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Sample records for rna polymerase sigma

  1. 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.

  2. Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase.

    Science.gov (United States)

    Jishage, M; Dasgupta, D; Ishihama, A

    2001-05-01

    Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase sigma(70) subunit. The contact site of Rsd on sigma(70) was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted sigma(70) and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter -35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and sigma(70) contact transcription factors.

  3. Mass spectrometry of Escherichia coli RNA polymerase: interactions of the core enzyme with sigma70 and Rsd protein.

    Science.gov (United States)

    Ilag, Leopold L; Westblade, Lars F; Deshayes, Caroline; Kolb, Annie; Busby, Stephen J W; Robinson, Carol V

    2004-02-01

    The E. coli RNA polymerase core enzyme is a multisubunit complex of 388,981 Da. To initiate transcription at promoters, the core enzyme associates with a sigma subunit to form holo RNA polymerase. Here we have used nanoflow electrospray mass spectrometry, coupled with tandem mass spectrometry, to probe the interaction of the RNA polymerase core enzyme with the most abundant sigma factor, sigma70. The results show remarkably well-resolved spectra for both the core and holo RNA polymerases. The regulator of sigma70, Rsd protein, has previously been identified as a protein that binds to free sigma70. We show that Rsd also interacts with core enzyme. In addition, by adding increasing amounts of Rsd, we show that sigma70 is displaced from holo RNA polymerase, resulting in complexes of Rsd with core and sigma70. The results argue for a model in which Rsd not only sequesters sigma70, but is also an effector of core RNA polymerase.

  4. The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme E sigma 70 and by the heat shock RNA polymerase E sigma 32.

    Science.gov (United States)

    Charpentier, B; Branlant, C

    1994-02-01

    Escherichia coli D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is produced by the gapA gene and is structurally related to eukaryotic GAPDHs. These facts led to the proposal that the gapA gene originated by a horizontal transfer of genetic information. The yields and start sites of gapA mRNAs produced in various fermentation conditions and genetic contexts were analyzed by primer extension. The transcriptional regulatory region of the gapA gene was found to contain four promoter sequences, three recognized by the vegetative RNA polymerase E sigma 70 and one recognized by the heat shock RNA polymerase E sigma 32. Transcription of gapA by E sigma 32 is activated in the logarithmic phase under conditions of starvation and of heat shock. Using a GAPDH- strain, we found that GAPDH production has a positive effect on cell growth at 43 degrees C. Thus, E. coli GAPDH displays some features of heat shock proteins. One of the gapA promoter sequences transcribed by E sigma 70 is subject to catabolic repression. Another one has growth phase-dependent efficiency. This complex area of differentially regulated promoters allows the production of large amounts of gapA transcripts in a wide variety of environmental conditions. On the basis of these data, the present view of E sigma 32 RNA polymerase function has to be enlarged, and the various hypotheses on E. coli gapA gene origin have to be reexamined.

  5. Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

    Science.gov (United States)

    Jishage, M; Ishihama, A

    1999-06-01

    The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

  6. A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response sigma(E) in Escherichia coli.

    Science.gov (United States)

    Rezuchova, B; Kormanec, J

    2001-06-01

    We have previously established a two-plasmid system in Escherichia coli for identification of promoters recognized by RNA polymerase containing a heterologous sigma factor. Attempts to optimize this system for identification of promoters recognized by RNA polymerase containing E. coli extracytoplasmic stress response sigma(E) failed owing to high toxicity of the expressed rpoE. A new system for identification of sigma(E)-cognate promoters was established, and verified using the two known sigma(E)-dependent promoters, rpoEp2 and degPp. Expression of the sigma(E)-encoding rpoE gene was under the control of the AraC-dependent P(BAD) promoter. A low level of arabinose induced a non-toxic, however, sufficient level of sigma(E) to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized both known sigma(E)-dependent promoters, rpoEp2 and degPp, which were cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZ alpha reporter gene. This new system has proved to be useful for identification of E. coli sigma(E)-cognate promoters. Moreover, the system could be used for identification of ECF sigma-cognate promoters from other bacteria.

  7. Mapping of the Rsd Contact Site on the Sigma 70 Subunit of Escherichia coli RNA Polymerase

    OpenAIRE

    Jishage, Miki; Dasgupta, Dipak; Ishihama, Akira

    2001-01-01

    Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase ς70 subunit. The contact site of Rsd on ς70 was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted ς70 and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter −35 recognition helix-turn-helix motif within region 4, overlapping with the regions in...

  8. The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum

    OpenAIRE

    Šilar, Radoslav

    2016-01-01

    Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster...

  9. Transcriptional Organization and In Vivo Role of the Escherichia coli rsd Gene, Encoding the Regulator of RNA Polymerase Sigma D

    OpenAIRE

    Jishage, Miki; Ishihama, Akira

    1999-01-01

    The regulator of sigma D (Rsd) was identified as an RNA polymerase ς70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of ς70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953–4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, ςS-dependent P1 and ς70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expressi...

  10. In silico Analysis and Molecular Modeling of RNA Polymerase, Sigma S (RpoS Protein in Pseudomonas aeruginosa PAO1

    Directory of Open Access Journals (Sweden)

    Mansour Sedighi

    2015-10-01

    Full Text Available Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S gene encodes sigma-38 (σ38, or RpoS, a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection. Methods: The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. Results: The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D structure was modeled using SWISS-MODEL Workspace. Conclusion: Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions.

  11. A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp.

    Science.gov (United States)

    Potúcková, L; Kelemen, G H; Findlay, K C; Lonetto, M A; Buttner, M J; Kormanec, J

    1995-07-01

    A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial sigma 70 family. Homologues were present in other Streptomyces spp., and that of the genetically well studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to sigma B of Bacillus subtilis. However, while sigma B is involved in stationary-phase gene expression and in the general stress response in B. subtilis, sigma F affects morphological differentiation in Streptomyces. Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. sigma F is proposed to control the late stages of spore development in Streptomyces.

  12. The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase

    Science.gov (United States)

    Shimada, Tomohiro; Tanaka, Kan

    2017-01-01

    The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the “constitutive promoters” that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters was achieved for RpoD (σ70), the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of “inducible promoters” in the presence of additional supporting factors. PMID:28666008

  13. The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase.

    Directory of Open Access Journals (Sweden)

    Tomohiro Shimada

    Full Text Available The promoter selectivity of Escherichia coli RNA polymerase (RNAP is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the "constitutive promoters" that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters was achieved for RpoD (σ70, the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38, heat-shock specific RpoH (σ32, flagellar-chemotaxis specific RpoF (σ28 and extra-cytoplasmic stress-response RpoE (σ24. The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of "inducible promoters" in the presence of additional supporting factors.

  14. Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Dostálová, Hana; Holátko, Jiří; Busche, T.; Rucká, Lenka; Rapoport, Andrey; Halada, Petr; Nešvera, Jan; Kalinowski, J.; Pátek, Miroslav

    2017-01-01

    Roč. 7, JUN 23 (2017), s. 1-13, č. článku 133. ISSN 2191-0855 R&D Projects: GA ČR(CZ) GA17-06991S Institutional support: RVO:61388971 Keywords : Corynebacterium glutamicum * Promoter * Sigma factor Subject RIV: EE - Microbiology, Virology Impact factor: 1.825, year: 2016

  15. The main early and late promoters of Bacillus subtilis phage phi 29 form unstable open complexes with sigma A-RNA polymerase that are stabilized by DNA supercoiling.

    Science.gov (United States)

    Rojo, F; Nuez, B; Mencía, M; Salas, M

    1993-02-25

    Most Escherichia coli promoters studied so far form stable open complexes with sigma 70-RNA polymerase which have relatively long half-lives and, therefore, are resistant to a competitor challenge. A few exceptions are nevertheless known. The analysis of a number of promoters in Bacillus subtilis has suggested that the instability of open complexes formed by the vegetative sigma A-RNA polymerase may be a more general phenomenon than in Escherichia coli. We show that the main early and late promoters from the Bacillus subtilis phage phi 29 form unstable open complexes that are stabilized either by the formation of the first phosphodiester bond between the initiating nucleoside triphosphates or by DNA supercoiling. The functional characteristics of these two strong promoters suggest that they are not optimized for a tight and stable RNA polymerase binding. Their high activity is probably the consequence of the efficiency of further steps leading to the formation of an elongation complex.

  16. Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions.

    OpenAIRE

    Jishage, M; Iwata, A.; Ueda, S.; Ishihama, A

    1996-01-01

    By a quantitative Western immunoblot analysis, the intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma S, the rpoS gene product), and of two minor sigma subunits, sigma 54 (sigma N, the rpoN gene product) and sigma 28 (sigma F, the rpoF gene product), were determined in two Escherichia coli strains, W3110 and MC4100. The results indicated that the levels of sigma 54 and sigma 28 are maintained at 10 and 50%, respectively, of the ...

  17. Differential mechanisms of binding of anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA to E. coli RNA polymerase lead to diverse physiological consequences.

    Science.gov (United States)

    Sharma, Umender K; Chatterji, Dipankar

    2008-05-01

    Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to sigma(70) with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

  18. The stress sigma factor of RNA polymerase RpoS/σS is a solvent exposed open molecule in solution.

    Science.gov (United States)

    Cavaliere, Paola; Brier, Sébastien; Filipenko, Petr; Sizun, Christina; Raynal, Bertrand; Bonneté, Françoise; Levi-Acobas, Fabienne; Bellalou, Jacques; England, Patrick; Chamot-Rooke, Julia; Mayer, Claudine; Norel, Françoise

    2017-12-11

    In bacteria, one primary and multiple alternative σ factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl. ©2017 The Author(s).

  19. Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa.

    Science.gov (United States)

    Dove, S L; Hochschild, A

    2001-11-01

    A number of transcriptional regulators mediate their effects through direct contact with the sigma(70) subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of sigma(70) that harbors conserved region 4. This region of sigma contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma(70)-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-sigma factor Rsd and the sigma(70) subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of sigma(70) and also that amino acid substitution R596H, within region 4 of sigma(70), weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between sigma and two other regulators shown previously to contact region 4 of sigma(70). We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the sigma(70) subunit of RNAP from this organism. We found that amino acid substitution R600H in sigma(70) from P. aeruginosa, corresponding to the R596H substitution in E. coli sigma(70), specifically weakens the interaction between AlgQ and sigma(70). Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of sigma(70) and probably regulate gene expression through this contact.

  20. Increased bioplastic production with an RNA polymerase sigma factor SigE during nitrogen starvation in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Osanai, Takashi; Numata, Keiji; Oikawa, Akira; Kuwahara, Ayuko; Iijima, Hiroko; Doi, Yoshiharu; Tanaka, Kan; Saito, Kazuki; Hirai, Masami Yokota

    2013-12-01

    Because cyanobacteria directly harvest CO2 and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.

  1. Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences▿

    Science.gov (United States)

    Sharma, Umender K.; Chatterji, Dipankar

    2008-01-01

    Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70. PMID:18359804

  2. Characterization of four nuclear-encoded plastid RNA polymerase sigma factor genes in the liverwort Marchantia polymorpha: blue-light- and multiple stress-responsive SIG5 was acquired early in the emergence of terrestrial plants.

    Science.gov (United States)

    Kanazawa, Takehiko; Ishizaki, Kimitsune; Kohchi, Takayuki; Hanaoka, Mitsumasa; Tanaka, Kan

    2013-10-01

    The plastids of plant cells each contain their own genome, and a bacterial-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP) is involved in transcription of this genome. While the catalytic core subunits are encoded by the plastid genome, the specificity subunit of PEP, sigma, is generally encoded by the nuclear genome and imported into plastids from the cytoplasm after translation. In this study, we identified and analyzed four sigma factor genes from the nuclear genome of a liverwort, Marchantia polymorpha. Phylogenetic analysis suggested that three of the four genes were orthologous to vascular plant genes and thus they were named MpSIG1, MpSIG2 and MpSIG5. The remaining gene was named MpSIGX. The gene products were predicted to localize to the plastid, and this prediction was experimentally demonstrated by expressing yellow fluorescent protein fusion genes in vivo. As with SIG5 genes of other plant species, expression of MpSIG5 was induced by blue-light irradiation and also under various stress conditions, indicating that the regulatory mechanism responsible is conserved among divergent plant species. However, while the major role of SIG5 in vascular plants is to repair the damaged PSII reaction center through psbD gene transcription, the relevant blue-light-responsive promoter (psbD-BLRP) was not found in M. polymorpha and psbD transcript accumulation did not occur in conjunction with MpSIG5 induction. Thus, the physiological role of SIG5 is probably divergent among plant phyla.

  3. A Perspective on the Enhancer Dependent Bacterial RNA Polymerase

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    Nan Zhang

    2015-05-01

    Full Text Available Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

  4. Disengaging polymerase: Terminating RNA polymerase II transcription in budding yeast☆

    Science.gov (United States)

    Mischo, Hannah E.; Proudfoot, Nick J.

    2013-01-01

    Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3′ end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. PMID:23085255

  5. RNA polymerase activity of Ustilago maydis virus

    Energy Technology Data Exchange (ETDEWEB)

    Yie, S.W.

    1986-01-01

    Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

  6. Structural Biology of Bacterial RNA Polymerase

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    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.

  7. RNA polymerase II collision interrupts convergent transcription

    DEFF Research Database (Denmark)

    Hobson, David J; Wei, Wu; Steinmetz, Lars M

    2012-01-01

    Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical...... and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo...... also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent...

  8. RNA Polymerase II–The Transcription Machine

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 3. RNA Polymerase II – The Transcription Machine - Nobel Prize in Chemistry 2006. Jiyoti Verma Aruna Naorem Anand Kumar Manimala Sen Parag Sadhale. General Article Volume 12 Issue 3 March 2007 pp 47-53 ...

  9. Structure and Function of Caliciviral RNA Polymerases

    Directory of Open Access Journals (Sweden)

    Ji-Hye Lee

    2017-11-01

    Full Text Available Caliciviruses are a leading agent of human and animal gastroenteritis and respiratory tract infections, which are growing concerns in immunocompromised individuals. However, no vaccines or therapeutics are yet available. Since the rapid rate of genetic evolution of caliciviruses is mainly due to the error-prone nature of RNA-dependent RNA polymerase (RdRp, this article focuses on recent studies of the structures and functions of RdRp from caliciviruses. It also provides recent advances in the interactions of RdRp with virion protein genome-linked (VPg and RNA and the structural and functional features of its precursor.

  10. Chemical fidelity of an RNA polymerase ribozyme

    DEFF Research Database (Denmark)

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

    2013-01-01

    The emergence of catalytically active RNA enzymes (ribozymes) is widely believed to have been an important transition in the origin of life. In the context of a likely heterogeneous chemical environment, substrate specificity and selectivity of these primordial enzymes would have been critical...... for function. Here we have explored the chemical fidelity, i.e. substrate selectivity and specificity for both single and multiple catalytic steps of the Z RNA polymerase ribozyme-a modern day analogue of the primordial RNA replicase. Using a wide range of nucleotide analogues and ionic conditions, we observe...

