Artículos de revistas: "Transcription by bacterial RNA polymerase"

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Djordjevic, Marko. "Modeling Transcription Initiation By Bacterial RNA Polymerase". Biophysical Journal 96, n.º 3 (febrero de 2009): 57a. http://dx.doi.org/10.1016/j.bpj.2008.12.193.

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Mosaei, Hamed y John Harbottle. "Mechanisms of antibiotics inhibiting bacterial RNA polymerase". Biochemical Society Transactions 47, n.º 1 (15 de enero de 2019): 339–50. http://dx.doi.org/10.1042/bst20180499.

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Abstract Transcription, the first phase of gene expression, is performed by the multi-subunit RNA polymerase (RNAP). Bacterial RNAP is a validated target for clinical antibiotics. Many natural and synthetic compounds are now known to target RNAP, inhibiting various stages of the transcription cycle. However, very few RNAP inhibitors are used clinically. A detailed knowledge of inhibitors and their mechanisms of action (MOA) is vital for the future development of efficacious antibiotics. Moreover, inhibitors of RNAP are often useful tools with which to dissect RNAP function. Here, we review the MOA of antimicrobial transcription inhibitors.

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3

Zhang, Nan, Vidya C. Darbari, Robert Glyde, Xiaodong Zhang y Martin Buck. "The bacterial enhancer-dependent RNA polymerase". Biochemical Journal 473, n.º 21 (27 de octubre de 2016): 3741–53. http://dx.doi.org/10.1042/bcj20160741c.

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Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.

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4

Szalewska-Pałasz, Agnieszka. "Properties of Escherichia coli RNA polymerase from a strain devoid of the stringent response alarmone ppGpp." Acta Biochimica Polonica 55, n.º 2 (14 de junio de 2008): 317–23. http://dx.doi.org/10.18388/abp.2008_3078.

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The stringent response alarmone guanosine tetraphosphate (ppGpp) affects transcription from many promoters. ppGpp binds directly to the transcription enzyme of Escherichia coli, RNA polymerase. Analysis of the crystal structure of RNA polymerase with ppGpp suggested that binding of this nucleotide may result in some conformational or post-translational alterations to the enzyme. These changes might affect in vitro performance of the enzyme. Here, a comparison of the in vitro properties of RNA polymerases isolated from wild type and ppGpp-deficient bacteria shows that both enzymes do not differ in i) transcription activity of various promoters (e.g. sigma(70)-rrnB P1, lambdapL, T7A1), ii) response to ppGpp, iii) promoter-RNA polymerase open complex stability. Thus, it may be concluded that ppGpp present in the bacterial cell prior to purification of the RNA polymerase does not result in the alterations to the enzyme that could be permanent and affect its in vitro transcription capacity.

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5

Agapov, Aleksei, Artem Ignatov, Matti Turtola, Georgiy Belogurov, Daria Esyunina y Andrey Kulbachinskiy. "Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase". Journal of Biological Chemistry 295, n.º 28 (21 de mayo de 2020): 9583–95. http://dx.doi.org/10.1074/jbc.ra119.011844.

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DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

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6

Harden, Timothy T., Christopher D. Wells, Larry J. Friedman, Robert Landick, Ann Hochschild, Jane Kondev y Jeff Gelles. "Bacterial RNA polymerase can retain σ70 throughout transcription". Proceedings of the National Academy of Sciences 113, n.º 3 (5 de enero de 2016): 602–7. http://dx.doi.org/10.1073/pnas.1513899113.

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Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ70, can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ70 retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ70–RNAP complex during initiation from the λ PR′ promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ70 and the kinetics of σ70 release from actively transcribing complexes. σ70 release from mature elongation complexes was slow (0.0038 s−1); a substantial subpopulation of elongation complexes retained σ70 throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ70 manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ70 during transcription.

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Ouhammouch, Mohamed, Finn Werner, Robert O. J. Weinzierl y E. Peter Geiduschek. "A Fully Recombinant System for Activator-dependent Archaeal Transcription". Journal of Biological Chemistry 279, n.º 50 (14 de octubre de 2004): 51719–21. http://dx.doi.org/10.1074/jbc.c400446200.

