Journal articles: 'RNA polymerases'

1

Serrano, Alicia, Martín Moret, Isabel Fernández-Parras, Aureliano Bombarely, Francisco Luque, and Francisco Navarro. "RNA Polymerases IV and V Are Involved in Olive Fruit Development." Genes 15, no. 1 (December 19, 2023): 1. http://dx.doi.org/10.3390/genes15010001.

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Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialise some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as those corresponding to RNA polymerase II in olive trees. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olives. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development through the synthesis of this type of RNAs.

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Shareef, Afzaal M., Barbara Ludeke, Paul Jordan, Jerome Deval, and Rachel Fearns. "Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases." PLOS Pathogens 17, no. 12 (December 16, 2021): e1010151. http://dx.doi.org/10.1371/journal.ppat.1010151.

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It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

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Crotty, Shane, David Gohara, Devin K. Gilligan, Sveta Karelsky, Craig E. Cameron, and Raul Andino. "Manganese-Dependent Polioviruses Caused by Mutations within the Viral Polymerase." Journal of Virology 77, no. 9 (May 1, 2003): 5378–88. http://dx.doi.org/10.1128/jvi.77.9.5378-5388.2003.

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ABSTRACT Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3Dpol, we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn2+-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.

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Egorova, Tatiana, Ekaterina Shuvalova, Sabina Mukba, Alexey Shuvalov, Peter Kolosov, and Elena Alkalaeva. "Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides." International Journal of Molecular Sciences 22, no. 10 (May 14, 2021): 5186. http://dx.doi.org/10.3390/ijms22105186.

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Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.

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Pan, Junhua, Vikram N. Vakharia, and Yizhi Jane Tao. "The structure of a birnavirus polymerase reveals a distinct active site topology." Proceedings of the National Academy of Sciences 104, no. 18 (April 24, 2007): 7385–90. http://dx.doi.org/10.1073/pnas.0611599104.

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Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.

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6

Bettiol, Michael F., Randall T. Irvin, and Paul A. Horgen. "Immunological analyses of selected eukaryotic RNA polymerases II." Canadian Journal of Biochemistry and Cell Biology 63, no. 12 (December 1, 1985): 1217–30. http://dx.doi.org/10.1139/o85-153.

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Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.

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7

Cramer, Patrick. "Multisubunit RNA polymerases." Current Opinion in Structural Biology 12, no. 1 (February 2002): 89–97. http://dx.doi.org/10.1016/s0959-440x(02)00294-4.

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8

Lysenko, E. A., and V. V. Kuznetsov. "Plastid RNA Polymerases." Molecular Biology 39, no. 5 (September 2005): 661–74. http://dx.doi.org/10.1007/s11008-005-0081-1.

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9

Blair, D. G. R. "Eukaryotic RNA polymerases." Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 89, no. 4 (January 1988): 647–70. http://dx.doi.org/10.1016/0305-0491(88)90306-9.

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10

Peramachi Palanivelu. "Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts." World Journal of Advanced Research and Reviews 17, no. 3 (March 30, 2023): 706–27. http://dx.doi.org/10.30574/wjarr.2023.17.3.0455.

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Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.

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11

Core, Leighton J., Joshua J. Waterfall, and John T. Lis. "Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters." Science 322, no. 5909 (December 4, 2008): 1845–48. http://dx.doi.org/10.1126/science.1162228.

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RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on ∼30% of human genes, transcription extends beyond pre-messenger RNA 3′ cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.

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12

Siegel, Robert W., Laurent Bellon, Leonid Beigelman, and C. Cheng Kao. "Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World." Journal of Virology 73, no. 8 (August 1, 1999): 6424–29. http://dx.doi.org/10.1128/jvi.73.8.6424-6429.1999.

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ABSTRACT All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.

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Korkhin, Yakov, Ulug M. Unligil, Otis Littlefield, Pamlea J. Nelson, David I. Stuart, Paul B. Sigler, Stephen D. Bell, and Nicola G. A. Abrescia. "Evolution of Complex RNA Polymerases: The Complete Archaeal RNA Polymerase Structure." PLoS Biology 7, no. 5 (May 5, 2009): e1000102. http://dx.doi.org/10.1371/journal.pbio.1000102.