  11. Origin and Evolution of RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    de Farias, Savio T; Dos Santos Junior, Ariosvaldo P; Rêgo, Thais G; José, Marco V

    2017-01-01

    RNA-dependent RNA polymerases (RdRp) are very ancient enzymes and are essential for all viruses with RNA genomes. We reconstruct the origin and evolution of this polymerase since the initial stages of the origin of life. The origin of the RdRp was traced back from tRNA ancestors. At the origin of the RdRp the most ancient part of the protein is the cofactor-binding site that had the capacity of binding to simple molecules as magnesium, calcium, and ribonucleotides. Our results suggest that RdRp originated from junctions of proto-tRNAs that worked as the first genes at the emergence of the primitive translation system, where the RNA was the informational molecule. The initial domain, worked as a building block for the emergence of the fingers and thumb domains. From the ancestral RdRp, we could establish the evolutionary stages of viral evolution from a rooted ancestor to modern viruses. It was observed that the selective pressure under the RdRp was the organization and functioning of the genome, where RNA double-stranded and RNA single-stranded virus formed a separate group. We propose an evolutionary route to the polymerases and the results suggest an ancient scenario for the origin of RNA viruses.

  12. Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA.

    Science.gov (United States)

    Chen, Tingjian; Romesberg, Floyd E

    2017-07-26

    There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

  13. Analyzing RNA polymerase III by electron cryomicroscopy.

    Science.gov (United States)

    Fernández-Tornero, Carlos; Böttcher, Bettina; Rashid, Umar Jan; Müller, Christoph W

    2011-01-01

    Recent electron cryomicroscopy reconstructions have provided new insights into the overall organization of yeast RNA polymerase (Pol) III, responsible for the synthesis of small, non-translated RNAs. The structure of the free Pol III enzyme at 10 Å resolution provides an accurate framework to better understand its overall architecture and the structural organization and functional role of two Pol III-specific subcomplexes. Cryo-EM structures of elongating Pol III bound to DNA/RNA scaffolds show the rearrangement of the Pol III-specific subcomplexes that enclose incoming DNA. In one reconstruction downstream DNA and newly transcribed RNA can be followed over considerably longer distances as in the crystal structure of elongating Pol II. The Pol III transcription machinery is increasingly recognized as a possible target for cancer therapy. The recent cryo-EM reconstructions contribute to the molecular understanding of Pol III transcription as a prerequisite for targeting its components.

  14. Solving the RNA polymerase I structural puzzle

    Energy Technology Data Exchange (ETDEWEB)

    Moreno-Morcillo, María [European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Taylor, Nicholas M. I. [Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid (Spain); Gruene, Tim [Georg-August-University, Tammannstrasse 4, 37077 Göttingen (Germany); Legrand, Pierre [SOLEIL Synchrotron, L’Orme de Merisiers, Saint Aubin, Gif-sur-Yvette (France); Rashid, Umar J. [European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Ruiz, Federico M. [Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid (Spain); Steuerwald, Ulrich; Müller, Christoph W. [European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid (Spain); European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany)

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  15. Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

    Science.gov (United States)

    Esyunina, D M; Kulbachinskiy, A V

    2015-10-01

    The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

  16. Identification of a sigma B-dependent small noncoding RNA in Listeria monocytogenes

    DEFF Research Database (Denmark)

    Nielsen, Jesper Sejrup; Olsen, Anders Steno; Bonde, Mette

    2008-01-01

    In Listeria monocytogenes, the alternative sigma factor sigma(B) plays important roles in stress tolerance and virulence. Here, we present the identification of SbrA, a novel small noncoding RNA that is produced in a sigma(B)-dependent manner. This finding adds the sigma(B) regulon to the growing...

  17. RNA polymerase III promoter elements enhance transcription of RNA polymerase II genes

    Energy Technology Data Exchange (ETDEWEB)

    Oliviero, S.; Monaci, P.

    1988-02-25

    Using transient expression assays in cultured human cells the authors have observed that RNA Polymerase III promoter sequences exert a positive cis-acting enhancer effect on RNA Polymerase II transcription. A DNA segment containing a copy of the Alu repeated element enhances transcription of the liver specific Haptoglobin related (Hpr) promoter in Hepatoma cell lines but not in HeLa cells. A tRNA/sup pro/ gene acts as enhancer of the SV40 promoter both in Hepatoma and in HeLa cell lines. Transcription from the SV40 promoter is also enhanced by DNA segments containing only the box A or the box B of the tRNA/sup pro/ promoter.

  18. The polymerase of negative-stranded RNA viruses.

    Science.gov (United States)

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

    2013-04-01

    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 nonsegmented (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 transiently dissociated from NP during RNA synthesis. Here we summarize and compare the polymerases of NNS and SNS RNA viruses, and the current structural data on the polymerases. Those comparisons inform us on the evolution of related RNA synthesis machines which use two distinct mechanisms for mRNA cap formation. Copyright © 2013. Published by Elsevier B.V.

  19. Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing

    Energy Technology Data Exchange (ETDEWEB)

    Haag, Jeremy R.; Pikaard, Craig S.

    2011-08-01

    In all eukaryotes, nuclear DNA-dependent RNA polymerases I, II and III synthesize the myriad RNAs that are essential for life. Remarkably, plants have evolved two additional multisubunit RNA polymerases, RNA polymerases IV and V, which orchestrate non-coding RNA-mediated gene silencing processes affecting development, transposon taming, antiviral defence and allelic crosstalk. Biochemical details concerning the templates and products of RNA polymerases IV and V are lacking. However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family.

  20. 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...

  1. C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7.

    Science.gov (United States)

    Sadhale, P P; Woychik, N A

    1994-09-01

    We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

  2. Modulation of RNA polymerase activity through trigger loop folding

    OpenAIRE

    Miropolskaya, Nataliya; Nikiforov, Vadim; Klimašauskas, Saulius; Artsimovitch, Irina; Kulbachinskiy, Andrey

    2010-01-01

    Folding of the trigger loop of RNA polymerase promotes nucleotide addition through creating a closed, catalytically competent conformation of the active center. Here, we discuss the impact of adjacent RNA polymerase elements, including the F loop and the jaw domain, as well as external regulatory factors on the trigger loop folding and catalysis.

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

    NARCIS (Netherlands)

    Dorssers, L.

    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

  4. Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases

    Directory of Open Access Journals (Sweden)

    Aravind L

    2003-01-01

    Full Text Available Abstract Background The eukaryotic RNA-dependent RNA polymerase (RDRP is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. Results Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP, showed that the RDRP and the β' subunit of DDRP (and its orthologs in archaea and eukaryotes contain a conserved double-psi β-barrel (DPBB domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue, which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP β' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the

  5. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

    Directory of Open Access Journals (Sweden)

    Aravind L

    2008-10-01

    Full Text Available Abstract Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. Reviewers This article was reviewed by Eugene Koonin and Mark Ragan.

  6. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    OpenAIRE

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cy...

  7. Dysregulation of RNA polymerase I transcription during disease.

    Science.gov (United States)

    Hannan, K M; Sanij, E; Rothblum, L I; Hannan, R D; Pearson, R B

    2013-01-01

    Transcription of the ribosomal RNA genes by the dedicated RNA polymerase I enzyme and subsequent processing of the ribosomal RNA are fundamental control steps in the synthesis of functional ribosomes. Dysregulation of Pol I transcription and ribosome biogenesis is linked to the etiology of a broad range of human diseases. Diseases caused by loss of function mutations in the molecular constituents of the ribosome, or factors intimately associated with RNA polymerase I transcription and processing are collectively termed ribosomopathies. Ribosomopathies are generally rare and treatment options are extremely limited tending to be more palliative than curative. Other more common diseases are associated with profound changes in cellular growth such as cardiac hypertrophy, atrophy or cancer. In contrast to ribosomopathies, altered RNA polymerase I transcriptional activity in these diseases largely results from dysregulated upstream oncogenic pathways or by direct modulation by oncogenes or tumor suppressors at the level of the RNA polymerase I transcription apparatus itself. Ribosomopathies associated with mutations in ribosomal proteins and ribosomal RNA processing or assembly factors have been covered by recent excellent reviews. In contrast, here we review our current knowledge of human diseases specifically associated with dysregulation of RNA polymerase I transcription and its associated regulatory apparatus, including some cases where this dysregulation is directly causative in disease. We will also provide insight into and discussion of possible therapeutic approaches to treat patients with dysregulated RNA polymerase I transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Gang Lu

    2016-05-01

    Full Text Available 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.

  9. The RNA polymerase II CTD coordinates transcription and RNA processing.

    Science.gov (United States)

    Hsin, Jing-Ping; Manley, James L

    2012-10-01

    The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity.

  10. RNA polymerase associated with human rotaviruses in diarrhea stools.

    Science.gov (United States)

    Hruska, J F; Notter, M F; Menegus, M A; Steinhoff, M C

    1978-01-01

    RNA polymerase activity was detected in six stools which were partially purified by high-speed centrifugation from infants with rotavirus gastroenteritis, but was not detected in five stools which were negative for rotavirus by counterimmunoelectrophoresis and radioimmunoassay. The polymerase activity was associated with the 1.38-g/ml rotavirus band after purification in a CsCl gradient. PMID:207902

  11. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. PMID:25623070

  12. RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression.

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J

    2015-03-20

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. DNA-dependent RNA polymerase from Crithidia oncopelti kinetoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Zaitseva, G.N.; Levchenko, I.V.; Tarasov, I.A.; Kuz' min, E.V.

    1986-03-10

    Mitochondrial DNA-dependent RNA polymerase was isolated from Crithidia oncopelti kinetoplasts, and its properties were studied. RNA polymerase was solubilized from the structures with 2% digitonin in 0.25 M KCl. The enzyme was purified 550-fold according to activity by gel filtration through Sephadex 4B, followed by chromatography on heparin-Sepharose 4B, phosphocellulose, and DEAE-Sephadex A-50. The optimum conditions of the RNA polymerase reaction (time of incubation, temperature, Mg/sup 2 +/, K/sup +/ concentrations, etc) were determined. It was established that the activity of the enzyme is not inhibited by ..cap alpha..-amanitin, rifampicin, and streptolidigin, but is strongly suppressed by Mn/sup 2 +/ ions, a high KCl concentrations, as well as ethidium bromide. The RNA polymerase isolated transcribes denatured DNA substantially better than the native form. The enzyme utilizes mtDNA (in hybrid plasmids) as a substrate appreciably more actively than the nuclear form. Among the substrates used, the greatest template activity is possessed by single-stranded poly(dAT). In all the properties studied, DNA-dependent RNA polymerase from C. oncopelti kinetoplasts is similar to the mitochondrial enzymes of other eukaryotes but differs from the nuclear enzymes of this organism and from bacterial RNA polymerases.

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

    Energy Technology Data Exchange (ETDEWEB)

    Brenowitz,M.; Erie, D.; Chance, M.

    2005-01-01

    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 ({alpha}, {alpha}2, {beta}, {beta}', and {omega}) that contain the amino acid residues required for the enzyme's catalytic activity. A sixth subunit ({sigma}) guides RNAP to specific sequences on the genomic DNA (promoters) that mark the beginning of a gene or group of genes.

  15. Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization.

    Science.gov (United States)

    Yagura, T; Kozu, T; Seno, T

    1982-02-01

    A mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase alpha, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly(dT) or poly(dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly(dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly(dC) did not when GTP was added at high concentrations. GDP could be substituted for GTP, but the activity with poly(dC) plus GDP depended largely on the stimulating factor. Involvement of known RNA polymerases in the activity with poly(dT) was excluded, because addition of purified mouse RNA polymerases I and II had no effect on the incorporation of dATP, and alpha-amanitin (100 micrograms/ml) did not inhibit the incorporations of dATP and ATP. Analysis of the inhibition by the nucleotide analog 2',3'-dideoxynucleoside 5'-triphosphate (ddNTP) further supported the involvement of new RNA polymerase; ddNTPs inhibited the activities with poly(dT) and poly(dC) significantly more than RNA polymerases I and II or DNA polymerase alpha activity with poly(dT) . oligo(rA) and poly(dC) . oligo(dG) as template. Lineweaver-Burk analysis of the inhibitions showed that ddATP inhibited competitively with respect to ATP, and ddGTP inhibited competitively with respect to GDP but noncompetitively with respect to GTP.

  16. RNA polymerase activity in isolated nuclei of Nicotiana sanderae ...

    Indian Academy of Sciences (India)

    tribpo

    elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA poly- merase II activities. ... activity levels undergo dramatic changes during developmental stages such as seed germination (Guilfoyle and ... contained the respective hormones used for the growth (1 mg/L 2,4-D for callus and 1 mg/L NAA as ...

  17. Rat1p maintains RNA polymerase II CTD phosphorylation balance

    DEFF Research Database (Denmark)

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco

    2014-01-01

    In S. cerevisiae, the 5'-3' exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understoo...

  18. Yeast mitochondrial RNA polymerase primes mitochondrial DNA polymerase at origins of replication and promoter sequences.

    Science.gov (United States)

    Sanchez-Sandoval, Eugenia; Diaz-Quezada, Corina; Velazquez, Gilberto; Arroyo-Navarro, Luis F; Almanza-Martinez, Norineli; Trasviña-Arenas, Carlos H; Brieba, Luis G

    2015-09-01

    Three proteins phylogenetically grouped with proteins from the T7 replisome localize to yeast mitochondria: DNA polymerase γ (Mip1), mitochondrial RNA polymerase (Rpo41), and a single-stranded binding protein (Rim1). Human and T7 bacteriophage RNA polymerases synthesize primers for their corresponding DNA polymerases. In contrast, DNA replication in yeast mitochondria is explained by two models: a transcription-dependent model in which Rpo41 primes Mip1 and a model in which double stranded breaks create free 3' OHs that are extended by Mip1. Herein we found that Rpo41 transcribes RNAs that can be extended by Mip1 on single and double-stranded DNA. In contrast to human mitochondrial RNA polymerase, which primes DNA polymerase γ using transcripts from the light-strand and heavy-strand origins of replication, Rpo41 primes Mip1 at replication origins and promoter sequences in vitro. Our results suggest that in ori1, short transcripts serve as primers, whereas in ori5 an RNA transcript longer than 29 nucleotides is used as primer. Copyright © 2015 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.

  19. Molecular architecture of the vesicular stomatitis virus RNA polymerase.

    Science.gov (United States)

    Rahmeh, Amal A; Schenk, Andreas D; Danek, Eric I; Kranzusch, Philip J; Liang, Bo; Walz, Thomas; Whelan, Sean P J

    2010-11-16

    Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Pup.05.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Pupae SRX...013069 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Pup.05.RNA_polymerase_II.AllCell.bed ...

  4. 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 hg19 RNA polymerase RNA polymerase II Bone SRX...,SRX351408 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bon.20.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II All cell...,SRX1013886,SRX1013900 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.ALL.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Unc.10.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Unclass...ified http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Unc.10.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Bon.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Bone ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Bon.05.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.20.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II All cell ...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.20.RNA_Polymerase_II.AllCell.bed ...

  13. File list: Pol.CDV.20.RNA_Polymerase_II.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)

    Full Text Available Pol.Emb.50.RNA_Polymerase_II.AllCell ce10 RNA polymerase RNA Polymerase II Embryo h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.50.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Liv.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Liver E...RX204069 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Liv.05.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Lng.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Lung S...RX016555,SRX150101,SRX150102 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Lng.05.RNA_polymerase_III.AllCell.bed ...

