression*

    OpenAIRE

    Wang, Zhibo; Cotney, Justin; Shadel, Gerald S.

    2007-01-01

    The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POLRMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcrip...

  14. RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA polymerase recycling in transcription

    OpenAIRE

    Sukhodolets, Maxim V.; Cabrera, Julio E.; Zhi, Huijun; Jin, Ding Jun

    2001-01-01

    We report that RapA, an Escherichia coli RNA polymerase (RNAP)-associated homolog of SWI2/SNF2, is capable of dramatic activation of RNA synthesis. The RapA-mediated transcriptional activation in vitro depends on supercoiled DNA and high salt concentrations, a condition that is likely to render the DNA superhelix tightly compacted. Moreover, RapA activates transcription by stimulating RNAP recycling. Mutational analyses indicate that the ATPase activity of RapA is essential for its function a...

  15. Human RNA Polymerase III transcriptomes and relationships to Pol II promoters, enhancer-binding factors and chromatin domains

    OpenAIRE

    Oler, Andrew J.; Alla, Ravi K.; Roberts, Douglas N.; Wong, Alexander; Hollenhorst, Peter C.; Chandler, Katherine J.; Cassiday, Patrick A.; Nelson, Cassie A.; Hagedorn, Curt H.; Graves, Barbara J.; Cairns, Bradley R

    2010-01-01

    RNA polymerase (Pol) III transcribes many noncoding RNAs (e.g. tRNAs) important for translational capacity and other functions. Here, we localized Pol III, alternative TFIIIB complexes (BRF1/2) and TFIIIC in HeLa cells, determining the Pol III transcriptome, defining gene classes, and revealing ‘TFIIIC-only’ sites. Pol III localization in other transformed and primary cell lines revealed novel and cell-type specific Pol III loci, and one miRNA. Surprisingly, only a fraction of the in silico-p...

  16. Use of Existing Diagnostic Reverse-Transcription Polymerase Chain Reaction Assays for Detection of Ebola Virus RNA in Semen.

    Science.gov (United States)

    Pettitt, James; Higgs, Elizabeth S; Adams, Rick D; Jahrling, Peter B; Hensley, Lisa E

    2016-04-15

    Sexual transmission of Ebola virus in Liberia has now been documented and associated with new clusters in regions previously declared Ebola free. Assays that have Emergency Use Authorization (EUA) and are routinely used to detect Ebola virus RNA in whole blood and plasma specimens at the Liberian Institute for Biomedical Research were tested for their suitability in detecting the presence of Ebola virus RNA in semen. Qiagen AVL extraction protocols, as well as the Ebola Zaire Target 1 and major groove binder quantitative reverse-transcription polymerase chain reaction assays, were demonstrably suitable for this purpose and should facilitate epidemiologic investigations, including those involving long-term survivors of Ebola. PMID:26374912

  17. The yeast TFB1 and SSL1 genes, which encode subunits of transcription factor IIH, are required for nucleotide excision repair and RNA polymerase II transcription.

    OpenAIRE

    Z. Wang; Buratowski, S; Svejstrup, J Q; Feaver, W J; Wu, X; Kornberg, R D; Donahue, T F; Friedberg, E C

    1995-01-01

    The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive tempera...

  18. The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase.

    Science.gov (United States)

    Gwynn, Emma J; Smith, Abigail J; Guy, Colin P; Savery, Nigel J; McGlynn, Peter; Dillingham, Mark S

    2013-01-01

    UvrD-like helicases play diverse roles in DNA replication, repair and recombination pathways. An emerging body of evidence suggests that their different cellular functions are directed by interactions with partner proteins that target unwinding activity to appropriate substrates. Recent studies in E. coli have shown that UvrD can act as an accessory replicative helicase that resolves conflicts between the replisome and transcription complexes, but the mechanism is not understood. Here we show that the UvrD homologue PcrA interacts physically with B. subtilis RNA polymerase, and that an equivalent interaction is conserved in E. coli where UvrD, but not the closely related helicase Rep, also interacts with RNA polymerase. The PcrA-RNAP interaction is direct and independent of nucleic acids or additional mediator proteins. A disordered but highly conserved C-terminal region of PcrA, which distinguishes PcrA/UvrD from otherwise related enzymes such as Rep, is both necessary and sufficient for interaction with RNA polymerase. PMID:24147116

  19. The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase.

    Directory of Open Access Journals (Sweden)

    Emma J Gwynn

    Full Text Available UvrD-like helicases play diverse roles in DNA replication, repair and recombination pathways. An emerging body of evidence suggests that their different cellular functions are directed by interactions with partner proteins that target unwinding activity to appropriate substrates. Recent studies in E. coli have shown that UvrD can act as an accessory replicative helicase that resolves conflicts between the replisome and transcription complexes, but the mechanism is not understood. Here we show that the UvrD homologue PcrA interacts physically with B. subtilis RNA polymerase, and that an equivalent interaction is conserved in E. coli where UvrD, but not the closely related helicase Rep, also interacts with RNA polymerase. The PcrA-RNAP interaction is direct and independent of nucleic acids or additional mediator proteins. A disordered but highly conserved C-terminal region of PcrA, which distinguishes PcrA/UvrD from otherwise related enzymes such as Rep, is both necessary and sufficient for interaction with RNA polymerase.

  20. Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription.

    Science.gov (United States)

    Banerjee, Rajeev; Weidman, Mary K; Navarro, Sonia; Comai, Lucio; Dasgupta, Asim

    2005-08-01

    Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3C(pro) appeared to cleave the TATA-binding protein-associated factor 110 (TAF(110)), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF(110) and purified 3C(pro) indicated that the Q(265)G(266) and Q(805)G(806) sites were cleaved by 3C(pro). Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells. PMID:16033979