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The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from thein vitroassembly of the 12-subunit RNA polymerase from the hyperthermophileMethanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

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8

Willkomm, Dagmar K. y Roland K. Hartmann. "6S RNA – an ancient regulator of bacterial RNA polymerase rediscovered". Biological Chemistry 386, n.º 12 (1 de diciembre de 2005): 1273–77. http://dx.doi.org/10.1515/bc.2005.144.

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AbstractThe bacterial riboregulator 6S RNA was one of the first non-coding RNAs to be discovered in the late 1960s, but its cellular role remained enigmatic until the year 2000. 6S RNA, only recognized to be ubiquitous among bacteria in 2005, binds to RNA polymerase in a σ factor-dependent manner to repress transcription from a subgroup of promoters. The common feature of a double-stranded rod with a central bulge has led to the proposal that 6S RNA may mimic an open promoter complex.

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9

Pupov, Danil, Daria Esyunina, Andrey Feklistov y Andrey Kulbachinskiy. "Single-strand promoter traps for bacterial RNA polymerase". Biochemical Journal 452, n.º 2 (10 de mayo de 2013): 241–48. http://dx.doi.org/10.1042/bj20130069.

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Besides canonical double-strand DNA promoters, multisubunit RNAPs (RNA polymerases) recognize a number of specific single-strand DNA and RNA templates, resulting in synthesis of various types of RNA transcripts. The general recognition principles and the mechanisms of transcription initiation on these templates are not fully understood. To investigate further the molecular mechanisms underlying the transcription of single-strand templates by bacterial RNAP, we selected high-affinity single-strand DNA aptamers that are specifically bound by RNAP holoenzyme, and characterized a novel class of aptamer-based transcription templates. The aptamer templates have a hairpin structure that mimics the upstream part of the open promoter bubble with accordingly placed specific promoter elements. The affinity of the RNAP holoenzyme to such DNA structures probably underlies its promoter-melting activity. Depending on the template structure, the aptamer templates can direct synthesis of productive RNA transcripts or effectively trap RNAP in the process of abortive synthesis, involving DNA scrunching, and competitively inhibit promoter recognition. The aptamer templates provide a novel tool for structure–function studies of transcription initiation by bacterial RNAP and its inhibition.

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Nielsen, Soren, Yulia Yuzenkova y Nikolay Zenkin. "Mechanism of Eukaryotic RNA Polymerase III Transcription Termination". Science 340, n.º 6140 (27 de junio de 2013): 1577–80. http://dx.doi.org/10.1126/science.1237934.

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Gene expression in organisms involves many factors and is tightly controlled. Although much is known about the initial phase of transcription by RNA polymerase III (Pol III), the enzyme that synthesizes the majority of RNA molecules in eukaryotic cells, termination is poorly understood. Here, we show that the extensive structure of Pol III–synthesized transcripts dictates the release of elongation complexes at the end of genes. The poly-T termination signal, which does not cause termination in itself, causes catalytic inactivation and backtracking of Pol III, thus committing the enzyme to termination and transporting it to the nearest RNA secondary structure, which facilitates Pol III release. Similarity between termination mechanisms of Pol III and bacterial RNA polymerase suggests that hairpin-dependent termination may date back to the common ancestor of multisubunit RNA polymerases.

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11

Warman, Emily A., Shivani S. Singh, Alicia G. Gubieda y David C. Grainger. "A non-canonical promoter element drives spurious transcription of horizontally acquired bacterial genes". Nucleic Acids Research 48, n.º 9 (16 de abril de 2020): 4891–901. http://dx.doi.org/10.1093/nar/gkaa244.

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Abstract RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase.

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12

Zorov, Savva, Yulia Yuzenkova, Vadim Nikiforov, Konstantin Severinov y Nikolay Zenkin. "Antibiotic Streptolydigin Requires Noncatalytic Mg2+for Binding to RNA Polymerase". Antimicrobial Agents and Chemotherapy 58, n.º 3 (16 de diciembre de 2013): 1420–24. http://dx.doi.org/10.1128/aac.02248-13.