  17. 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 mm9 RNA polymerase RNA Polymerase III Embryo ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Utr.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Uterus... SRX017001,SRX018606,SRX017002 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Utr.05.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Kid.20.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Kidney S...SRX1206072,SRX1206066,SRX326423,SRX1206067,SRX003883,SRX003882,SRX367323 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Kid.20.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Pup.10.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Pupae SRX...013069 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Pup.10.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Myo.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Muscle h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Myo.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Dig.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Digestiv...//dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Dig.10.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.50.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Adipocyte... SRX800011,SRX800010,SRX341031,SRX341032,SRX341029,SRX800016,SRX800017,SRX341030 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.50.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Pancreas... SRX190244 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Pan.10.RNA_polymerase_II.AllCell.bed ...

  7. File list: Pol.Lng.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Lng.05.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Lung SRX0...62976,SRX143816,SRX020252 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Lng.05.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Embryo SR...7582,SRX013077,SRX050604,SRX050605 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Emb.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Epd.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Epidermi...245,SRX663247,SRX807622 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Epd.05.RNA_polymerase_II.AllCell.bed ...

  10. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Lng.10.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Lung SRX1...43816,SRX062976,SRX020252 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Lng.10.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Bon.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Bone h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bon.20.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.50.RNA_Polymerase_II.AllCell ce10 RNA polymerase RNA Polymerase II All cell...X1388766,SRX1388757,SRX1388763 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.ALL.50.RNA_Polymerase_II.AllCell.bed ...

  14. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Spl.10.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Spleen ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Spl.10.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.50.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Pancrea...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Pan.50.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.50.RNA_polymerase_II.AllCell ce10 RNA polymerase RNA polymerase II Embryo S...,SRX043866 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.50.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Epd.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Epidermi...247,SRX080162,SRX807622 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Epd.50.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Myo.05.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Muscle SR.../dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Myo.05.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.PSC.50.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Pluripote...SRX213760,SRX213764 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.PSC.50.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Gonad h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Gon.05.RNA_Polymerase_III.AllCell.bed ...

  3. 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 hg19 RNA polymerase RNA polymerase II Cardiova...,SRX346933,SRX346936,SRX367018,SRX367016 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.20.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Liv.20.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Liver SRX...7,SRX020172,SRX020174,ERX204060,ERX204064,SRX547085,SRX020178,SRX193437,SRX193438 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Liv.20.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.PSC.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Pluripo...tent stem cell http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.PSC.05.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.PSC.20.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Pluripo...tent stem cell http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.PSC.20.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Dig.50.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Digestive... tract SRX112957,SRX143802 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Dig.50.RNA_Polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Unc.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Unclassi...fied http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Unc.10.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Lng.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Lung S...RX016555,SRX150101,SRX150102 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Lng.50.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Bon.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Bone SRX...,SRX351408 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bon.10.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Pancre...as http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Pan.20.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adl.05.RNA_polymerase_II.AllCell ce10 RNA polymerase RNA polymerase II Adult SR...SRX005635,SRX554718,SRX043963,SRX043965 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Adl.05.RNA_polymerase_II.AllCell.bed ...

  14. 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 ...

  15. File list: Pol.Myo.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Lar.05.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Larvae SR...SRX151962,SRX182775,SRX661503,SRX013070,SRX013072,SRX013113,SRX013082,SRX151961 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Lar.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Epd.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Epider...mis SRX016997 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Epd.50.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Dig.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Digest...ive tract http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Dig.50.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.10.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II All c...ell types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.ALL.10.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.50.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Embryo... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.50.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.20.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Gonad ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Gon.20.RNA_polymerase_II.AllCell.bed ...

  2. File list: Pol.Gon.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Gonad ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Gon.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Plc.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Placen...ta http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Plc.05.RNA_polymerase_III.AllCell.bed ...

  4. File list: Pol.Bld.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

  1. File list: Pol.Liv.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Liv.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Liver SR...1013886 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Liv.50.RNA_polymerase_II.AllCell.bed ...

  2. File list: Pol.Epd.10.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.Myo.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

  5. File list: Pol.Lar.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

  6. File list: Pol.Unc.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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  8. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.50.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II All c...ell types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.ALL.50.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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  11. File list: Pol.Kid.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Kid.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Kidney S...SRX128201,SRX128200,SRX003882,SRX1206065,SRX1206066,SRX1206067,SRX367323 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Kid.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Oth.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Others... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Oth.10.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Dig.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Digest...ive tract http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Dig.20.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.CDV.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Cardiova...,SRX367018,SRX367016,SRX112014,SRX112013 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.50.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.PSC.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Plurip...otent stem cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.PSC.10.RNA_polymerase_III.AllCell.bed ...

  18. File list: Pol.Adp.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Adipoc...yte http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.10.RNA_polymerase_III.AllCell.bed ...

  19. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Adipoc...yte http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.05.RNA_polymerase_III.AllCell.bed ...

  1. File list: Pol.Bld.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Blood ...SRX017006,SRX015143,SRX150560,SRX018610,SRX150396,SRX015144 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.20.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III All ce...,SRX018606,SRX150396,SRX015144,SRX017002 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.ALL.10.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Unc.05.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II Uncla...ssified http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.Unc.05.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adl.05.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Adult ...SRX395531,SRX331268,SRX331270,SRX395532 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Adl.05.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Kid.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Kidney... SRX016996 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Kid.05.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Blood ...SRX017006,SRX150560,SRX015143,SRX018610,SRX150396,SRX015144 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.10.RNA_polymerase_III.AllCell.bed ...

  11. File list: Pol.Lng.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Lng.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Lung S...RX016555,SRX150102,SRX150101 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Lng.10.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.CDV.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Cardio...vascular http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.10.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Prs.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Prosta...te http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Prs.05.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.10.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Embryo SR...7582,SRX050604,SRX050605,SRX013077 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Emb.10.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Unc.20.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II Uncla...ssified http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.Unc.20.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Epd.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Epider...mis SRX016997 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Epd.20.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.CDV.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Cardio...vascular http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.05.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III All ce...,SRX017001,SRX018606,SRX150396,SRX015144 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.ALL.50.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Gonad ...http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Gon.10.RNA_polymerase_III.AllCell.bed ...

  1. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.05.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III All ce...ll types SRX395531,SRX331268,SRX331270,SRX395532 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.ALL.05.RNA_polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.20.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II All c...ell types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.ALL.20.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.YSt.50.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II Yeast... strain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.YSt.50.RNA_polymerase_II.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III All ce...,SRX150396,SRX015144,SRX150101,SRX150102 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.ALL.05.RNA_polymerase_III.AllCell.bed ...

  7. File list: Pol.Adp.50.RNA_polymerase_II.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.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  12. File list: Pol.Dig.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  19. File list: Pol.Unc.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  1. File list: Pol.Bld.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  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.Oth.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  6. File list: Pol.Plc.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.Dig.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  9. File list: Pol.Myo.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Pol.Utr.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Pol.Brs.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  13. File list: Pol.Neu.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.Epd.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Epd.05.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Epidermis... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Epd.05.RNA_Polymerase_II.AllCell.bed ...

  15. File list: Pol.Plc.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  17. File list: Pol.Emb.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Bon.10.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Bone ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Bon.10.RNA_Polymerase_III.AllCell.bed ...

  19. File list: Pol.ALL.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.Lar.05.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.Gon.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Plc.10.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Placent...a http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Plc.10.RNA_Polymerase_III.AllCell.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  9. File list: Pol.Liv.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  11. File list: Pol.Lar.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  18. File list: Pol.Lar.10.RNA_Polymerase_II.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.Utr.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  2. File list: Pol.Spl.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  5. File list: Pol.Oth.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  16. File list: Pol.PSC.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Pol.Lar.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Myo.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Dig.50.RNA_polymerase_II.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.Neu.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Neu.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Neural S...6,SRX743834,SRX743838,SRX743840,SRX743832 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Neu.50.RNA_polymerase_II.AllCell.bed ...

  2. Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate

    NARCIS (Netherlands)

    Vilfan, I.D.; Candelli, A.; Hage, S.; Aalto, A.P.; Poranen, M.M.; Bamford, D.H.; Dekker, N.H.

    2008-01-01

    RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important

  3. Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.

    Science.gov (United States)

    Yu, Hsiang-Ping; Hsiao, Yi-Ling; Pan, Hung-Yin; Huang, Chih-Hung; Hou, Shao-Yi

    2011-12-15

    A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Targeting structural dynamics of the RNA-dependent RNA polymerase for anti-viral strategies.

    Science.gov (United States)

    Boehr, David D; Liu, Xinran; Yang, Xiaorong

    2014-12-01

    The RNA-dependent RNA polymerase is responsible for genome replication of RNA viruses. Nuclear magnetic resonance experiments and molecular dynamics simulations have indicated that efficient and faithful polymerase function requires highly coordinated internal protein motions. Interference with these motions, either through amino acid substitutions or small molecule binding, can disrupt polymerase and virus function. In particular, these studies have pointed toward highly conserved structural elements, like the motif-D active-site loop, that can be modified to generate polymerases with desired properties. Viruses encoding engineered polymerases might serve as live, attenuated vaccine strains. Further elucidation of polymerase structural dynamics will also provide new avenues for anti-viral drug design. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. FACT facilitates chromatin transcription by RNA polymerases I and III

    DEFF Research Database (Denmark)

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle....... The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results...... in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III...

  6. Studies of the Escherichia coli Rsd-sigma70 complex.

    Science.gov (United States)

    Westblade, Lars F; Ilag, Leopold L; Powell, Andrew K; Kolb, Annie; Robinson, Carol V; Busby, Stephen J W

    2004-01-16

    Escherichia coli Rsd protein was previously identified on the basis of its binding to the RNA polymerase sigma(70) subunit. The Rsd-sigma(70) complex has been studied using different methods. Our data show that Rsd associates with sigma(70) to form a complex with a stoichiometry of 1:1. Alanine scanning and deletion mutagenesis were used to locate regions of sigma(70) that are required for the formation of the Rsd-sigma(70) complex.

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

    Directory of Open Access Journals (Sweden)

    Argyris Papantonis

    2010-07-01

    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.

  8. Ubiquitylation and degradation of elongating RNA polymerase II

    DEFF Research Database (Denmark)

    Wilson, Marcus D; Harreman, Michelle; Svejstrup, Jesper Q

    2013-01-01

    evolved to ensure that transcription stalling or arrest does not occur. If, however, the polymerase cannot be restarted, it becomes poly-ubiquitylated and degraded by the proteasome. This process is highly regulated, ensuring that only RNAPII molecules that cannot otherwise be salvaged are degraded....... In this review, we describe the mechanisms and factors responsible for the last resort mechanism of transcriptional elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.......During its journey across a gene, RNA polymerase II has to contend with a number of obstacles to its progression, including nucleosomes, DNA-binding proteins, DNA damage, and sequences that are intrinsically difficult to transcribe. Not surprisingly, a large number of elongation factors have...

  9. Poised RNA polymerase II gives pause for thought.

    Science.gov (United States)

    Margaritis, Thanasis; Holstege, Frank C P

    2008-05-16

    Transcription by RNA polymerase II (Pol II) is thought to be predominantly regulated by recruitment of Pol II to promoters. Recent genome-wide analyses demonstrate that many genes are in fact regulated after recruitment of Pol II, by mechanisms such as pausing of Pol II proximal to promoters.

  10. Small RNA Deep Sequencing Reveals Role for Arabidopsis thaliana RNA-Dependent RNA Polymerases in Viral siRNA Biogenesis

    OpenAIRE

    Qi, Xiaopeng; Bao, Forrest Sheng; Xie, Zhixin

    2009-01-01

    RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (T...

  11. 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. Copyright © 2015 by the Genetics Society of America.

  12. Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

    Directory of Open Access Journals (Sweden)

    David Dulin

    2015-02-01

    Full Text Available RNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage Φ6 RNA-dependent RNA polymerase. We extract large datasets fingerprinting real-time polymerase dynamics over four magnitudes in time, in the presence of nucleotide analogs, and under varying NTP and divalent cation concentrations and fork stability. Quantitative analysis reveals a new pause state that modulates polymerase fidelity and so ties viral polymerase pausing to the biological function of optimizing virulence. Adjusting the frequency of such pauses offers a target for therapeutics and may also reflect an evolutionary strategy for virus populations to track the gradual evolution of their hosts.

  13. The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Tomohiro Shimada

    Full Text Available The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of "constitutive promoters" that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35 and TATAAT(-10, were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent "inducible promoters". One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature.

  14. Cell growth- and differentiation-dependent regulation of RNA polymerase III transcription.

    Science.gov (United States)

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; Da Silva, Daniel; Roeder, Robert G; Teichmann, Martin

    2010-09-15

    RNA polymerase III transcribes small untranslated RNAs that fulfill essential cellular functions in regulating transcription, RNA processing, translation and protein translocation. RNA polymerase III transcription activity is tightly regulated during the cell cycle and coupled to growth control mechanisms. Furthermore, there are reports of changes in RNA polymerase III transcription activity during cellular differentiation, including the discovery of a novel isoform of human RNA polymerase III that has been shown to be specifically expressed in undifferentiated human H1 embryonic stem cells. Here, we review major regulatory mechanisms of RNA polymerase III transcription during the cell cycle, cell growth and cell differentiation.

  15. 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

    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...

  16. The Dicer-like, Argonaute and RNA-dependent RNA polymerase ...

    Indian Academy of Sciences (India)

    The Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in Populus trichocarpa: gene structure, gene expression, phylogenetic analysis and evolution. Kang Zhao Hualin Zhao Zhu Chen Lin Feng Jie Ren Ronghao Cai Yan Xiang. Research Note Volume 94 Issue 2 June 2015 pp 317-321 ...

  17. A conserved RNA polymerase III promoter required for gammaherpesvirus TMER transcription and microRNA processing.

    Science.gov (United States)

    Diebel, Kevin W; Claypool, David J; van Dyk, Linda F

    2014-07-01

    Canonical RNA polymerase III (pol III) type 2 promoters contain a single A and B box and are well documented for their role in tRNA and SINE transcription in eukaryotic cells. The genome of Murid herpesvirus 4 (MuHV-4) contains eight polycistronic tRNA-microRNA encoded RNA (TMER) genes that are transcribed from a RNA pol III type 2-like promoter containing triplicated A box elements. Here, we demonstrate that the triplicated A box sequences are required in their entirety to produce functional MuHV-4 miRNAs. We also identify that these RNA pol III type 2-like promoters are conserved in eukaryotic genomes. Human and mouse predicted tRNA genes containing these promoters also show enrichment of alternative RNA pol III transcription termination sequences and are predicted to give rise to longer tRNA primary transcripts. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. RNA polymerases IV and V influence the 3' boundaries of Polymerase II transcription units in Arabidopsis.