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14

Cohen, Susan E., Veronica G. Godoy, and Graham C. Walker. "Transcriptional Modulator NusA Interacts with Translesion DNA Polymerases in Escherichia coli." Journal of Bacteriology 191, no. 2 (November 7, 2008): 665–72. http://dx.doi.org/10.1128/jb.00941-08.

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ABSTRACT NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.

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Peramachi Palanivelu. "Identification of DEDD- and PHP-Superfamilies of Proofreading Exonucleases in the Acidic Protein Subunit PA of RNA Polymerase of Human Influenza Viruses." World Journal of Advanced Research and Reviews 16, no. 2 (November 30, 2022): 804–24. http://dx.doi.org/10.30574/wjarr.2022.16.2.1260.

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RNA polymerase from human influenza viruses A, B and C is a heterotrimric enzyme, made up of three different subunits. It performs the crucial function of both genome replication as well as transcription. One of the RNA polymerase subunits, the polymerase acidic protein subunit (PA), is suggested to function as an endonuclease in a ‘cap-snatching’ mechanism, unique to influenza viruses. However, by multiple sequence alignment (MSA) analysis, it was found that the PA subunits of the polymerases do harbour typical proofreading (PR) DEDD-superfamily of exonuclease active site in all three viruses. However, in human influenza A virus, an additional putative PR exonuclease active site amino acids belonging to Polymerase-Histidinol Phosphatase (PHP)-superfamily are identified. The identified active site amino acids data are in close agreement with the similar DEDD- and PHP-superfamilies of PR exonucleases, already reported from both DNA-dependent RNA polymerases (DdRps) and RNA-dependent RNA polymerases (RdRps) from prokaryotes, eukaryotes and RNA viruses. The putative PHP–family PR exonuclease active site, identified by MSA analysis, is also in close agreement to the already reported PHP–family of PR exonuclease active sites from DdDps of replicases and pol X polymerases of the bacterial kingdom. Only in the pandemic causing human influenza A virus, the putative PHP-family PR exonuclease domain is found along with the DEDD-family PR exonuclease domain.

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Fraenkel‐Conrat, Heinz, and Albert Van Kammen. "RNA‐Directed rna polymerases of plants." Critical Reviews in Plant Sciences 4, no. 3 (January 1986): 213–26. http://dx.doi.org/10.1080/07352688609382224.

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Choi, Kyung H., and Michael G. Rossmann. "RNA-dependent RNA polymerases from Flaviviridae." Current Opinion in Structural Biology 19, no. 6 (December 2009): 746–51. http://dx.doi.org/10.1016/j.sbi.2009.10.015.

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Kawaguchi, Atsushi, Tadasuke Naito, and Kyosuke Nagata. "Involvement of Influenza Virus PA Subunit in Assembly of Functional RNA Polymerase Complexes." Journal of Virology 79, no. 2 (January 15, 2005): 732–44. http://dx.doi.org/10.1128/jvi.79.2.732-744.2005.

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ABSTRACT The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear. ts53 virus is a temperature-sensitive (ts) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology 65:363-373, 1975). We confirmed that the mRNA synthesis level of ts53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding ts53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In ts53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.

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Ahlquist, P. "RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing." Science 296, no. 5571 (May 17, 2002): 1270–73. http://dx.doi.org/10.1126/science.1069132.

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Naryshkina, Tatyana, Adrian Bruning, Olivier Gadal, and Konstantin Severinov. "Role of Second-Largest RNA Polymerase I Subunit Zn-Binding Domain in Enzyme Assembly." Eukaryotic Cell 2, no. 5 (October 2003): 1046–52. http://dx.doi.org/10.1128/ec.2.5.1046-1052.2003.

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ABSTRACT The second-largest subunits of eukaryal RNA polymerases are similar to the β subunits of prokaryal RNA polymerases throughout much of their lengths. The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini. The domain is also present in archaeal homologs but is absent from prokaryal homologs. Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I. Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex. Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn2+ binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function. Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.

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Jeng, Shih-Tong, Sheue-Hwey Lay, and Hsi-Mei Lai. "Transcription termination by bacteriophage T3 and SP6 RNA polymerases at Rho-independent terminators." Canadian Journal of Microbiology 43, no. 12 (December 1, 1997): 1147–56. http://dx.doi.org/10.1139/m97-163.