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ABSTRACTMultisubunit RNA polymerase, an enzyme that accomplishes transcription in all living organisms, is a potent target for antibiotics. The antibiotic streptolydigin inhibits RNA polymerase by sequestering the active center in a catalytically inactive conformation. Here, we show that binding of streptolydigin to RNA polymerase strictly depends on a noncatalytic magnesium ion which is likely chelated by the aspartate of the bridge helix of the active center. Substitutions of this aspartate may explain different sensitivities of bacterial RNA polymerases to streptolydigin. These results provide the first evidence for the role of noncatalytic magnesium ions in the functioning of RNA polymerase and suggest new routes for the modification of existing and the design of new inhibitors of transcription.

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13

Lee, Shun Jin y Jay D. Gralla. "Osmo-Regulation of Bacterial Transcription via Poised RNA Polymerase". Molecular Cell 14, n.º 2 (abril de 2004): 153–62. http://dx.doi.org/10.1016/s1097-2765(04)00202-3.

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14

Vassylyev, Dmitry G., Marina N. Vassylyeva, Anna Perederina, Tahir H. Tahirov y Irina Artsimovitch. "Structural basis for transcription elongation by bacterial RNA polymerase". Nature 448, n.º 7150 (20 de junio de 2007): 157–62. http://dx.doi.org/10.1038/nature05932.

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15

Tagami, S., S. Sekine, T. Kumarevel, M. Yamamoto y S. Yokoyama. "Crystallography of bacterial RNA polymerase complexed with transcription factors". Acta Crystallographica Section A Foundations of Crystallography 64, a1 (23 de agosto de 2008): C351—C352. http://dx.doi.org/10.1107/s0108767308088764.

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16

Santos, Joana A. y Meindert H. Lamers. "Novel Antibiotics Targeting Bacterial Replicative DNA Polymerases". Antibiotics 9, n.º 11 (4 de noviembre de 2020): 776. http://dx.doi.org/10.3390/antibiotics9110776.

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Multidrug resistance is a worldwide problem that is an increasing threat to global health. Therefore, the development of new antibiotics that inhibit novel targets is of great urgency. Some of the most successful antibiotics inhibit RNA transcription, RNA translation, and DNA replication. Transcription and translation are inhibited by directly targeting the RNA polymerase or ribosome, respectively. DNA replication, in contrast, is inhibited indirectly through targeting of DNA gyrases, and there are currently no antibiotics that inhibit DNA replication by directly targeting the replisome. This contrasts with antiviral therapies where the viral replicases are extensively targeted. In the last two decades there has been a steady increase in the number of compounds that target the bacterial replisome. In particular a variety of inhibitors of the bacterial replicative polymerases PolC and DnaE have been described, with one of the DNA polymerase inhibitors entering clinical trials for the first time. In this review we will discuss past and current work on inhibition of DNA replication, and the potential of bacterial DNA polymerase inhibitors in particular as attractive targets for a new generation of antibiotics.

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17

Magill, Christine P., Stephen P. Jackson y Stephen D. Bell. "Identification of a Conserved Archaeal RNA Polymerase Subunit Contacted by the Basal Transcription Factor TFB". Journal of Biological Chemistry 276, n.º 50 (17 de octubre de 2001): 46693–96. http://dx.doi.org/10.1074/jbc.c100567200.

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Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promotersin vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (ω-subunit).

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18

Szafranski, Przemyslaw y W. Jerzy Smagowicz. "Relative Affinities of Nucleotide Substrates for the Yeast tRNA Gene Transcription Complex". Zeitschrift für Naturforschung C 47, n.º 3-4 (1 de abril de 1992): 320–22. http://dx.doi.org/10.1515/znc-1992-3-426.

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Abstract Apparent Michaelis constants for nucleotides in transcription of yeast tRN Agene by hom ologous RNA polymerase III with auxiliary protein factors, were found to be remarkably higher in initiation than in elongation of RNA chain. This supports presumptions regarding topological similarities between catalytic centers of bacterial and eukaryotic RNA polymerases.