    Science.gov (United States)

    McKinlay, Anastasia; Podicheti, Ram; Wendte, Jered M; Cocklin, Ross; Rusch, Douglas B

    2017-12-21

    Nuclear multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved in plants as specialized forms of Pol II. Their functions are best understood in the context of RNA-directed DNA methylation (RdDM), a process in which Pol IV-dependent 24 nt siRNAs direct the de novo cytosine methylation of regions transcribed by Pol V. Pol V has additional functions, independent of Pol IV and 24 nt siRNA biogenesis, in maintaining the repression of transposons and genomic repeats whose silencing depends on maintenance cytosine methylation. Here we report that Pol IV and Pol V play unexpected roles in defining the 3' boundaries of Pol II transcription units. Nuclear run-on assays reveal that in the absence of Pol IV or Pol V, Pol II occupancy downstream of poly A sites increases for approximately 12% of protein-coding genes. This effect is most pronounced for convergently transcribed gene pairs. Although Pols IV and V are detected near transcript ends of the affected Pol II - transcribed genes, their role in limiting Pol II read-through is independent of siRNA biogenesis or cytosine methylation for the majority of these genes. Interestingly, we observed that splicing was less efficient in pol IV or pol V mutant plants, compared to wild-type plants, suggesting that Pol IV or Pol V might affect pre-mRNA processing. We speculate that Pols IV and V (and/or their associated factors) play roles in Pol II transcription termination and pre-mRNA splicing by influencing polymerase elongation rates and/or release at collision sites for convergent genes.

  19. Architecture of initiation-competent 12-subunit RNA polymerase II.

    Science.gov (United States)

    Armache, Karim-Jean; Kettenberger, Hubert; Cramer, Patrick

    2003-06-10

    RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core and the two-subunit Rpb4/7 complex that is required for transcription initiation. Previous structures of the Pol II core revealed a "clamp," which binds the DNA template strand via three "switch regions," and a flexible "linker" to the C-terminal repeat domain (CTD). Here we derived a model of the complete Pol II by fitting structures of the core and Rpb4/7 to a 4.2-A crystallographic electron density map. Rpb4/7 protrudes from the polymerase "upstream face," on which initiation factors assemble for promoter DNA loading. Rpb7 forms a wedge between the clamp and the linker, restricting the clamp to a closed position. The wedge allosterically prevents entry of the promoter DNA duplex into the active center cleft and induces in two switch regions a conformation poised for template-strand binding. Interaction of Rpb4/7 with the linker explains Rpb4-mediated recruitment of the CTD phosphatase to the CTD during Pol II recycling. The core-Rpb7 interaction and some functions of Rpb4/7 are apparently conserved in all eukaryotic and archaeal RNA polymerases but not in the bacterial enzyme.

  20. Structural bases of norovirus RNA dependent RNA polymerase inhibition by novel suramin-related compounds

    OpenAIRE

    Croci, R.; Pezzullo, M.; Tarantino, D.; Milani, M.; Tsay, S.C.; Sureshbabu, R.; Tsai, Y.J.; Mastrangelo, E.; Rohayem, J.; Bolognesi, M.; Hwu, J.R.

    2014-01-01

    Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, wa...

  1. Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

    OpenAIRE

    Dulin, David; Vilfan, Igor D.; Berghuis, Bojk A.; Hage, Susanne; Bamford, Dennis H.; Poranen, Minna M.; Depken, Martin; Dekker, Nynke H.

    2015-01-01

    RNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage ?6 RNA-dependent RNA polymerase. We extra...

  2. Transcribing RNA polymerase III observed by electron cryomicroscopy.

    Science.gov (United States)

    Hoffmann, Niklas A; Jakobi, Arjen J; Vorländer, Matthias K; Sachse, Carsten; Müller, Christoph W

    2016-08-01

    Electron cryomicroscopy reconstructions of elongating RNA polymerase (Pol) III at 3.9 Å resolution and of unbound Pol III (apo Pol III) in two distinct conformations at 4.6 Å and 4.7 Å resolution allow the construction of complete atomic models of Pol III and provide new functional insights into the adaption of Pol III to fulfill its specific transcription tasks. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  3. Hinge action versus grip in translocation by RNA polymerase.

    Science.gov (United States)

    Nedialkov, Yuri A; Opron, Kristopher; Caudill, Hailey L; Assaf, Fadi; Anderson, Amanda J; Cukier, Robert I; Wei, Guowei; Burton, Zachary F

    2018-01-01

    Based on molecular dynamics simulations and functional studies, a conformational mechanism is posited for forward translocation by RNA polymerase (RNAP). In a simulation of a ternary elongation complex, the clamp and downstream cleft were observed to close. Hinges within the bridge helix and trigger loop supported generation of translocation force against the RNA-DNA hybrid resulting in opening of the furthest upstream i-8 RNA-DNA bp, establishing conditions for RNAP sliding. The β flap tip helix and the most N-terminal β' Zn finger engage the RNA, indicating a path of RNA threading out of the exit channel. Because the β flap tip connects to the RNAP active site through the β subunit double-Ψ-β-barrel and the associated sandwich barrel hybrid motif (also called the flap domain), the RNAP active site is coupled to the RNA exit channel and to the translocation of RNA-DNA. Using an exonuclease III assay to monitor translocation of RNAP elongation complexes, we show that K + and Mg 2+ and also an RNA 3'-OH or a 3'-H 2 affect RNAP sliding. Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K + and Mg 2+ concentration, biochemical assays are consistent with a conformational change that drives forward translocation as observed in simulations. Mutational analysis of the bridge helix indicates that 778-GARKGL-783 (Escherichia coli numbering) is a homeostatic hinge that undergoes multiple bends to compensate for complex conformational dynamics during phosphodiester bond formation and translocation.

  4. Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro.

    Science.gov (United States)

    Pilsl, Michael; Merkl, Philipp E; Milkereit, Philipp; Griesenbeck, Joachim; Tschochner, Herbert

    2016-01-01

    RNA polymerase I (Pol I) activity is crucial to provide cells with sufficient amounts of ribosomal RNA (rRNA). Synthesis of rRNA takes place in the nucleolus, is tightly regulated and is coordinated with synthesis and assembly of ribosomal proteins, finally resulting in the formation of mature ribosomes. Many studies on Pol I mechanisms and regulation in the model organism S. cerevisiae were performed using either complex in vitro systems reconstituted from more or less purified fractions or genetic analyses. While providing many valuable insights these strategies did not always discriminate between direct and indirect effects in transcription initiation and termination, when mutated forms of Pol I subunits or transcription factors were investigated. Therefore, a well-defined minimal system was developed which allows to reconstitute highly efficient promoter-dependent Pol I initiation and termination of transcription. Transcription can be initiated at a minimal promoter only in the presence of recombinant core factor and extensively purified initiation competent Pol I. Addition of recombinant termination factors triggers transcriptional pausing and release of the ternary transcription complex. This minimal system represents a valuable tool to investigate the direct impact of (lethal) mutations in components of the initiation and termination complexes on the mechanism and regulation of rRNA synthesis.

  5. Conformational flexibility of RNA polymerase III during transcriptional elongation

    Science.gov (United States)

    Fernández-Tornero, Carlos; Böttcher, Bettina; Rashid, Umar Jan; Steuerwald, Ulrich; Flörchinger, Beate; Devos, Damien P; Lindner, Doris; Müller, Christoph W

    2010-01-01

    RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III at 9.9 Å resolution and its elongation complex at 16.5 Å resolution. Particle sub-classification reveals prominent EM densities for the two Pol III-specific subcomplexes, C31/C82/C34 and C37/C53, that can be interpreted using homology models. While the winged-helix-containing C31/C82/C34 subcomplex initiates transcription from one side of the DNA-binding cleft, the C37/C53 subcomplex accesses the transcription bubble from the opposite side of this cleft. The transcribing Pol III enzyme structure not only shows the complete incoming DNA duplex, but also reveals the exit path of newly synthesized RNA. During transcriptional elongation, the Pol III-specific subcomplexes tightly enclose the incoming DNA duplex, which likely increases processivity and provides structural insights into the conformational switch between Pol III-mediated initiation and elongation. PMID:20967027

  6. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. In vitro transcription of Sonchus yellow net virus RNA by a virus-associated RNA-dependent RNA polymerase

    NARCIS (Netherlands)

    Flore, P.H.

    1986-01-01

    The aim of the investigation presented in this thesis was to elucidate the nature of the RNA- dependent RNA polymerase, thought to be associated with Sonchus yellow net virus (SYNV), a rhabdovirus infecting plants. This research was initiated to shed light on the

  8. 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 hg19 RNA polymerase RNA polymerase II No descr...iption http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.50.RNA_polymerase_II.AllCell.bed ...

  9. 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 ...

  10. 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 ...

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

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  6. File list: Pol.EmF.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. 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 ...

  8. Cyclin-dependent kinase 9 links RNA polymerase II transcription to processing of ribosomal RNA.

    Science.gov (United States)

    Burger, Kaspar; Mühl, Bastian; Rohrmoser, Michaela; Coordes, Britta; Heidemann, Martin; Kellner, Markus; Gruber-Eber, Anita; Heissmeyer, Vigo; Strässer, Katja; Eick, Dirk

    2013-07-19

    Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

  9. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Brian [Laboratory for Molecular Biophysics, The Rockefeller University, New York, United States; Feklistov, Andrey [Laboratory for Molecular Biophysics, The Rockefeller University, New York, United States; Lass-Napiorkowska, Agnieszka [Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, United States; Landick, Robert [Department of Biochemistry, University of Wisconsin-madison, Madison, United States; Department of Bacteriology, University of Wisconsin-Madison, Madison, United States; Darst, Seth A. [Laboratory for Molecular Biophysics, The Rockefeller University, New York, United States

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

  10. A method for quantifying the force dependence of initiation by T7 RNA polymerase

    NARCIS (Netherlands)

    Kalafut, B.S.; Skinner, G.M.; Visscher, K.

    2009-01-01

    To access the genetic code to be transcribed to RNA, RNA polymerases must first open a “transcription bubble” in the DNA. Structural studies suggest that the minimal model of initiation by T7 bacterophage RNA polymerase (T7 RNAP) consists of two distinct steps: initial binding, in which the T7 RNAP

  11. BRCA1/BARD1 ubiquitinate phosphorylated RNA polymerase II.

    Science.gov (United States)

    Starita, Lea M; Horwitz, Andrew A; Keogh, Michael-Christopher; Ishioka, Chikashi; Parvin, Jeffrey D; Chiba, Natsuko

    2005-07-01

    The breast- and ovarian-specific tumor suppressor BRCA1, when associated with BARD1, is an ubiquitin ligase. We have shown here that this heterodimer ubiquitinates a hyperphosphorylated form of Rpb1, the largest subunit of RNA polymerase II. Two major phosphorylation sites have been identified in the Rpb1 carboxyl terminal domain, serine 2 (Ser-2) or serine 5 (Ser-5) of the YSPTSPS heptapeptide repeat. Only the Ser-5 hyperphosphorylated form is ubiquitinated by BRCA1/BARD1. Overexpression of BRCA1 in cells stimulated the DNA damage-induced ubiquitination of Rpb1. Similar to the in vitro reaction, the stimulation of Rpb1 ubiquitination by BRCA1 in cells occurred only on those molecules hyperphosphorylated on Ser-5 of the heptapeptide repeat. In vitro, the carboxyl terminus of BRCA1 (amino acids 501-1863) was dispensable for the ubiquitination of hyperphosphorylated Rpb1. In cells, however, efficient Rpb1 ubiquitination required the carboxyl terminus of BRCA1, suggesting that interactions mediated by this region were essential in the complex milieu of the nucleus. These results link the BRCA1-dependent ubiquitination of the polymerase with DNA damage.

  12. Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.

    Directory of Open Access Journals (Sweden)

    Xiaopeng Qi

    Full Text Available RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs requires RNA-dependent RNA Polymerase (RDR activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4% and 22-nucleotide (12.9% in size and originating predominately (79.9% from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30 and an unknown protein similar to translocon-associated protein alpha (TRAP alpha, respectively, yielded a positive result in cleavage validation by 5'RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

  13. Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

    DEFF Research Database (Denmark)

    Helbo, Alexandra Søgaard; Lay, Fides D; Jones, Peter A

    2017-01-01

    Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high......-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies...... the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide....

  14. DNA structure in human RNA polymerase II promoters

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

    1998-01-01

    The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters...... with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low...... in a region upstream of the transcriptional start point and significantly higher downstream. Investigation of the sequence composition in the two regions shows that the bendability profile originates from the sequential structure of the DNA, rather than the general nucleotide composition...

  15. Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Dulin, David; Vilfan, Igor D; Berghuis, Bojk A; Hage, Susanne; Bamford, Dennis H; Poranen, Minna M; Depken, Martin; Dekker, Nynke H

    2015-02-11

    RNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage Φ6 RNA-dependent RNA polymerase. We extract large datasets fingerprinting real-time polymerase dynamics over four magnitudes in time, in the presence of nucleotide analogs, and under varying NTP and divalent cation concentrations and fork stability. Quantitative analysis reveals a new pause state that modulates polymerase fidelity and so ties viral polymerase pausing to the biological function of optimizing virulence. Adjusting the frequency of such pauses offers a target for therapeutics and may also reflect an evolutionary strategy for virus populations to track the gradual evolution of their hosts. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases.

    Science.gov (United States)

    Miropolskaya, Nataliya; Esyunina, Daria; Kulbachinskiy, Andrey

    2017-04-21

    RNA cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA cleavage. RNA cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli, Deinococcus radiodurans, and Thermus aquaticus, we show that the functions of the TL and Gre factors in RNA cleavage are conserved in various species, with important variations that may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA cleavage by all three RNAPs and eliminate the interspecies differences in the reaction rates. GreA factors activate RNA cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent cleavage are lower for ΔTL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA cleavage in certain types of backtracked transcription complexes, suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. 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.

  18. sigma54-Mediated control of the mannose phosphotransferase sytem in Lactobacillus plantarum impacts on carbohydrate metabolism

    NARCIS (Netherlands)

    Stevens, M.J.A.; Molenaar, D.; Jong, de A.; Vos, de W.M.; Kleerebezem, M.

    2010-01-01

    Sigma factors direct specific binding of the bacterial RNA polymerase to the promoter. Here we present the elucidation of the sigma(54 ) regulon in Lactobacillus plantarum. A sequence-based regulon prediction of sigma(54)-dependent promoters revealed an operon encoding a mannose phosphotransferase

  19. sigma(54)-mediated control of the mannose phosphotransferase sytem in Lactobacillus plantarum impacts on carbohydrate metabolism

    NARCIS (Netherlands)

    Stevens, Marc J. A.; Molenaar, Douwe; de Jong, Anne; De Vos, Willem M.; Kleerebezem, Michiel

    Sigma factors direct specific binding of the bacterial RNA polymerase to the promoter. Here we present the elucidation of the sigma(54) regulon in Lactobacillus plantarum. A sequence-based regulon prediction of sigma(54)-dependent promoters revealed an operon encoding a mannose phosphotransferase

  20. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.

    Science.gov (United States)

    Artsimovitch, Irina; Vassylyeva, Marina N; Svetlov, Dmitri; Svetlov, Vladimir; Perederina, Anna; Igarashi, Noriyuki; Matsugaki, Naohiro; Wakatsuki, Soichi; Tahirov, Tahir H; Vassylyev, Dmitry G

    2005-08-12

    Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.