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Transcription termination of T3 and SP6 DNA-dependent RNA polymerases have been studied on the DNA templates containing the threonine (thr) attenuator and its variants. The thr attenuator is from the regulatory region of the thr operon of Escherichia coli. The DNA template, encoding the thr attenuator, contains specific features of the rho-independent terminators. It comprises a dG + dC rich dyad symmetry, encoding a stem-and-loop RNA, which is followed by a poly(U) region at the 3′-end. Thirteen attenuator variants have been analyzed for their ability to terminate transcription and the results indicated that the structure as well as the sequence in the G + C rich region of RNA hairpin affect termination of both RNA polymerases. Also, a single base change in the A residues of the hairpin failed to influence termination, whereas changes in the poly(U) region significantly reduced the termination of both T3 and SP6 RNA polymerases. The requirement of a poly(U) region for termination by T3 and SP6 RNA polymerases was studied with nested deletion mutants in this region. The minimum number of U residues required for termination of SP6 and T3 RNA polymerases was five and three, respectively. However, both RNA polymerases needed at least eight U residues to reach a termination efficiency close to that achieved by wild-type thr attenuator encoding nine U residues. In addition, the orientation of the loop sequences of the RNA hairpin did not affect the transcription termination of either of the bacteriophage RNA polymerases.Key words: transcription termination, bacteriophage RNA polymerase.

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Thomm, Michael, Christoph Reich, Sebastian Grünberg, and Souad Naji. "Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 18–22. http://dx.doi.org/10.1042/bst0370018.

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The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure–function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the α subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the ΔH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

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Kulbachinskiy, A., I. Bass, E. Bogdanova, A. Goldfarb, and V. Nikiforov. "Cold Sensitivity of Thermophilic and Mesophilic RNA Polymerases." Journal of Bacteriology 186, no. 22 (November 15, 2004): 7818–20. http://dx.doi.org/10.1128/jb.186.22.7818-7820.2004.

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ABSTRACT RNA polymerase from mesophilic Deinococcus radiodurans displays the same cold sensitivity of promoter opening as RNA polymerase from the closely related thermophilic Thermus aquaticus. This suggests that, contrary to the accepted view, cold sensitivity of promoter opening by thermophilic RNA polymerases may not be a consequence of their thermostability.

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Mirkovitch, J., and J. E. Darnell. "Mapping of RNA polymerase on mammalian genes in cells and nuclei." Molecular Biology of the Cell 3, no. 10 (October 1992): 1085–94. http://dx.doi.org/10.1091/mbc.3.10.1085.

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The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.

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Willis, S. H., K. M. Kazmierczak, R. H. Carter, and L. B. Rothman-Denes. "N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases." Journal of Bacteriology 184, no. 18 (September 15, 2002): 4952–61. http://dx.doi.org/10.1128/jb.184.18.4952-4961.2002.

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ABSTRACT Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

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Martin, M., and F. J. Medina. "A Drosophila anti-RNA polymerase II antibody recognizes a plant nucleolar antigen, RNA polymerase I, which is mostly localized in fibrillar centres." Journal of Cell Science 100, no. 1 (September 1, 1991): 99–107. http://dx.doi.org/10.1242/jcs.100.1.99.

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The distribution of nucleolar RNA polymerase in the nucleolus of onion root meristematic cells has been studied by means of an antibody originally raised against Drosophila RNA polymerase II. This antibody recognizes the homologous domains of the large subunit of the enzyme, which are highly conserved throughout evolution in the three classes of eucaryotic RNA polymerases. Given that RNA polymerase I is confined to the nucleolus, and that the onion cell nucleolus lacks digitations of extranucleolar chromatin, we conclude that the nucleolar enzyme localized is RNA polymerase I. A quantitative approach, independent of the existence of borderlines between nucleolar fibrillar centres and the dense fibrillar component, allowed us to show that the enzyme is localized in fibrillar centres and in the transition area between them and the dense fibrillar component, in parallel with the nucleolar DNA. These results, together with previous autoradiographic, cytochemical and immunocytochemical results, in this and other species, lead us to conclude that the activation of rDNA for transcription occurs in the fibrillar centres and pre-rRNA synthesis is expressed at the transition area between fibrillar centres and the dense fibrillar component. Fibrillar centres are connected to each other by extended RNA polymerase-bound DNA fibres, presumably active in transcription. This work provides evidence of the high evolutionary conservation of some domains of the large subunit of RNA polymerases and of the existence of fibrillar centres in the nucleolus of plant cells, totally homologous to those described in mammalian cells.