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19

Kim, Ju-Sim, Lin Liu, Liam F. Fitzsimmons, Yang Wang, Matthew A. Crawford, Mauricio Mastrogiovanni, Madia Trujillo et al. "DksA–DnaJ redox interactions provide a signal for the activation of bacterial RNA polymerase". Proceedings of the National Academy of Sciences 115, n.º 50 (14 de noviembre de 2018): E11780—E11789. http://dx.doi.org/10.1073/pnas.1813572115.

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RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA.

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20

Luciano, Daniel J. y Joel G. Belasco. "Np4A alarmones function in bacteria as precursors to RNA caps". Proceedings of the National Academy of Sciences 117, n.º 7 (4 de febrero de 2020): 3560–67. http://dx.doi.org/10.1073/pnas.1914229117.

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Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

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21

Thomm, Michael, Christoph Reich, Sebastian Grünberg y Souad Naji. "Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases". Biochemical Society Transactions 37, n.º 1 (20 de enero de 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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Ceyssens, Pieter-Jan, Jeroen De Smet, Jeroen Wagemans, Natalia Akulenko, Evgeny Klimuk, Subray Hedge, Marleen Voet et al. "The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit". Viruses 12, n.º 9 (2 de septiembre de 2020): 976. http://dx.doi.org/10.3390/v12090976.

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In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host Pseudomonas aeruginosa. From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.

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Esyunina, Daria, Aleksei Agapov y Andrey Kulbachinskiy. "Regulation of transcriptional pausing through the secondary channel of RNA polymerase". Proceedings of the National Academy of Sciences 113, n.º 31 (18 de julio de 2016): 8699–704. http://dx.doi.org/10.1073/pnas.1603531113.

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Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stress-resistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg2+ or Mn2+) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.

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Tuteja, Renu, Abulaish Ansari y Virander Singh Chauhan. "Emerging Functions of Transcription Factors in Malaria Parasite". Journal of Biomedicine and Biotechnology 2011 (2011): 1–6. http://dx.doi.org/10.1155/2011/461979.

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Transcription is a process by which the genetic information stored in DNA is converted into mRNA by enzymes known as RNA polymerase. Bacteria use only one RNA polymerase to transcribe all of its genes while eukaryotes contain three RNA polymerases to transcribe the variety of eukaryotic genes. RNA polymerase also requires other factors/proteins to produce the transcript. These factors generally termed as transcription factors (TFs) are either associated directly with RNA polymerase or add in building the actual transcription apparatus. TFs are the most common tools that our cells use to control gene expression.Plasmodium falciparumis responsible for causing the most lethal form of malaria in humans. It shows most of its characteristics common to eukaryotic transcription but it is assumed that mechanisms of transcriptional control inP. falciparumsomehow differ from those of other eukaryotes. In this article we describe the studies on the main TFs such as myb protein, high mobility group protein and ApiA2 family proteins from malaria parasite. These studies show that these TFs are slowly emerging to have defined roles in the regulation of gene expression in the parasite.

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Basu, Ritwika S., Brittany A. Warner, Vadim Molodtsov, Danil Pupov, Daria Esyunina, Carlos Fernández-Tornero, Andrey Kulbachinskiy y Katsuhiko S. Murakami. "Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme". Journal of Biological Chemistry 289, n.º 35 (27 de junio de 2014): 24549–59. http://dx.doi.org/10.1074/jbc.m114.584037.

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26

Neußer, Thomas, Nina Gildehaus, Reinhild Wurm y Rolf Wagner. "Studies on the expression of 6S RNA from E. coli: involvement of regulators important for stress and growth adaptation". Biological Chemistry 389, n.º 3 (1 de marzo de 2008): 285–97. http://dx.doi.org/10.1515/bc.2008.023.