  1. Conservation of RNA polymerase during maturation of the Rana pipiens oocyte

    Energy Technology Data Exchange (ETDEWEB)

    Hollinger, T.G.; Smith, L.D.

    1976-01-01

    DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5 percent of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.

  2. The proteomes of transcription factories containing RNA polymerases I, II or III.

    Science.gov (United States)

    Melnik, Svitlana; Deng, Binwei; Papantonis, Argyris; Baboo, Sabyasachi; Carr, Ian M; Cook, Peter R

    2011-09-25

    Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.

  3. Evolution and distribution of RNA polymerase II regulatory sites from RNA polymerase III dependant mobile Alu elements

    Directory of Open Access Journals (Sweden)

    Brahmachari Samir K

    2004-10-01

    Full Text Available Abstract Background The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting. Results We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes. Conclusions Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of

  4. Processing of a phosphoglycerate kinase reporter mRNA in Trypanosoma brucei is not coupled to transcription by RNA polymerase II.

    Science.gov (United States)

    Stewart, Mhairi; Haile, Simon; Jha, Bhaskar Anand; Cristodero, Marina; Li, Chi-Ho; Clayton, Christine

    2010-08-01

    Capping of mRNAs is strictly coupled to RNA polymerase II transcription and there is evidence, mainly from metazoans, that other steps in pre-mRNA processing show a similar linkage. In trypanosomes, however, the mRNA cap is supplied by a trans spliced leader sequence. Thus pre-mRNAs transcribed by RNA Polymerase I are capped by trans splicing, and translation-competent transgenic mRNAs can be produced by RNA Polymerase I and T7 RNA polymerase so long as the primary transcript has a splice acceptor signal. We quantified the efficiency of processing of trypanosome pre-mRNAs produced from a plasmid integrated either at the tubulin locus, or in an rRNA spacer, and transcribed by RNA polymerase II, RNA polymerase I or T7 RNA polymerase. The processing efficiencies were similar for primary transcripts from the tubulin locus, produced by RNA polymerase II, and for RNA from an rRNA spacer, transcribed by RNA polymerase I. Primary transcripts produced by T7 RNA polymerase from the tubulin locus were processed almost as well. There was therefore no evidence for recruitment of the 3'-splicing apparatus by the RNA polymerase. Abundant transcripts transcribed from the rRNA locus by T7 RNA polymerase were somewhat less efficiently processed.

  5. Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.

    Science.gov (United States)

    Maffioli, Sonia I; Zhang, Yu; Degen, David; Carzaniga, Thomas; Del Gatto, Giancarlo; Serina, Stefania; Monciardini, Paolo; Mazzetti, Carlo; Guglierame, Paola; Candiani, Gianpaolo; Chiriac, Alina Iulia; Facchetti, Giuseppe; Kaltofen, Petra; Sahl, Hans-Georg; Dehò, Gianni; Donadio, Stefano; Ebright, Richard H

    2017-06-15

    Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Recombinant Thermus aquaticus RNA Polymerase for Structural Studies

    Energy Technology Data Exchange (ETDEWEB)

    Juznedelov,K.; Lamour, V.; Patikoglou, G.; Chlenov, M.; Darst, S.; Severinov, K.

    2006-01-01

    Advances in the structural biology of bacterial transcription have come from studies of RNA polymerases (RNAPs) from the thermophilic eubacteria Thermus aquaticus (Taq) and Thermus thermophilus (Tth). These structural studies have been limited by the fact that only endogenous Taq or Tth RNAP, laboriously purified from large quantities of Taq or Tth cell paste and offering few options for genetic modification, is suitable for structural studies. Recombinant systems for the preparation of Taq RNAP by co-overexpression and assembly in the heterologous host, Escherichia coli, have been described, but these did not yield enzyme suitable for crystallographic studies. Here we describe recombinant systems for the preparation of Taq RNAP harboring full or partial deletions of the Taq {beta}' non-conserved domain (NCD), yielding enzyme suitable for crystallographic studies. This opens the way for structural studies of genetically manipulated enzymes, allowing the preparation of more crystallizable enzymes and facilitating detailed structure/function analysis. Characterization of the Taq{beta}'NCD deletion mutants generated in this study showed that the {beta}'NCD is important for the efficient binding of the s subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1 Angstroms resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the {beta}-flap, further defining the range of conformations accessible to this flexible structural element.

  7. Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases.

    Science.gov (United States)

    Esyunina, Daria; Turtola, Matti; Pupov, Danil; Bass, Irina; Klimašauskas, Saulius; Belogurov, Georgiy; Kulbachinskiy, Andrey

    2016-02-18

    RNA cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription.

    Science.gov (United States)

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription.

  9. The rearrangement of motif F in the flavivirus RNA-directed RNA polymerase.

    Science.gov (United States)

    Potapova, Ulyana; Feranchuk, Sergey; Leonova, Galina; Belikov, Sergei

    2017-11-04

    In the flavivirus genus, the non-structural protein NS5 plays a central role in RNA viral replication and constitutes a major target for drug discovery. One of the prime challenges in the study of NS5 protein is to investigate the interplay between the two protein domains, namely, the RNA-dependent RNA polymerase (RdRp) domain and the methyltransferase (MTase) domain. These investigations could clarify the multiple roles of NS5 protein in the virus life cycle. Here we present the results of sequence analyses and structural bioinformatics studies of NS5 protein, which suggest that the conserved motif F in the NS5 protein could act as a lock which controls the rearrangement of the domains and as a switch in the protein enzymatic activity. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Ferron, François; Bussetta, Cécile; Dutartre, Hélène; Canard, Bruno

    2005-10-14

    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. 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. 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 polymerases.

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

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    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

  12. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

    OpenAIRE

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcript...

  13. 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

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

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    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.

  15. Transcription inactivation through local refolding of the RNA polymerase structure

    Energy Technology Data Exchange (ETDEWEB)

    Belogurov, Georgiy A.; Vassylyeva, Marina N.; Sevostyanova, Anastasiya; Appleman, James R.; Xiang, Alan X.; Lira, Ricardo; Webber, Stephen E.; Klyuyev, Sergiy; Nudler, Evgeny; Artsimovitch, Irina; Vassylyev, Dmitry G.; (OSU); (UAB); (Anadys); (NYUSM)

    2009-02-12

    Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx - a desmethyl derivative of myxopyronin B - complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the {beta}'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex - the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

  16. Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

    NARCIS (Netherlands)

    Dulin, D.; Arnold, Jamie J.; van Laar, T.; Oh, Hyung Suk; Lee, Cheri; Perkins, Angela L.; Harki, Daniel A.; Depken, S.M.; Cameron, Craig E.; Dekker, N.H.

    2017-01-01

    RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites.

  17. Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein-Protein Interaction.

    Science.gov (United States)

    Banda, Srikanth; Cao, Nan; Tse-Dinh, Yuk-Ching

    2017-09-15

    We report here a distinct mechanism of interaction between topoisomerase I and RNA polymerase in Mycobacterium tuberculosis and Mycobacterium smegmatis that has evolved independently from the previously characterized interaction between bacterial topoisomerase I and RNA polymerase. Bacterial DNA topoisomerase I is responsible for preventing the hyper-negative supercoiling of genomic DNA. The association of topoisomerase I with RNA polymerase during transcription elongation could efficiently relieve transcription-driven negative supercoiling. Our results demonstrate a direct physical interaction between the C-terminal domains of topoisomerase I (TopoI-CTDs) and the β' subunit of RNA polymerase of M. smegmatis in the absence of DNA. The TopoI-CTDs in mycobacteria are evolutionarily unrelated in amino acid sequence and three-dimensional structure to the TopoI-CTD found in the majority of bacterial species outside Actinobacteria, including Escherichia coli. The functional interaction between topoisomerase I and RNA polymerase has evolved independently in mycobacteria and E. coli, with distinctively different structural elements of TopoI-CTD utilized for this protein-protein interaction. Zinc ribbon motifs in E. coli TopoI-CTD are involved in the interaction with RNA polymerase. For M. smegmatis TopoI-CTD, a 27-amino-acid tail that is rich in basic residues at the C-terminal end is responsible for the interaction with RNA polymerase. Overexpression of recombinant TopoI-CTD in M. smegmatis competed with the endogenous topoisomerase I for protein-protein interactions with RNA polymerase. The TopoI-CTD overexpression resulted in decreased survival following treatment with antibiotics and hydrogen peroxide, supporting the importance of the protein-protein interaction between topoisomerase I and RNA polymerase during stress response of mycobacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Possible roles of σ-dependent RNA polymerase pausing in transcription regulation.

    Science.gov (United States)

    Petushkov, Ivan; Esyunina, Daria; Kulbachinskiy, Andrey

    2017-12-02

    The σ subunit of bacterial RNA polymerase is required for promoter recognition during transcription initiation but may also regulate transcription elongation. The principal σ 70 subunit of Escherichia coli was shown to travel with RNA polymerase and induce transcriptional pausing at promoter-like motifs, with potential regulatory output. We recently demonstrated that an alternative σ 38 subunit can also induce RNA polymerase pausing. Here, we outline proposed regulatory roles of σ-dependent pausing in bacteria and discuss possible interplay between alternative σ variants and regulatory factors during transcription elongation.

  19. Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family.

    Science.gov (United States)

    Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S

    1998-02-01

    RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

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

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    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.

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

    Science.gov (United States)

    Gao, Zhihuan; Liu, Hai-Liang; Daxinger, Lucia; Pontes, Olga; He, Xinjian; Qian, Weiqiang; Lin, Huixin; Xie, Mingtang; Lorkovic, Zdravko J; Zhang, Shoudong; Miki, Daisuke; Zhan, Xiangqiang; Pontier, Dominique; Lagrange, Thierry; Jin, Hailing; Matzke, Antonius J M; Matzke, Marjori; Pikaard, Craig S; Zhu, Jian-Kang

    2010-05-06

    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.

  2. Analysis of Ribonucleotide 5'-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.

    Science.gov (United States)

    Lu, Gaofei; Bluemling, Gregory R; Collop, Paul; Hager, Michael; Kuiper, Damien; Gurale, Bharat P; Painter, George R; De La Rosa, Abel; Kolykhalov, Alexander A

    2017-03-01

    Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors. Copyright © 2017 American Society for Microbiology.

  3. Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II.

    Science.gov (United States)

    Arndt, Karen M; Reines, Daniel

    2015-01-01

    The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1-Nab3-Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes.

  4. A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme.

    Science.gov (United States)

    Suh, Man-Hee; Meyer, Peter A; Gu, Meigang; Ye, Ping; Zhang, Mincheng; Kaplan, Craig D; Lima, Christopher D; Fu, Jianhua

    2010-10-29

    RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5'-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.

  5. A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

    Science.gov (United States)

    Suh, Man-Hee; Meyer, Peter A.; Gu, Meigang; Ye, Ping; Zhang, Mincheng; Kaplan, Craig D.; Lima, Christopher D.; Fu, Jianhua

    2010-01-01

    RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5′-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes. PMID:20720002

  6. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.

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    Peng Gong

    Full Text Available RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle

  7. Structural bases of norovirus RNA dependent RNA polymerase inhibition by novel suramin-related compounds.

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    Romina Croci

    Full Text Available Noroviruses (NV are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9 as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp. Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM; the compound inhibits also murine NV (mNV RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3-7 hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

  8. Norovirus RNA-dependent RNA polymerase: A computational study of metal-binding preferences.

    Science.gov (United States)

    Shaik, Md Munan; Bhattacharjee, Nicholus; Feliks, Mikolaj; Ng, Kenneth K-S; Field, Martin J

    2017-08-01

    Norovirus (NV) RNA-dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer-template duplex was investigated using X-ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back-tracked state of the enzyme, in which the 3'-end of the primer occupies the position expected for the post-incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435-1445. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    Science.gov (United States)

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  10. Loss of human ribosomal gene CpG methylation enhances cryptic RNA polymerase II transcription and disrupts ribosomal RNA processing.

    Science.gov (United States)

    Gagnon-Kugler, Thérèse; Langlois, Frédéric; Stefanovsky, Victor; Lessard, Frédéric; Moss, Tom

    2009-08-28

    Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to downregulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an explanation for the cytotoxic effects of loss of CpG methylation.

  11. The P1 promoter of the Escherichia coli rpoH gene is utilized by sigma 70 -RNAP or sigma s -RNAP depending on growth phase.

    Science.gov (United States)

    Janaszak, Anna; Nadratowska-Wesołowska, Beata; Konopa, Grazyna; Taylor, Alina

    2009-02-01

    The P1 promoter of the Escherichia coli rpoH gene has been known as a sigma(70)-dependent promoter (RNAP). We show here that it is also recognized by sigma(S). The sigma(70) and sigma(S) RNA polymerase subunits direct transcription from the P1 promoter in the exponential and stationary growth phases, respectively, and transcriptional start sites for the two holoenzymes differ by 1 nt. The transcription after heat shock is sigma(70)-dependent. The results are based on (1) sequence analysis that revealed features of promoters responsive to both, sigma(70)- and sigma(S)-RNAP, (2) in vitro transcription experiments verifying that both holoenzymes are able to transcribe the promoter, (3) electron microscopy results indicating that both holoenzymes bind the same site, (4) primer extension test performed with RNA isolated from the wild-type and rpoS mutant strains, demonstrating that transcription from the P1 promoter in the stationary phase is sigma(S)-dependent. These and previous results point to cooperation of sigma(70), sigma(24), sigma(S) and sigma(54) regulons in the expression of the rpoH gene, coding for the main regulator of the stress response, thus rendering it active in a variety of conditions.

  12. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

    DEFF Research Database (Denmark)

    Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ss......RNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

  13. Screen Anti-influenza Lead Compounds That Target the PAC Subunit of H5N1 Viral RNA Polymerase

    Science.gov (United States)

    Xiang, Junfeng; Li, Qian; Liang, Huanhuan; Tang, Yalin; Liu, Yingfang

    2012-01-01

    The avian influenza (H5N1) viral RNA polymerase protein PAC was used as a target to screen nine chlorogenic acid derivatives for their polymerase inhibitor activity. Among them, seven compounds were PAC ligands, and four inhibited influenza RNA polymerase activity. These results aid in the design of anti-influenza agents based on caffeoylquinic acid. PMID:22936968

  14. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6.

    Directory of Open Access Journals (Sweden)

    Anthony Devert

    Full Text Available Cellular RNA-dependent RNA polymerases (RDRs are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA from single stranded RNA (ssRNA targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA. However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants.

  15. Studying transcription initiation by RNA polymerase with diffusion-based single-molecule fluorescence.

    Science.gov (United States)

    Alhadid, Yazan; Chung, SangYoon; Lerner, Eitan; Taatjes, Dylan J; Borukhov, Sergei; Weiss, Shimon

    2017-07-01

    Over the past decade, fluorescence-based single-molecule studies significantly contributed to characterizing the mechanism of RNA polymerase at different steps in transcription, especially in transcription initiation. Transcription by bacterial DNA-dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. In this review, we describe how single-molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging. © 2017 The Protein Society.