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Laurila, Minni R. L., Paula S. Salgado, David I. Stuart, Jonathan M. Grimes, and Dennis H. Bamford. "Back-priming mode of ϕ6 RNA-dependent RNA polymerase." Journal of General Virology 86, no. 2 (February 1, 2005): 521–26. http://dx.doi.org/10.1099/vir.0.80492-0.

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The RNA-dependent RNA polymerase of the double-stranded RNA bacteriophage ϕ6 is capable of primer-independent initiation, as are many RNA polymerases. The structure of this polymerase revealed an initiation platform, composed of a loop in the C-terminal domain (QYKW, aa 629–632), that was essential for de novo initiation. A similar element has been identified in hepatitis C virus RNA-dependent RNA polymerase. Biochemical studies have addressed the role of this platform, revealing that a mutant version can utilize a back-priming initiation mechanism, where the 3′ terminus of the template adopts a hairpin-like conformation. Here, the mechanism of back-primed initiation is studied further by biochemical and structural methods.

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Makeyev, Eugene V., and Jonathan M. Grimes. "RNA-dependent RNA polymerases of dsRNA bacteriophages." Virus Research 101, no. 1 (April 2004): 45–55. http://dx.doi.org/10.1016/j.virusres.2003.12.005.

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Jensen, Grant J., Gavin Meredith, David A. Bushnell, and Roger D. Kornberg. "Structure of Wild Type Yeast RNA Polymerase II and Location of RPB4 and RPB7." Microscopy and Microanalysis 4, S2 (July 1998): 972–73. http://dx.doi.org/10.1017/s1431927600024983.

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Nucleic acid polymerase structure has been studied by both X-ray and electron crystallography. To date, only the smaller, single subunit polymerases have been subjected to X-ray analysis, including the bacteriophage T7 RNA polymerase, which is the only RNA polymerase whose structure is known to atomic resolution. Lower resolution structures of several multisubunit polymerases have been determined by electron crystallography, including a mutant form of yeast RNA polymerase II which lacks subunits Rpb4 and Rpb7 (denoted A4/7 polymerase). All polymerase structures obtained by both X-ray and electron crystallography show a large cleft appropriate in size for binding duplex DNA, and further appear to contain a mobile arm allowing open and closed conformations of the cleft, presumably permitting entry and retention of DNA. Subunits Rpb4 and Rpb7 of RNA polymerase II form a dissociable subcomplex that has been implicated in the stress response and in the initiation of transcription. Human homologs of Rpb4 and Rpb7 have been identified.

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Zhang, Jiayou. "Host RNA polymerase II makes minimal contributions to retroviral frame-shift mutations." Journal of General Virology 85, no. 8 (August 1, 2004): 2389–95. http://dx.doi.org/10.1099/vir.0.80081-0.

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The rate of mutation during retrovirus replication is high. Mutations can occur during transcription of the viral genomic RNA from the integrated provirus or during reverse transcription from viral RNA to form viral DNA or during replication of the proviral DNA as the host cell is dividing. Therefore, three polymerases may all contribute to retroviral evolution: host RNA polymerase II, viral reverse transcriptases and host DNA polymerases, respectively. Since the rate of mutation for host DNA polymerase is very low, mutations are more likely to be caused by the host RNA polymerase II and/or the viral reverse transcriptase. A system was established to detect the frequency of frame-shift mutations caused by cellular RNA polymerase II, as well as the rate of retroviral mutation during a single cycle of replication in vivo. In this study, it was determined that RNA polymerase II contributes less than 3 % to frame-shift mutations that occur during retrovirus replication. Therefore, the majority of frame-shift mutations detected within the viral genome are the result of errors during reverse transcription.