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AbstractThe small bacterial 6S RNA has been recognized as a transcriptional regulator, facilitating the transition from exponential to stationary growth phase by preferentially inhibiting Eσ70RNA polymerase holoenzyme transcription. Consistent with this function, the cellular concentration of 6S RNA increases with stationary phase. We have studied the underlying mechanisms responsible for the growth phase-dependent differences in 6S RNA concentration. To this aim, we have analyzed the effects of the typical bacterial growth phase and stress regulators FIS, H-NS, LRP and StpA on 6S RNA expression. Measurements of 6S RNA accumulation in strains deficient in each one of these proteins support their contribution as potential regulators. Specific binding of the four proteins to DNA fragments containing 6S RNA promoters was demonstrated by gel retardation and DNase I footprinting. Moreover,in vitrotranscription analysis with both RNA polymerase holoenzymes, Eσ70and Eσ38, demonstrated a direct inhibition of 6S RNA transcription by H-NS, StpA and LRP, while FIS seems to act as a dual regulator.In vitrotranscription in the presence of ppGpp indicates that 6S RNA promoters are not stringently regulated. Our results underline that regulation of 6S RNA transcription depends on a complex network, involving a set of bacterial regulators with general importance in the adaptation to changing growth conditions.

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Graczyk, Damian, Robert J. White y Kevin M. Ryan. "Involvement of RNA Polymerase III in Immune Responses". Molecular and Cellular Biology 35, n.º 10 (16 de marzo de 2015): 1848–59. http://dx.doi.org/10.1128/mcb.00990-14.

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Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

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Shepherd, N., P. Dennis y H. Bremer. "Cytoplasmic RNA Polymerase inEscherichia coli". Journal of Bacteriology 183, n.º 8 (15 de abril de 2001): 2527–34. http://dx.doi.org/10.1128/jb.183.8.2527-2534.2001.

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ABSTRACT To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase β and β′ subunits in DNA-free minicells from the minicell-producingEscherichia coli strain χ925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase β and β′ subunits in minicells produced by this strain corresponded to about 17% (or 2.5 μM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.

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29

Macadlo, Lauren A., Iskander M. Ibrahim y Sujith Puthiyaveetil. "Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation". Journal of Experimental Botany 71, n.º 3 (23 de octubre de 2019): 1029–38. http://dx.doi.org/10.1093/jxb/erz464.

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Abstract Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.

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30

Liu, Bin, Chuan Hong, Rick K. Huang, Zhiheng Yu y Thomas A. Steitz. "Structural basis of bacterial transcription activation". Science 358, n.º 6365 (16 de noviembre de 2017): 947–51. http://dx.doi.org/10.1126/science.aao1923.

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In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3′,5′-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo–electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ70–RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.

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31

Blombach, Fabian, Tina Daviter, Daniel Fielden, Dina Grohmann, Katherine Smollett y Finn Werner. "Archaeology of RNA polymerase: factor swapping during the transcription cycle". Biochemical Society Transactions 41, n.º 1 (29 de enero de 2013): 362–67. http://dx.doi.org/10.1042/bst20120274.

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All RNAPs (RNA polymerases) repeatedly make use of their DNA template by progressing through the transcription cycle multiple times. During transcription initiation and elongation, distinct sets of transcription factors associate with multisubunit RNAPs and modulate their nucleic-acid-binding and catalytic properties. Between the initiation and elongation phases of the cycle, the factors have to be exchanged by a largely unknown mechanism. We have shown that the binding sites for initiation and elongation factors are overlapping and that the binding of the factors to RNAP is mutually exclusive. This ensures an efficient exchange or ‘swapping’ of factors and could furthermore assist RNAP during promoter escape, enabling robust transcription. A similar mechanism applies to the bacterial RNAP system. The elongation factors are evolutionarily conserved between the bacterial (NusG) and archaeo-eukaryotic (Spt5) systems; however, the initiation factors [σ and TBP (TATA-box-binding protein)/TF (transcription factor) B respectively] are not. Therefore we propose that this factor-swapping mechanism, operating in all three domains of life, is the outcome of convergent evolution.