  16. The RNA-dependent RNA polymerase of Citrus tristeza virus forms oligomers.

    Science.gov (United States)

    Cevik, Bayram

    2013-12-01

    The RNA-dependent RNA polymerases (RdRp) from Citrus tristeza virus (CTV) were tagged with HA and FLAG epitopes. Differentially tagged proteins were expressed either individually or concomitantly in Escherichia coli. Immunoprecipitation of the expressed proteins with anti-FLAG antibody followed by Western blot with anti-HA antibody demonstrated that molecules of RdRp from CTV interact to form oligomers. Yeast two-hybrid assays showed that molecules of RdRp interact in eukaryotic cells. Co-immunoprecipitation with anti-FLAG antibody of truncated HA-tagged RdRps (RdRpΔ1-166-HA, RdRpΔ1-390-HA, RdRp1-169-HA) co-expressed with full-length RdRp-FLAG showed that only RdRp1-169-HA interacted with the full-length FLAG-RdRp. Yeast two-hybrid assays with truncated RdRp constructs confirmed that the oligomerization site resides in the N-terminal region and that the first 169 aa of CTV RdRp are necessary and sufficient for oligomerization both in bacterial and yeast cells. Development of control strategies targeting viral RdRp oligomer formation may inhibit virus replication and prove useful in control of CTV. © 2013 Elsevier Inc. All rights reserved.

  17. Aquarius, a novel gene isolated by gene trapping with an RNA-dependent RNA polymerase motif.

    Science.gov (United States)

    Sam, M; Wurst, W; Klüppel, M; Jin, O; Heng, H; Bernstein, A

    1998-06-01

    In a retinoic acid (RA) gene trap screen of mouse embryonic stem (ES) cells, a novel gene, named Aquarius (Aqr), was identified and characterized. The promoterless lacZ marker was used to trap the genomic locus and to determine the expression pattern of the gene. Aqr transcripts are strongly induced in response to RA in vitro. During embryogenesis, Aqr is expressed in mesoderm, in the neural crest and its target tissues, and in neuroepithelium. Expression was first detected at 8.5 days postcoitum, when neural crest cells are visible at the lateral ridges of the neural plate. The gene-trapped Aqr locus was transmitted through the mouse germ line in three genetic backgrounds. In the F2 generation, the expected mendelian ratio of 1:2:1 was observed in all backgrounds, indicating that homozygous mice are viable. Homozygotes are normal in size and weight and breed normally. The gene trap insertion, however, does not seem to generate a null mutation, because Aqr transcripts are still present in the homozygous mutant animals. The Aqr open reading frame has weak homology to RNA-dependent RNA polymerases (RRPs) of the murine hepatitis viruses and contains an RRP motif. Aqr was mapped to mouse chromosome 2 between regions E5 through F2 by using fluorescence in situ hybridization analysis.

  18. Arenavirus Z protein controls viral RNA synthesis by locking a polymerase-promoter complex.

    Science.gov (United States)

    Kranzusch, Philip J; Whelan, Sean P J

    2011-12-06

    Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.

  19. Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the Sigma54 (RpoN) regulon of Salmonella Typhimurium LT2

    Science.gov (United States)

    Background: Sigma54, or RpoN, is an alternative s factor found widely in eubacteria. A significant complication in analysis of the global sigma54 regulon in a bacterium is that the sigma54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to init...

  20. Uncovering layers of human RNA polymerase II transcription

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In recent years DNA microarray and high-throughput sequencing technologies have challenged the “gene-centric” view that pre-mRNA is the only RNA species transcribed off protein-coding genes. Instead unorthodox transcription from within genic- and intergenic regions has been demonstrated to occur ...... and unstable RNAs emitted from within, and immediately upstream human protein-coding genes. Mechanisms of their production and turn-over as well as their possible functions will be discussed...

  1. 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

  2. Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

    Science.gov (United States)

    Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

    2015-05-26

    DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.

    Science.gov (United States)

    Subissi, Lorenzo; Posthuma, Clara C; Collet, Axelle; Zevenhoven-Dobbe, Jessika C; Gorbalenya, Alexander E; Decroly, Etienne; Snijder, Eric J; Canard, Bruno; Imbert, Isabelle

    2014-09-16

    In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3'-5' exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5'-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

  4. Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea.

    NARCIS (Netherlands)

    Blombach, F.; Makarova, K.S.; Marrero, J.; Siebers, B.G.; Koonin, E.V.; Oost, J. van der

    2009-01-01

    One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA

  5. Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea

    NARCIS (Netherlands)

    Blombach, F.; Makarova, K.S.; Marrero, J.; Siebers, B.; Koonin, E.V.; Oost, van der J.

    2009-01-01

    One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA

  6. Rifampicin-resistance, rpoB polymorphism and RNA polymerase genetic engineering.

    Science.gov (United States)

    Alifano, Pietro; Palumbo, Carla; Pasanisi, Daniela; Talà, Adelfia

    2015-05-20

    Following its introduction in 1967, rifampicin has become a mainstay of therapy in the treatment of tuberculosis, leprosy and many other widespread diseases. Its potent antibacterial activity is due to specific inhibition of bacterial RNA polymerase. However, resistance to rifampicin was reported shortly after its introduction in the medical practice. Studies in the model organism Escherichia coli helped to define the molecular mechanism of rifampicin-resistance demonstrating that resistance is mostly due to chromosomal mutations in rpoB gene encoding the RNA polymerase β chain. These studies also revealed the amazing potential of the molecular genetics to elucidate the structure-function relationships in bacterial RNA polymerase. The scope of this paper is to illustrate how rifampicin-resistance has been recently exploited to better understand the regulatory mechanisms that control bacterial cell physiology and virulence, and how this information has been used to maneuver, on a global scale, gene expression in bacteria of industrial interest. In particular, we reviewed recent literature regarding: (i) the effects of rpoB mutations conferring rifampicin-resistance on transcription dynamics, bacterial fitness, physiology, metabolism and virulence; (ii) the occurrence in nature of "mutant-type" or duplicated rifampicin-resistant RNA polymerases; and (iii) the RNA polymerase genetic engineering method for strain improvement and drug discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. The proximal sequence element (PSE) plays a major role in establishing the RNA polymerase specificity of Drosophila U-snRNA genes.

    Science.gov (United States)

    Jensen, R C; Wang, Y; Hardin, S B; Stumph, W E

    1998-01-15

    Most small nuclear RNA (snRNA) genes are transcribed by RNA polymerase II, but some (e.g., U6) are transcribed by RNA polymerase III. In vertebrates a TATA box at a fixed distance downstream of the proximal sequence element (PSE) acts as a dominant determinant for recruiting RNA polymerase III to U6 gene promoters. In contrast, vertebrate snRNA genes that contain a PSE but lack a TATA box are transcribed by RNA polymerase II. In plants, transcription of both classes of snRNA genes requires a TATA box in addition to an upstream sequence element (USE), and polymerase specificity is determined by the spacing between these two core promoter elements. In these examples, the PSE (or USE) is interchangeable between the two classes of snRNA genes. Here we report the surprising finding that the Drosophila U1 and U6 PSEs cannot functionally substitute for each other; rather, determination of RNA polymerase specificity is an intrinsic property of the PSE sequence itself. The alteration of two or three base pairs near the 3'-end of the U1 and U6 PSEs was sufficient to switch the RNA polymerase specificity of Drosophila snRNA promoters in vitro. These findings reveal a novel mechanism for achieving RNA polymerase specificity at insect snRNA promoters.

  8. Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template

    Science.gov (United States)

    2015-01-01

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D 1H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3′(−)SL by 1H NMR, the base pair at the stem’s edge was identified as the most stable component of the structure. 1H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3′(−)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process. PMID:25310724

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Shilpa Aggarwal

    2010-04-01

    Full Text Available 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.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.Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of

  11. The RNA-dependent-RNA polymerase, an emerging antiviral drug target for the Hendra virus.

    Science.gov (United States)

    Velkov, Tony; Carbone, Vincenzo; Akter, Jesmin; Sivanesan, Sivashangarie; Li, Jian; Beddoe, Travis; Marsh, Glenn A

    2014-01-01

    Australia is facing a major national medical challenge with the emergence of the Hendra virus (HeV) as a medically and economically important pathogen of humans and animals. Clinical symptoms of human HeV infection can include fever, hypotension, dizziness, encephalitis, respiratory haemorrhage and edema. The window of opportunity for successful patient treatment remains unknown, but is likely to be very narrow. Currently, very few effective therapeutic options are available for the case management of severe HeV infections or the rapid silencing of local outbreaks. This underscores the need for more activity in the drug discovery arena to develop much needed therapeutics that specifically targets this deadly disease. The structural analysis of HeV is very much in its infancy, which leaves many gaps in our understanding of the biology of HeV and makes structure-guided drug design difficult. Structural studies of the viral RNAdependent- RNA polymerase (RdRp), which is the heart of the viral replication machinery, will set the stage for rational drug design and fill a major gap in our understanding of the HeV replication machinery. This review examines the current knowledge based on the multi-domain architecture of the Hendra RdRp and highlights which essential domain functions represent tangible targets for drug development against this deadly disease.

  12. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Directory of Open Access Journals (Sweden)

    Galiano V

    2016-10-01

    Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

  13. MicroRNA-297 promotes cardiomyocyte hypertrophy via targeting sigma-1 receptor.

    Science.gov (United States)

    Bao, Qinxue; Zhao, Mingyue; Chen, Li; Wang, Yu; Wu, Siyuan; Wu, Wenchao; Liu, Xiaojing

    2017-04-15

    Sigma-1 receptor (Sig-1R) is a ligand-regulated endoplasmic reticulum (ER) chaperone involved in cardiac hypertrophy, but it is not known whether Sig-1R is regulated by microRNAs (miRNAs). According to bioinformatic analysis, miR-297 was suggested as a potential target miRNA for Sig-1R. Therefore, we verified whether miR-297 could target Sig-1R and investigated the possible mechanisms underlying the role of miR-297 in cardiac hypertrophy. Bioinformatic analysis combined with laboratory experiments, including quantitative RT-PCR, Western blotting, and luciferase assay, were performed to identify the target miRNA of Sig-1R. Transverse aortic constriction (TAC) model and neonatal rat cardiomyocytes (NCMs) stimulated with angiotensin II (AngII) were used to explore the relationship between miR-297 and Sig-1R. Additionally, the function of miR-297 in cardiomyocyte hypertrophy and ER stress/unfolded protein response (UPR) signaling pathway was investigated by transfecting miR-297 mimics/inhibitor. miR-297 levels were increased in both TAC-induced hypertrophic heart tissue and AngII-induced cardiomyocyte hypertrophy. Up-regulation of miR-297 by specific mimics exacerbated AngII-induced cardiomyocyte hypertrophy, whereas inhibition of miR-297 suppressed the process. During cardiomyocyte hypertrophy, Sig-1R expression, which was negatively regulated by miR-297 by directly targeting its 3'untranslated region (UTR), was decreased. Furthermore, attenuation of miR-297 inhibited the activation of X-box binding protein 1 (Xbp1) and activating transcriptional factor 4 (ATF4) signaling pathways in NCMs. Our data demonstrate that miR-297 promotes cardiomyocyte hypertrophy by inhibiting the expression of Sig-1R and activation of ER stress signaling, which provides a novel interpretation for cardiac hypertrophy. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Transcription Elongation by RNA Polymerase I Is Linked to Efficient rRNA Processing and Ribosome Assembly

    OpenAIRE

    Schneider, David A.; Michel, Antje; Sikes, Martha L.; Vu, Loan; Dodd, Jonathan A.; Salgia, Shilpa; Osheim, Yvonne N.; Beyer, Ann L.; Nomura, Masayasu

    2007-01-01

    The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant sh...

  15. Genetic and genomic analysis of RNA polymerase II backtracking in saccharomyces cerevisiae

    OpenAIRE

    Miguel Jiménez, María Dolores de

    2016-01-01

    Genetic and genomic analysis of RNA polymerase II backtracking in Saccharomyces cerevisiae   El trabajo de esta tesis se divide en dos capítulos: ¿The role of TFIIS in nucleolar stress¿ y ¿RNA polymerase II backtracking across the genome¿. En el primer capítulo se describe como  el factor transcripcional TFIIS es especialmente requerido para la trascripción de los genes de proteínas ribosómicas (genes RPs) en condi...

  16. Metal A and metal B sites of nuclear RNA polymerases Pol IV and Pol V are required for siRNA-dependent DNA methylation and gene silencing.

    Directory of Open Access Journals (Sweden)

    Jeremy R Haag

    Full Text Available Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb. Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active.

  17. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

    Science.gov (United States)

    Pai, Dave A.; Kaplan, Craig D.; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C.; Engelke, David R.

    2014-01-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template. PMID:24614752

  18. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

    Science.gov (United States)

    Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C; Engelke, David R

    2014-05-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

  19. Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

    Science.gov (United States)

    Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel

    2017-01-01

    ABSTRACT Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the

  20. 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.

  1. 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

  2. Microprocessor Recruitment to Elongating RNA Polymerase II Is Required for Differential Expression of MicroRNAs

    Directory of Open Access Journals (Sweden)

    Victoria A. Church

    2017-09-01

    Full Text Available The cellular abundance of mature microRNAs (miRNAs is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor’s differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha directly associates with the C-terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb. When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.

  3. Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo

    2012-01-01

    and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining...

  4. Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II

    NARCIS (Netherlands)

    B. Steurer (Barbara); J.A. Marteijn (Jurgen)

    2016-01-01

    textabstractThe faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The

  5. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

    NARCIS (Netherlands)

    Dauwerse, J.G.; Dixon, J.; Seland, S.; Ruivenkamp, C.A.; Haeringen, A. van; Hoefsloot, L.H.; Peters, D.J.; Boers, A.C.; Daumer-Haas, C.; Maiwald, R.; Zweier, C.; Kerr, B.; Cobo, A.M.; Toral, J.F.; Hoogeboom, A.J.M.; Lohmann, D.R.; Hehr, U.; Dixon, M.J.; Breuning, M.H.; Wieczorek, D.

    2011-01-01

    We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both

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

    NARCIS (Netherlands)

    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, 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

  7. Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea

    Directory of Open Access Journals (Sweden)

    Siebers Bettina

    2009-10-01

    Full Text Available Abstract One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA polymerase II with respect to the subunit composition. Here we identify archaeal orthologs of the eukaryotic RNA polymerase III subunit RPC34. Genome context analysis supports a function of this archaeal protein in the transcription of non-coding RNAs. These findings suggest that functional separation of RNA polymerases for protein-coding genes and non-coding RNAs might predate the origin of the Eukaryotes. Reviewers: This article was reviewed by Andrei Osterman and Patrick Forterre (nominated by Purificación López-García

  8. Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea.

    Science.gov (United States)

    Blombach, Fabian; Makarova, Kira S; Marrero, Jeannette; Siebers, Bettina; Koonin, Eugene V; van der Oost, John

    2009-10-14

    One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA polymerase II with respect to the subunit composition. Here we identify archaeal orthologs of the eukaryotic RNA polymerase III subunit RPC34. Genome context analysis supports a function of this archaeal protein in the transcription of non-coding RNAs. These findings suggest that functional separation of RNA polymerases for protein-coding genes and non-coding RNAs might predate the origin of the Eukaryotes.