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Huang, Kun, Xiao-Xian Wu, Cheng-Li Fang, Zhou-Geng Xu, Hong-Wei Zhang, Jian Gao, Chuan-Miao Zhou, et al. "Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA." Science 374, no. 6575 (December 24, 2021): 1579–86. http://dx.doi.org/10.1126/science.abj9184.

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A tunnel between RNA polymerases Eukaryotes encode five multiple-subunit, DNA-dependent RNA polymerases, of which Pol I, Pol II, and Pol III function as single units to produce cellular single-stranded RNA. The plant-specific Pol IV forms a complex with RDR2 (an RNA-dependent RNA polymerase) to produce double-stranded precursors of small interfering RNA essential for genomic DNA methylation. Huang et al . determined the cryo–electron microscopy structures of the Pol IV-RDR2 complex. The structures show that Pol IV and RDR2 connect their active centers through an inner RNA transfer channel and that Pol IV reverses transcription direction and hands over its transcript directly through the channel to RDR2 for the production of the second strand of the double-stranded RNA. —DJ

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Peil, Kadri, Signe Värv, Marko Lõoke, Kersti Kristjuhan, and Arnold Kristjuhan. "Uniform Distribution of Elongating RNA Polymerase II Complexes in Transcribed Gene Locus." Journal of Biological Chemistry 286, no. 27 (May 23, 2011): 23817–22. http://dx.doi.org/10.1074/jbc.m111.230805.

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The intensity of gene transcription is generally reflected by the level of RNA polymerase II (RNAPII) recruitment to the gene. However, genome-wide studies of polymerase occupancy indicate that RNAPII distribution varies among genes. In some loci more polymerases are found in the 5′ region, whereas in other loci, in the 3′ region of the gene. We studied the distribution of elongating RNAPII complexes at highly transcribed GAL-VPS13 locus in Saccharomyces cerevisiae and found that in the cell population the amount of polymerases gradually decreased toward the 3′ end of the gene. However, the conventional chromatin immunoprecipitation assay averages the signal from the cell population, and no data on single cell level can be gathered. To study the spacing of elongating polymerases on single chromosomes, we used a sequential chromatin immunoprecipitation assay for the detection of multiple RNAPII complexes on the same DNA fragment. Our results demonstrate uniform distribution of elongating polymerases throughout all regions of the GAL-VPS13 gene.

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Campagnola, Grace, Seth McDonald, Stéphanie Beaucourt, Marco Vignuzzi, and Olve B. Peersen. "Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases." Journal of Virology 89, no. 1 (October 15, 2014): 275–86. http://dx.doi.org/10.1128/jvi.01574-14.

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ABSTRACTViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates andin vivofidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects onin vitronucleotide discrimination, and these effects are strongly correlated with elongation rates andin vivomutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects onin vitronucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growthin vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

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Steffen, Pierre, and Agnes Ullmann. "Hybrid Bordetella pertussis-Escherichia coli RNA Polymerases: Selectivity of Promoter Activation." Journal of Bacteriology 180, no. 6 (March 15, 1998): 1567–69. http://dx.doi.org/10.1128/jb.180.6.1567-1569.1998.

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ABSTRACT We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lacpromoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli α subunit. This suggests that activator contact sites involve a high degree of selectivity.

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Potisopon, Supanee, Stéphane Priet, Axelle Collet, Etienne Decroly, Bruno Canard, and Barbara Selisko. "The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain." Nucleic Acids Research 42, no. 18 (September 10, 2014): 11642–56. http://dx.doi.org/10.1093/nar/gku666.

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Abstract Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.

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Suphaphiphat, Pirada, Bjoern Keiner, Heidi Trusheim, Stefania Crotta, Annunziata Barbara Tuccino, Pu Zhang, Philip R. Dormitzer, Peter W. Mason, and Michael Franti. "Human RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus in Canine Cells." Journal of Virology 84, no. 7 (January 13, 2010): 3721–25. http://dx.doi.org/10.1128/jvi.01925-09.

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ABSTRACT We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a nonendogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.