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32

Harbottle, John y Nikolay Zenkin. "Ureidothiophene inhibits interaction of bacterial RNA polymerase with –10 promotor element". Nucleic Acids Research 48, n.º 14 (11 de julio de 2020): 7914–23. http://dx.doi.org/10.1093/nar/gkaa591.

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Abstract Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter –10 element, while not affecting interactions with –35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of –10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with –10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

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33

Steuten, Benedikt y Rolf Wagner. "A conformational switch is responsible for the reversal of the 6S RNA-dependent RNA polymerase inhibition in Escherichia coli". Biological Chemistry 393, n.º 12 (1 de diciembre de 2012): 1513–22. http://dx.doi.org/10.1515/hsz-2012-0237.

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Abstract 6S RNA is a bacterial transcriptional regulator, which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ70-containing RNA polymerase. This inhibitory RNA polymerase~6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs. Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6S RNA~pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem (conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented.

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34

Shin, Yeonoh, Mark Hedglin y Katsuhiko S. Murakami. "Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase". Nucleic Acids Research 48, n.º 4 (22 de enero de 2020): 2144–55. http://dx.doi.org/10.1093/nar/gkz1221.

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Abstract Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak–trigger–freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.

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35

Malik, S., D. K. Lee y R. G. Roeder. "Potential RNA polymerase II-induced interactions of transcription factor TFIIB." Molecular and Cellular Biology 13, n.º 10 (octubre de 1993): 6253–59. http://dx.doi.org/10.1128/mcb.13.10.6253.

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The ubiquitous transcription factor TFIIB is required for initiation by RNA polymerase II and serves as a target of some regulatory factors. The carboxy-terminal portion of TFIIB contains a large imperfect direct repeat reminiscent of the structural organization of the TATA-binding component (TBP) of TFIID, as well as sequence homology to conserved regions of bacterial sigma factors. The present study shows that the carboxy-terminal portion of TFIIB, like that of TBP, is folded into a compact protease-resistant core. The TFIIB core, unlike the TBP core, is inactive in transcription but retains structural features that enable it to form a complex with promoter-bound TFIID. The protease-susceptible amino terminus appears to contain components responsible for direct interaction with RNA polymerase II (in association with TFIIF) either on the promoter (in association with TFIID) or independently. In addition, core TFIIB (but not intact TFIIB) extends the footprint of TBP on promoter DNA, suggesting that TFIIB has a cryptic DNA-binding potential. These results are consistent with a model in which TFIIB, in a manner functionally analogous to that of bacterial sigma factors, undergoes an RNA polymerase II-dependent conformational change with resultant DNA interactions during the pathway leading to a functional preinitiation complex.

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36

Malik, S., D. K. Lee y R. G. Roeder. "Potential RNA polymerase II-induced interactions of transcription factor TFIIB". Molecular and Cellular Biology 13, n.º 10 (octubre de 1993): 6253–59. http://dx.doi.org/10.1128/mcb.13.10.6253-6259.1993.

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The ubiquitous transcription factor TFIIB is required for initiation by RNA polymerase II and serves as a target of some regulatory factors. The carboxy-terminal portion of TFIIB contains a large imperfect direct repeat reminiscent of the structural organization of the TATA-binding component (TBP) of TFIID, as well as sequence homology to conserved regions of bacterial sigma factors. The present study shows that the carboxy-terminal portion of TFIIB, like that of TBP, is folded into a compact protease-resistant core. The TFIIB core, unlike the TBP core, is inactive in transcription but retains structural features that enable it to form a complex with promoter-bound TFIID. The protease-susceptible amino terminus appears to contain components responsible for direct interaction with RNA polymerase II (in association with TFIIF) either on the promoter (in association with TFIID) or independently. In addition, core TFIIB (but not intact TFIIB) extends the footprint of TBP on promoter DNA, suggesting that TFIIB has a cryptic DNA-binding potential. These results are consistent with a model in which TFIIB, in a manner functionally analogous to that of bacterial sigma factors, undergoes an RNA polymerase II-dependent conformational change with resultant DNA interactions during the pathway leading to a functional preinitiation complex.