  9. A dynamic model for transcription elongation and sequence-dependent short pauses by RNA polymerase.

    Science.gov (United States)

    Xie, Ping

    2008-09-01

    RNA polymerase is an enzyme that transcribes genes from DNA onto strands of RNA and the transcription is a processive, accurate but discontinuous process. Despite extensive structural, biochemical and biophysical studies, the transcription elongation mechanism by the RNA polymerase is still not well determined. Here a new Brownian ratchet model is presented for this transcription elongation by the RNA polymerase. The structure's conformational changes observed in the RNAP translocation cycle are incorporated into the model. Using the model, the dynamic behaviors of continuous transcription elongation between two pauses and inhibition of next nucleotide addition after misincorporation are well explained. Moreover, the sequence-dependent short pauses result from site-specific interactions of RNAP with dsDNA and/or RNA-DNA hybrid. With this model, it is demonstrated that, at a given sequence, the lifetime distribution of the short pause has the single-exponential form at saturating nucleotide concentration, which is in contrast to the multi-exponential distribution of the dwell time during the continuous transcription elongation.

  10. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications.

    Science.gov (United States)

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2015-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress toward understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation.

  11. A method for quantifying the force dependence of initiation by T7 RNA polymerase

    Science.gov (United States)

    Kalafut, Bennett S.; Skinner, Gary M.; Visscher, Koen

    2009-08-01

    To access the genetic code to be transcribed to RNA, RNA polymerases must first open a "transcription bubble" in the DNA. Structural studies suggest that the minimal model of initiation by T7 bacterophage RNA polymerase (T7 RNAP) consists of two distinct steps: initial binding, in which the T7 RNAP binds to and bends the DNA, and opening, achieved by "scrunching" of the DNA. Since both steps involve mechanical deformation of the DNA, both may be affected by downstream DNA tension. Using an oscillating two-bead optical tweezers assay, we have measured the lifetime of single T7 RNAP-DNA initation complexes under tension. Global maximumlikelihood fitting of force-dependent and non-force-dependent versions of this minimal model shows that there is no conclusively discernible force-dependence of initiation in the measured 0-2 pN DNA tension range.

  12. The interplay between polymerase organization and nucleosome occupancy along DNA : How dynamic roadblocks on the DNA induce the formation of RNA polymerase pelotons

    NARCIS (Netherlands)

    van den Berg, A.A.

    2017-01-01

    During transcription RNA polymerase (RNAP) moves along a DNA molecule to copy the information on the DNA to an RNA molecule. Many textbook pictures show an RNAP sliding along empty DNA, but in reality it is crowded on the DNA and RNAP competes for space with many proteins such as other RNAP’s and

  13. The Escherichia coli regulator of sigma 70 protein, Rsd, can up-regulate some stress-dependent promoters by sequestering sigma 70.

    Science.gov (United States)

    Mitchell, Jennie E; Oshima, Taku; Piper, Sarah E; Webster, Christine L; Westblade, Lars F; Karimova, Gouzel; Ladant, Daniel; Kolb, Annie; Hobman, Jon L; Busby, Stephen J W; Lee, David J

    2007-05-01

    The Escherichia coli Rsd protein forms complexes with the RNA polymerase sigma(70) factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative sigma(38) factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with sigma(70). Our experiments support a model in which the role of Rsd is primarily to sequester sigma(70), thereby increasing the levels of RNA polymerase containing the alternative sigma(38) factor.

  14. Structure and function of the polymerase core of TRAMP, a RNA surveillance complex

    OpenAIRE

    Hamill, Stephanie; Wolin, Sandra L.; Reinisch, Karin M.

    2010-01-01

    The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ß superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ß superfamily both in sequence and in...

  15. Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments

    Directory of Open Access Journals (Sweden)

    Hafner Mathias

    2004-04-01

    Full Text Available Abstract Background The requirement of a large amount of high-quality RNA is a major limiting factor for microarray experiments using biopsies. An average microarray experiment requires 10–100 μg of RNA. However, due to their small size, most biopsies do not yield this amount. Several different approaches for RNA amplification in vitro have been described and applied for microarray studies. In most of these, systematic analyses of the potential bias introduced by the enzymatic modifications are lacking. Results We examined the sources of error introduced by the T7 RNA polymerase based RNA amplification method through hybridisation studies on microarrays and performed statistical analysis of the parameters that need to be evaluated prior to routine laboratory use. The results demonstrate that amplification of the RNA has no systematic influence on the outcome of the microarray experiment. Although variations in differential expression between amplified and total RNA hybridisations can be observed, RNA amplification is reproducible, and there is no evidence that it introduces a large systematic bias. Conclusions Our results underline the utility of the T7 based RNA amplification for use in microarray experiments provided that all samples under study are equally treated.

  16. Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

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    Guillaume Fournier

    2014-06-01

    Full Text Available Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

  17. NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble.

    Science.gov (United States)

    Turtola, Matti; Belogurov, Georgiy A

    2016-10-04

    Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.

  18. A Comparative Study of RNA Polymerase II Transcription Machinery in Yeasts

    Science.gov (United States)

    Sharma, Nimisha; Mehta, Surbhi

    The control of gene expression, predominantly at the level of transcription, plays a fundamental role in biological processes determining the phenotypic changes in cells and organisms. The eukaryotes have evolved a complex and sophisticated transcription machinery to transcribe DNA into RNA. RNA polymerase II enzyme lies at the centre of the transcription apparatus that comprises nearly 60 polypeptides and is responsible for the expression and regulation of proteinencoding genes. Much of our present understanding and knowledge of the RNA polymerase II transcription apparatus in eukaryotes has been derived from studies in Saccharomyces cerevisiae. More recently, Schizosaccharomyces pombe has emerged as a better model system to study transcription because the transcription mechanism in this yeast is closer to that in higher eukaryotes. Also, studies on components of the basal transcription machinery have revealed a number of properties that are common with other eukaryotes, but have also highlighted some features unique to S. pombe. In fact, the fungal transcription associated protein families show greater species specificity and only 15% of these proteins contain homologues shared between both S. cerevisiae and S. pombe. In this chapter, we compare the RNA polymerase II transcription apparatus in different yeasts.

  19. Cockayne syndrome group B protein enhances elongation by RNA polymerase II.

    Science.gov (United States)

    Selby, C P; Sancar, A

    1997-10-14

    Cockayne syndrome (CS) is characterized by impaired physical and mental development. Two complementation groups, CSA and CSB, have been identified. Here we report that the CSB gene product enhances elongation by RNA polymerase II. CSB stimulated the rate of elongation on an undamaged template by a factor of about 3. A thymine-thymine cyclobutane dimer located in the template strand is known to be a strong block to transcription. Addition of CSB to the blocked polymerase resulted in addition of one nucleotide to the nascent transcript. Finally, addition of transcription factor IIS is known to cause polymerase blocked at a thymine-thymine cyclobutane dimer to digest its nascent transcript, and CSB counteracted this transcript shortening action of transcription factor IIS. Thus a deficiency in transcription elongation may contribute to the CS phenotype.

  20. A Mechanistic Model for Cooperative Behavior of Co-transcribing RNA Polymerases.

    Science.gov (United States)

    Heberling, Tamra; Davis, Lisa; Gedeon, Jakub; Morgan, Charles; Gedeon, Tomáš

    2016-08-01

    In fast-transcribing prokaryotic genes, such as an rrn gene in Escherichia coli, many RNA polymerases (RNAPs) transcribe the DNA simultaneously. Active elongation of RNAPs is often interrupted by pauses, which has been observed to cause RNAP traffic jams; yet some studies indicate that elongation seems to be faster in the presence of multiple RNAPs than elongation by a single RNAP. We propose that an interaction between RNAPs via the torque produced by RNAP motion on helically twisted DNA can explain this apparent paradox. We have incorporated the torque mechanism into a stochastic model and simulated transcription both with and without torque. Simulation results illustrate that the torque causes shorter pause durations and fewer collisions between polymerases. Our results suggest that the torsional interaction of RNAPs is an important mechanism in maintaining fast transcription times, and that transcription should be viewed as a cooperative group effort by multiple polymerases.

  1. A Mechanistic Model for Cooperative Behavior of Co-transcribing RNA Polymerases.

    Directory of Open Access Journals (Sweden)

    Tamra Heberling

    2016-08-01

    Full Text Available In fast-transcribing prokaryotic genes, such as an rrn gene in Escherichia coli, many RNA polymerases (RNAPs transcribe the DNA simultaneously. Active elongation of RNAPs is often interrupted by pauses, which has been observed to cause RNAP traffic jams; yet some studies indicate that elongation seems to be faster in the presence of multiple RNAPs than elongation by a single RNAP. We propose that an interaction between RNAPs via the torque produced by RNAP motion on helically twisted DNA can explain this apparent paradox. We have incorporated the torque mechanism into a stochastic model and simulated transcription both with and without torque. Simulation results illustrate that the torque causes shorter pause durations and fewer collisions between polymerases. Our results suggest that the torsional interaction of RNAPs is an important mechanism in maintaining fast transcription times, and that transcription should be viewed as a cooperative group effort by multiple polymerases.

  2. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.

    Science.gov (United States)

    Giraud, Matthieu; Yoshida, Hideyuki; Abramson, Jakub; Rahl, Peter B; Young, Richard A; Mathis, Diane; Benoist, Christophe

    2012-01-10

    Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

  3. High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop.

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    Chenxi Qiu

    2016-11-01

    Full Text Available The active sites of multisubunit RNA polymerases have a "trigger loop" (TL that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

  4. The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

    Science.gov (United States)

    Brody, Yehuda; Neufeld, Noa; Bieberstein, Nicole; Causse, Sebastien Z.; Böhnlein, Eva-Maria; Neugebauer, Karla M.; Darzacq, Xavier; Shav-Tal, Yaron

    2011-01-01

    RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end. PMID:21264352

  5. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

    Directory of Open Access Journals (Sweden)

    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  6. Sigma A recognition sites in the Bacillus subtilis genome

    DEFF Research Database (Denmark)

    Jarmer, Hanne Østergaard; Larsen, Thomas Schou; Krogh, Anders Stærmose

    2001-01-01

    A hidden Markov model of sigma (A) RNA polymerase cofactor recognition sites in Bacillus subtilis, containing either the common or the extended -10 motifs, has been constructed based on experimentally verified sigma (A) recognition sites. This work suggests that more information exists...... at the initiation site of transcription in both types of promoters than previously thought. When tested on the entire B. subtilis genome, the model predicts that approximately half of the sigma (A) recognition sites are of the extended type. Some of the response-regulator aspartate phosphatases were among...

  7. Cooperative activities of hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin domain

    OpenAIRE

    Johnson, Kirby D.; Grass, Jeffrey A.; Boyer, Meghan E; Kiekhaefer, Carol M.; Blobel, Gerd A.; Weiss, Mitchell J.; Bresnick, Emery H.

    2002-01-01

    The hematopoietic transcription factor GATA-1 regulates erythropoiesis and β-globin expression. Although consensus GATA-1 binding sites exist throughout the murine β-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the β-globin promoters. The hematopoietic factor NF-E2 cooper...

  8. A Genetic Assay for Transcription Errors Reveals Multilayer Control of RNA Polymerase II Fidelity

    OpenAIRE

    Irvin, Jordan D.; Kireeva, Maria L.; Gotte, Deanna R.; Shafer, Brenda K.; Ingold Huang; Mikhail Kashlev; Strathern, Jeffrey N.

    2014-01-01

    Author Summary Mistakes made during the synthesis of messenger RNAs have been difficult to detect, both because mRNAs can be short lived, and because the translation of mRNAs into proteins has a much higher error rate that masks transcription errors. We present here a highly sensitive genetic screen that detects transcription errors and use it to identify mutations that increase the error rate of RNA polymerase II. The screen incorporates a new principle that allows transient transcription er...

  9. Pseudouridimycin: The First Nucleoside Analogue That Selectively Inhibits Bacterial RNA Polymerase.

    Science.gov (United States)

    Chellat, Mathieu F; Riedl, Rainer

    2017-10-16

    Seek, and ye shall find: After years of focusing research on synthetic antibiotics out of fear that all the useful natural ones had already been found, a novel antibacterial compound has been discovered through conventional microbial extract screening. The broad-spectrum nucleoside-analogue inhibitor pseudouridimycin is selective for bacterial RNA polymerase and elicits very low resistance rates. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Nature of the Nucleosomal Barrier to RNA Polymerase II | Center for Cancer Research

    Science.gov (United States)

    In the cell, RNA polymerase II (pol II) efficiently transcribes DNA packaged into nucleosomes, but in vitro encounters with the nucleosomes induce catalytic inactivation (arrest) of the pol II core enzyme. To determine potential mechanisms making nucleosomes transparent to transcription in vivo, we analyzed the nature of the nucleosome-induced arrest. We found that the arrests have been detected mostly at positions of strong intrinsic pause sites of DNA.

  11. RNA polymerase I-Rrn3 complex at 4.8 Å resolution

    Science.gov (United States)

    Engel, Christoph; Plitzko, Jürgen; Cramer, Patrick

    2016-07-01

    Transcription of ribosomal DNA by RNA polymerase I (Pol I) requires the initiation factor Rrn3. Here we report the cryo-EM structure of the Pol I-Rrn3 complex at 4.8 Å resolution. The structure reveals how Rrn3 binding converts an inactive Pol I dimer into an initiation-competent monomeric complex and provides insights into the mechanisms of Pol I-specific initiation and regulation.

  12. Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

    OpenAIRE

    Mariconti, Luisa; Loll, Bernhard; Schlinkmann, Karola; Wengi, Agnieszka; Meinhart, Anton; Dichtl, Bernhard

    2010-01-01

    RNA polymerase II (RNAP II) transcription and pre-mRNA 3′ end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3′ end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of trans...

  13. RNA Polymerase Structure, Function, Regulation, Dynamics, Fidelity, and Roles in GENE EXPRESSION | Center for Cancer Research

    Science.gov (United States)

    Multi-subunit RNA polymerases (RNAP) are ornate molecular machines that translocate on a DNA template as they generate a complementary RNA chain. RNAPs are highly conserved in evolution among eukarya, eubacteria, archaea, and some viruses. As such, multi-subunit RNAPs appear to be an irreplaceable advance in the evolution of complex life on earth. Because of their stepwise movement on DNA, RNAPs are considered to be molecular motors, and because RNAPs catalyze a templated polymerization reaction, they are central to biological information flow.

  14. Quantitative analysis of transcription elongation by RNA polymerase I in vitro.

    Science.gov (United States)

    Schneider, David Alan

    2012-01-01

    The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come.

  15. Stress induces changes in the phosphorylation of Trypanosoma cruzi RNA polymerase II, affecting its association with chromatin and RNA processing.