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Lisica, Ana, and Stephan W. Grill. "Optical tweezers studies of transcription by eukaryotic RNA polymerases." Biomolecular Concepts 8, no. 1 (March 1, 2017): 1–11. http://dx.doi.org/10.1515/bmc-2016-0028.

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AbstractTranscription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

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Cramer, P., K. J. Armache, S. Baumli, S. Benkert, F. Brueckner, C. Buchen, G. E. Damsma, et al. "Structure of Eukaryotic RNA Polymerases." Annual Review of Biophysics 37, no. 1 (June 2008): 337–52. http://dx.doi.org/10.1146/annurev.biophys.37.032807.130008.

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39

Studitsky, V. "Chromatin remodeling by RNA polymerases." Trends in Biochemical Sciences 29, no. 3 (March 2004): 127–35. http://dx.doi.org/10.1016/j.tibs.2004.01.003.

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Wild, Thomas, and Patrick Cramer. "Biogenesis of multisubunit RNA polymerases." Trends in Biochemical Sciences 37, no. 3 (March 2012): 99–105. http://dx.doi.org/10.1016/j.tibs.2011.12.001.

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Cramer, Patrick, and Eddy Arnold. "Proteins: how RNA polymerases work." Current Opinion in Structural Biology 19, no. 6 (December 2009): 680–82. http://dx.doi.org/10.1016/j.sbi.2009.10.013.

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42

Howe, Christopher J. "RNA polymerases and plastid evolution." Trends in Plant Science 1, no. 10 (October 1996): 323–24. http://dx.doi.org/10.1016/s1360-1385(96)82586-0.

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Gomez-Roman, Natividad, Zoë A. Felton-Edkins, Niall S. Kenneth, Sarah J. Goodfellow, Dimitris Athineos, Jingxin Zhang, Ben A. Ramsbottom, et al. "Activation by c-Myc of transcription by RNA polymerases I, II and III." Biochemical Society Symposia 73 (January 1, 2006): 141–54. http://dx.doi.org/10.1042/bss0730141.

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The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.

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Jain, Nimit, Lucas R. Blauch, Michal R. Szymanski, Rhiju Das, Sindy K. Y. Tang, Y. Whitney Yin, and Andrew Z. Fire. "Transcription polymerase–catalyzed emergence of novel RNA replicons." Science 368, no. 6487 (March 26, 2020): eaay0688. http://dx.doi.org/10.1126/science.aay0688.

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Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.

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Steitz, T., S. Smerdon, J. Jager, and C. Joyce. "A unified polymerase mechanism for nonhomologous DNA and RNA polymerases." Science 266, no. 5193 (December 23, 1994): 2022–25. http://dx.doi.org/10.1126/science.7528445.

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Coggins, Si'Ana A., Bijan Mahboubi, Raymond F. Schinazi, and Baek Kim. "Mechanistic cross-talk between DNA/RNA polymerase enzyme kinetics and nucleotide substrate availability in cells: Implications for polymerase inhibitor discovery." Journal of Biological Chemistry 295, no. 39 (July 31, 2020): 13432–43. http://dx.doi.org/10.1074/jbc.rev120.013746.

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Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.

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Shatskaya, G. S., and T. M. Dmitrieva. "Structural organization of viral RNA-dependent RNA polymerases." Biochemistry (Moscow) 78, no. 3 (March 2013): 231–35. http://dx.doi.org/10.1134/s0006297913030036.

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Wassenegger, Michael, and Gabi Krczal. "Nomenclature and functions of RNA-directed RNA polymerases." Trends in Plant Science 11, no. 3 (March 2006): 142–51. http://dx.doi.org/10.1016/j.tplants.2006.01.003.

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FERRERORTA, C., A. ARIAS, C. ESCARMIS, and N. VERDAGUER. "A comparison of viral RNA-dependent RNA polymerases." Current Opinion in Structural Biology 16, no. 1 (February 2006): 27–34. http://dx.doi.org/10.1016/j.sbi.2005.12.002.

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Lescar, Julien, and Bruno Canard. "RNA-dependent RNA polymerases from flaviviruses and Picornaviridae." Current Opinion in Structural Biology 19, no. 6 (December 2009): 759–67. http://dx.doi.org/10.1016/j.sbi.2009.10.011.

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Journal articles on the topic 'RNA polymerases'

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