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37

Li, Lingting, Vadim Molodtsov, Wei Lin, Richard H. Ebright y Yu Zhang. "RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription". Proceedings of the National Academy of Sciences 117, n.º 11 (3 de marzo de 2020): 5801–9. http://dx.doi.org/10.1073/pnas.1920747117.

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All organisms—bacteria, archaea, and eukaryotes—have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the “σ-finger” of the bacterial transcription initiation factor σ. The structures reveal that—for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5′-triphosphate RNA and 5′-hydroxy RNA—the “σ-finger” is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5′-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.

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38

Prajapati, Ranjit K., Petja Rosenqvist, Kaisa Palmu, Janne J. Mäkinen, Anssi M. Malinen, Pasi Virta, Mikko Metsä-Ketelä y Georgiy A. Belogurov. "Oxazinomycin arrests RNA polymerase at the polythymidine sequences". Nucleic Acids Research 47, n.º 19 (9 de septiembre de 2019): 10296–312. http://dx.doi.org/10.1093/nar/gkz782.

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Abstract Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.

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39

Bateman, E. y M. R. Paule. "Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates." Molecular and Cellular Biology 8, n.º 5 (mayo de 1988): 1940–46. http://dx.doi.org/10.1128/mcb.8.5.1940.

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Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

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40

Bateman, E. y M. R. Paule. "Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates". Molecular and Cellular Biology 8, n.º 5 (mayo de 1988): 1940–46. http://dx.doi.org/10.1128/mcb.8.5.1940-1946.1988.

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Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

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41

Westblade, Lars F., Elizabeth A. Campbell, Chirangini Pukhrambam, Julio C. Padovan, Bryce E. Nickels, Valerie Lamour y Seth A. Darst. "Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction". Nucleic Acids Research 38, n.º 22 (10 de agosto de 2010): 8357–69. http://dx.doi.org/10.1093/nar/gkq692.

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42

Tagami, Shunsuke, Shun-ichi Sekine, Thirumananseri Kumarevel, Nobumasa Hino, Yuko Murayama, Syunsuke Kamegamori, Masaki Yamamoto, Kensaku Sakamoto y Shigeyuki Yokoyama. "Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein". Nature 468, n.º 7326 (diciembre de 2010): 978–82. http://dx.doi.org/10.1038/nature09573.

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43

Vassylyev, D. G., M. Vassylyeva, A. Perederina, J. Zhang, M. Palangat, R. Landick, T. Tahirov y I. Artsimovitch. "Structural basis of transcription: structures of the bacterial RNA polymerase elongation complexes". Acta Crystallographica Section A Foundations of Crystallography 64, a1 (23 de agosto de 2008): C15—C16. http://dx.doi.org/10.1107/s0108767308099558.

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44

Davis, Maria C., Christopher A. Kesthely, Emily A. Franklin y Shawn R. MacLellan. "The essential activities of the bacterial sigma factor". Canadian Journal of Microbiology 63, n.º 2 (febrero de 2017): 89–99. http://dx.doi.org/10.1139/cjm-2016-0576.

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Transcription is the first and most heavily regulated step in gene expression. Sigma (σ) factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. σ Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when σ factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the σ70 family of σ proteins and the essential roles they play in the transcription process.

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45

Delaby, Marie, Lydia M. Varesio, Laurence Degeorges, Sean Crosson y Patrick H. Viollier. "The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics". Proceedings of the National Academy of Sciences 118, n.º 8 (18 de febrero de 2021): e2010357118. http://dx.doi.org/10.1073/pnas.2010357118.