    Science.gov (United States)

    Rocha, Antônio Augusto; Moretti, Nilmar Silvio; Schenkman, Sergio

    2014-07-01

    The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Recent advances in understanding transcription termination by RNA polymerase II [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Travis J. Loya

    2016-06-01

    Full Text Available Transcription termination is a fundamental process in which RNA polymerase ceases RNA chain extension and dissociates from the chromatin template, thereby defining the end of the transcription unit. Our understanding of the biological role and functional importance of termination by RNA polymerase II and the range of processes in which it is involved has grown significantly in recent years. A large set of nucleic acid-binding proteins and enzymes have been identified as part of the termination machinery. A greater appreciation for the coupling of termination to RNA processing and metabolism has been recognized. In addition to serving as an essential step at the end of the transcription cycle, termination is involved in the regulation of a broad range of cellular processes. More recently, a role for termination in pervasive transcription, non-coding RNA regulation, genetic stability, chromatin remodeling, the immune response, and disease has come to the fore. Interesting mechanistic questions remain, but the last several years have resulted in significant insights into termination and an increasing recognition of its biological importance.

  17. 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.

  18. Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

    Directory of Open Access Journals (Sweden)

    Trina Ekawati Tallei

    2015-10-01

    Full Text Available Mitochondria of Paramecium caudatum stock GT704 has a set of four kinds of linear plasmids with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The plasmids of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomers, respectively. The plasmid 2.8 kb, designated as pGT704-2.8, contains an open reading frame encodes for putative DNA-dependent RNA polymerase (RNAP. This study reveals that this RNAP belongs to superfamily of DNA/RNA polymerase and family of T7/T3 single chain RNA polymerase and those of mitochondrial plasmid of fungi belonging to Basidiomycota and Ascomycota. It is suggested that RNAP of pGT704-2.8 can perform transcription without transcription factor as promoter recognition. Given that only two motifs were found, it could not be ascertained whether this RNAP has a full function independently or integrated with mtDNA in carrying out its function.

  19. Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data.

    Directory of Open Access Journals (Sweden)

    David L Corcoran

    Full Text Available MicroRNAs (miRNAs are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II promoter. However, the length of the primary transcripts and promoter organization is currently unknown.We performed Pol II chromatin immunoprecipitation (ChIP-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP. We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters.miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex.

  20. Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila

    Directory of Open Access Journals (Sweden)

    Kavi Harsh H

    2009-11-01

    Full Text Available Abstract Background Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure. Results The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls. Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites. Conclusion Our experiments show a genetic and biochemical interaction between RNA Pol II (largest

  1. Crystal structure of the Escherichia coli regulator of sigma70, Rsd, in complex with sigma70 domain 4.

    Science.gov (United States)

    Patikoglou, Georgia A; Westblade, Lars F; Campbell, Elizabeth A; Lamour, Valérie; Lane, William J; Darst, Seth A

    2007-09-21

    The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.

  2. Structure and function of the polymerase core of TRAMP, a RNA surveillance complex

    Science.gov (United States)

    Hamill, Stephanie; Wolin, Sandra L.; Reinisch, Karin M.

    2010-01-01

    The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ß superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ß superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-Å resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol β RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3′ end plays a role in substrate recognition. PMID:20696927

  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. Mammalian Rrn3 is required for the formation of a transcription competent preinitiation complex containing RNA polymerase I.

    Science.gov (United States)

    Cavanaugh, Alice H; Evans, Ann; Rothblum, Lawrence I

    2008-01-01

    Mammalian Rrn3, an essential, polymerase-associated protein, is inactivated when cells are treated with cycloheximide, resulting in the inhibition of transcription by RNA polymerase I. Although Rrn3 is essential for transcription, its function in rDNA transcription has not been determined. For example, it is unclear whether Rrn3 is required for initiation or elongation by RNA polymerase I. Rrn3 has been shown to interact with the 43-kDa subunit of RNA polymerase I and with two of the subunits of SL1. In the current model for transcription, Rrn3 functions to recruit RNA polymerase I to the committed complex formed by SL1 and the rDNA promoter. To examine the question as to whether Rrn3 is required for the recruitment of RNA polymerase I to the template, we developed a novel assay similar to chromatin immunoprecipitation assays. We found that RNA polymerase I can be recruited to a template in the absence of active Rrn3. However, that complex will not initiate transcription, even after Rrn3 is added to the reaction. Interestingly, the complex that forms in the presence of active Rrn3 is biochemically distinguishable from that which forms in the absence of active Rrn3. For example, the functional complex is fivefold more resistant to heparin than that which forms in the absence of Rrn3. Our data demonstrate that Rrn3 must be present when the committed template complex is forming for transcription to occur.

  5. Comparative analysis of hepatitis B virus polymerase sequences required for viral RNA binding, RNA packaging, and protein priming.

    Science.gov (United States)

    Jones, Scott A; Clark, Daniel N; Cao, Feng; Tavis, John E; Hu, Jianming

    2014-02-01

    Hepatitis B virus replicates a DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific association with a viral RNA signal, termed ε (Hε), located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. HP is made up of four domains, including the terminal protein (TP), the spacer, the reverse transcriptase (RT), and the RNase H domains. A recently developed, Hε-dependent, in vitro protein priming assay was used in this study to demonstrate that almost the entire TP and RT domains and most of the RNase H domain were required for protein priming. Specific residues within TP, RT, and the spacer were identified as being critical for HP-Hε binding and/or protein priming. Comparison of HP sequence requirements for Hε binding, pgRNA packaging, and protein priming allowed the classification of the HP mutants into five groups, each with distinct effects on these complex and related processes. Detailed characterization of HP requirements for these related and essential functions of HP will further elucidate the mechanisms of its multiple functions and aid in the targeting of these functions for antiviral therapy.

  6. 3D Molecular Modelling Study of the H7N9 RNA-Dependent RNA Polymerase as an Emerging Pharmacological Target.

    Science.gov (United States)

    Vlachakis, Dimitrios; Karozou, Argiro; Kossida, Sophia

    2013-01-01

    Currently not much is known about the H7N9 strain, and this is the major drawback for a scientific strategy to tackle this virus. Herein, the 3D complex structure of the H7N9 RNA-dependent RNA polymerase has been established using a repertoire of molecular modelling techniques including homology modelling, molecular docking, and molecular dynamics simulations. Strikingly, it was found that the oligonucleotide cleft and tunnel in the H7N9 RNA-dependent RNA polymerase are structurally very similar to the corresponding region on the hepatitis C virus RNA-dependent RNA polymerase crystal structure. A direct comparison and a 3D postdynamics analysis of the 3D complex of the H7N9 RNA-dependent RNA polymerase provide invaluable clues and insight regarding the role and mode of action of a series of interacting residues on the latter enzyme. Our study provides a novel and efficiently intergraded platform with structural insights for the H7N9 RNA-dependent RNA Polymerase. We propose that future use and exploitation of these insights may prove invaluable in the fight against this lethal, ongoing epidemic.

  7. Rsd family proteins make simultaneous interactions with regions 2 and 4 of the primary sigma factor.

    Science.gov (United States)

    Yuan, Andy H; Gregory, Brian D; Sharp, Josh S; McCleary, Katherine D; Dove, Simon L; Hochschild, Ann

    2008-12-01

    Bacterial anti-sigma factors typically regulate sigma factor function by restricting the access of their cognate sigma factors to the RNA polymerase (RNAP) core enzyme. The Escherichia coli Rsd protein forms a complex with the primary sigma factor, sigma(70), inhibits sigma(70)-dependent transcription in vitro, and has been proposed to function as a sigma(70)-specific anti-sigma factor, thereby facilitating the utilization of alternative sigma factors. In prior work, Rsd has been shown to interact with conserved region 4 of sigma(70), but it is not known whether this interaction suffices to account for the regulatory functions of Rsd. Here we show that Rsd and the Rsd orthologue AlgQ, a global regulator of gene expression in Pseudomonas aeruginosa, interact with conserved region 2 of sigma(70). We show further that Rsd and AlgQ can interact simultaneously with regions 2 and 4 of sigma(70). Our findings establish that the abilities of Rsd and AlgQ to interact with sigma(70) region 2 are important determinants of their in vitro and in vivo activities.

  8. 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

  9. Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters.

    Science.gov (United States)

    Krebs, Arnaud R; Imanci, Dilek; Hoerner, Leslie; Gaidatzis, Dimos; Burger, Lukas; Schübeler, Dirk

    2017-08-03

    Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. 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.

  11. Kinetic competition between RNA Polymerase II and Sen1-dependent transcription termination

    DEFF Research Database (Denmark)

    Hazelbaker, Dane Z; Marquardt, Sebastian; Wlotzka, Wiebke

    2013-01-01

    processed. Sen1 mutants or faster-transcribing Pol II increase the average lengths of preprocessed snoRNA, CUT, and SUT transcripts, while slowed Pol II transcription produces shorter transcripts. These connections between transcription rate and Sen1 activity support a model whereby kinetic competition......The essential helicase-like protein Sen1 mediates termination of RNA Polymerase II (Pol II) transcription at snoRNAs and other noncoding RNAs in yeast. A mutation in the Pol II subunit Rpb1 that increases the elongation rate increases read-through transcription at Sen1-mediated terminators....... Termination and growth defects in sen1 mutant cells are partially suppressed by a slowly transcribing Pol II mutant and are exacerbated by a faster-transcribing Pol II mutant. Deletion of the nuclear exosome subunit Rrp6 allows visualization of noncoding RNA intermediates that are terminated but not yet...

  12. Involvement of phosphatidylinositol 4,5-bisphosphate in RNA polymerase I transcription.

    Science.gov (United States)

    Yildirim, Sukriye; Castano, Enrique; Sobol, Margarita; Philimonenko, Vlada V; Dzijak, Rastislav; Venit, Tomás; Hozák, Pavel

    2013-06-15

    RNA polymerase I (Pol I) transcription is essential for the cell cycle, growth and protein synthesis in eukaryotes. In the present study, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) is a part of the protein complex on the active ribosomal promoter during transcription. PIP2 makes a complex with Pol I and the Pol I transcription factor UBF in the nucleolus. PIP2 depletion reduces Pol I transcription, which can be rescued by the addition of exogenous PIP2. In addition, PIP2 also binds directly to the pre-rRNA processing factor fibrillarin (Fib), and co-localizes with nascent transcripts in the nucleolus. PIP2 binding to UBF and Fib modulates their binding to DNA and RNA, respectively. In conclusion, PIP2 interacts with a subset of Pol I transcription machinery, and promotes Pol I transcription.

  13. Bacterial Transcription Inhibitor of RNA Polymerase Holoenzyme Formation by Structure-Based Drug Design: From in Silico Screening to Validation.

    Science.gov (United States)

    Ma, Cong; Yang, Xiao; Lewis, Peter J

    2016-01-08

    Bacterial transcription is a proven target for antibacterial research. However, most of the known inhibitors targeting transcription are from natural extracts or are hits from screens where the binding site remains unidentified. Using an RNA polymerase holoenzyme homology structure from the model Gram-positive organism Bacillus subtilis, we created a pharmacophore model and used it for in silico screening of a publicly available library for compounds able to inhibit holoenzyme formation. The hits demonstrated specific affinity to bacterial RNA polymerase and excellent activity using in vitro assays and showed no binding to the equivalent structure from human RNA polymerase II. The target specificity in live cells and antibacterial activity was demonstrated in microscopy and growth inhibition experiments. This is the first example of targeted inhibitor development for a bacterial RNA polymerase, outlining a complete discovery process from virtual screening to biochemical validation. This approach could serve as an appropriate platform for the future identification of inhibitors of bacterial transcription.

  14. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

    Science.gov (United States)

    Gilmour, D S; Lis, J T

    1986-11-01

    By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

  15. An RNA polymerase III-dependent heterochromatin barrier at fission yeast centromere 1.

    Directory of Open Access Journals (Sweden)

    Kristin C Scott

    Full Text Available Heterochromatin formation involves the nucleation and spreading of structural and epigenetic features along the chromatin fiber. Chromatin barriers and associated proteins counteract the spreading of heterochromatin, thereby restricting it to specific regions of the genome. We have performed gene expression studies and chromatin immunoprecipitation on strains in which native centromere sequences have been mutated to study the mechanism by which a tRNA(Alanine gene barrier (cen1 tDNA(Ala blocks the spread of pericentromeric heterochromatin at the centromere of chromosome 1 (cen1 in the fission yeast, Schizosaccharomyces pombe. Within the centromere, barrier activity is a general property of tDNAs and, unlike previously characterized barriers, requires the association of both transcription factor IIIC and RNA Polymerase III. Although the cen1 tDNA(Ala gene is actively transcribed, barrier activity is independent of transcriptional orientation. These findings provide experimental evidence for the involvement of a fully assembled RNA polymerase III transcription complex in defining independent structural and functional domains at a eukaryotic centromere.

  16. 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.

  17. Sequence and structure prediction of RNA-dependent RNA polymerase of lily symptomless virus isolated from L. × 'Casablanca'.

    Science.gov (United States)

    Xu, Pinsan; Li, Huangai; Liu, Jiwen; Luan, Yushi; Yin, Yalei; Bai, Jianfang

    2011-06-01

    The DNA sequence of the RNA-dependent RNA polymerase (RdRp) gene of lily symptomless virus (LSV), a lily-infecting member of the genus Carlavirus, was determined from nine overlapping cDNA fragments of different sizes. The complete sequence of this RdRp gene (HM070294) consisted of 5,847 nucleotides coding for a protein of 220 kDa. It had 97-98% sequence identity with RdRps of other known isolates at both the DNA and the amino acid level. Phylogenetic analysis indicated that this RdRp (designated as RdRp-DL) was closely related to the RdRp of the Korean isolate (AM516059), as well as to the RdRps from Passiflora latent virus (PLV) and Kalanchoe latent virus (KLV) of the genus Carlavirus. Hydrophobic analysis of RdRp-DL revealed a hydrophobic N-terminus and a hydrophilic C-terminus. Helices and Loops were the major secondary structures of RdRp-DL. In addition, RdRp-DL also had three coil structures. Four conserved domains were identified: typoviral methyltransferase, RNA-dependent RNA polymerase, P-loop-containing nucleoside triphosphate hydrolases and carlavirus endopeptidase. A model of the tertiary structure predicted by I-TASSER was obtained for each of these conserved domains. This is the first report of a detailed phylogenetic analysis of LSV RdRp with those of other members of the genus Carlavirus, and the first to predict the domain structures of LSV RdRp.

  18. Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase*

    Science.gov (United States)

    Moustafa, Ibrahim M.; Korboukh, Victoria K.; Arnold, Jamie J.; Smidansky, Eric D.; Marcotte, Laura L.; Gohara, David W.; Yang, Xiaorong; Sánchez-Farrán, María Antonieta; Filman, David; Maranas, Janna K.; Boehr, David D.; Hogle, James M.; Colina, Coray M.; Cameron, Craig E.

    2014-01-01

    RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity. PMID:25378410

  19. Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.

    Science.gov (United States)

    Moustafa, Ibrahim M; Korboukh, Victoria K; Arnold, Jamie J; Smidansky, Eric D; Marcotte, Laura L; Gohara, David W; Yang, Xiaorong; Sánchez-Farrán, María Antonieta; Filman, David; Maranas, Janna K; Boehr, David D; Hogle, James M; Colina, Coray M; Cameron, Craig E

    2014-12-26

    RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

    Science.gov (United States)

    Rijal, Keshab; Maraia, Richard J

    2016-08-01

    The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.