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How DNA-dependent RNA polymerase (RNAP) acts on bacterial cell cycle progression during transcription elongation is poorly investigated. A forward genetic selection for Caulobacter crescentus cell cycle mutants unearthed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR promotes the accumulation of the essential cell cycle transcriptional activator CtrA in late S-phase but also affects transcription at a global level to protect cells from the quinolone antibiotic nalidixic acid that induces a multidrug efflux pump and from the RNAP inhibitor rifampicin that blocks transcription elongation. We show that TrcR associates with promoters and coding sequences in vivo in a rifampicin-dependent manner and that it interacts physically and genetically with RNAP. We show that TrcR function and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella spp. Thus, TrcR represents a hitherto unknown antibiotic target and the founding member of the DUF1013 family, an uncharacterized class of transcriptional regulators that track with RNAP during the elongation phase to promote transcription during the cell cycle.

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46

Bergendahl, Veit, Tomasz Heyduk y Richard R. Burgess. "Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase". Applied and Environmental Microbiology 69, n.º 3 (marzo de 2003): 1492–98. http://dx.doi.org/10.1128/aem.69.3.1492-1498.2003.

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ABSTRACT The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

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47

Stracy, Mathew, Christian Lesterlin, Federico Garza de Leon, Stephan Uphoff, Pawel Zawadzki y Achillefs N. Kapanidis. "Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid". Proceedings of the National Academy of Sciences 112, n.º 32 (29 de julio de 2015): E4390—E4399. http://dx.doi.org/10.1073/pnas.1507592112.

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Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

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48

Conn, Adam B., Stephen Diggs, Timothy K. Tam y Gregor M. Blaha. "Two Old Dogs, One New Trick: A Review of RNA Polymerase and Ribosome Interactions during Transcription-Translation Coupling". International Journal of Molecular Sciences 20, n.º 10 (27 de mayo de 2019): 2595. http://dx.doi.org/10.3390/ijms20102595.

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The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed. The discovery of physical interactions between RNA polymerase and ribosomes has spurred renewed interest into this long-standing paradigm of bacterial molecular biology. Here, we provide a concise presentation of recent insights gained from super-resolution microscopy, biochemical, and structural work, including cryo-EM studies. Based on the presented data, we put forward a dynamic model for the interaction between RNA polymerase and ribosomes, in which the interactions are repeatedly formed and broken. Furthermore, we propose that long intervening nascent RNA will loop out and away during the forming the interactions between the RNA polymerase and ribosomes. By comparing the effect of the direct interactions between RNA polymerase and ribosomes with those that transcription factors NusG and RfaH mediate, we submit that two distinct modes of coupling exist: Factor-free and factor-mediated coupling. Finally, we provide a possible framework for transcription-translation coupling and elude to some open questions in the field.

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49

Dorjsuren, Dorjbal, Yong Lin, Wenxiang Wei, Tatsuya Yamashita, Takahiro Nomura, Naoyuki Hayashi y Seishi Murakami. "RMP, a Novel RNA Polymerase II Subunit 5-Interacting Protein, Counteracts Transactivation by Hepatitis B Virus X Protein". Molecular and Cellular Biology 18, n.º 12 (1 de diciembre de 1998): 7546–55. http://dx.doi.org/10.1128/mcb.18.12.7546.

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ABSTRACT To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.

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50

Bell, S. D., C. P. Magill y S. P. Jackson. "Basal and regulated transcription in Archaea". Biochemical Society Transactions 29, n.º 4 (1 de agosto de 2001): 392–95. http://dx.doi.org/10.1042/bst0290392.

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The basal transcription machinery of Archaea is fundamentally related to the eucaryal RNA polymerase (RNAP) II apparatus. In addition to a 12-subunit RNAP, Archaea possess two general transcription factors, the activities of which are required for accurate and efficient in vitro transcription. These factors, TBP and TFB, are homologues of the eucaryal TATA-box binding protein and TFIIB respectively. Archaea also possess TFE, a homologue of the eucaryal RNAP II general transcription factor TFIIE. Although not absolutely required for transcription in vitro, TFE nonetheless plays a stimulatory role under conditions where promoter recognition by TBP is sub-optimal. The basal transcription apparatus of Archaea is closely related to that of Eucarya but archaeal transcriptional regulators resemble those of bacteria. The mode of action of two such regulators has been characterized to determine how these ‘bacterial-like’ regulators impinge on the ‘eucaryal-like’ basal machinery.

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