The Experts below are selected from a list of 55929 Experts worldwide ranked by ideXlab platform
Richard H. Ebright - One of the best experts on this subject based on the ideXlab platform.
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Discovery, properties, and biosynthesis of pseudouridimycin, an antiBacterial nucleoside-analog inhibitor of Bacterial RNA polymerase
Journal of Industrial Microbiology & Biotechnology, 2019Co-Authors: Sonia I Maffioli, Richard H. Ebright, Margherita Sosio, Stefano DonadioAbstract:Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits Bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of the rifamycin and lipiarmycin (fidaxomicin) classes. PUM was discovered by screening microbial fermentation extracts for RNAP inhibitors. In this review, we describe the discovery and characterization of PUM. We also describe the RNAP-inhibitory and antiBacterial properties of PUM. Finally, we review available information on the gene cluster and pathway for PUM biosynthesis and on the potential for discovering additional novel pseudouridine-containing nucleoside antibiotics by searching Bacterial genome and metagenome sequences for sequences similar to pumJ , the pseudouridine-synthase gene of the PUM biosynthesis gene cluster.
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salinamide f new depsipeptide antibiotic and inhibitor of Bacterial RNA polymerase from a marine derived streptomyces sp
The Journal of Antibiotics, 2015Co-Authors: David Degen, Kyoung Hwa Jang, Hossam M. Hassan, Richard H. Ebright, William FenicalAbstract:Salinamide F, new depsipeptide antibiotic and inhibitor of Bacterial RNA polymerase from a marine-derived Streptomyces sp.
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New target for inhibition of Bacterial RNA polymerase: ‘switch region’
Current opinion in microbiology, 2011Co-Authors: Aashish Srivastava, David Degen, Richard H. Ebright, Meliza Talaue, Shuang Liu, Elena V. Sineva, Anirban Chakraborty, Sergey Y Druzhinin, Sujoy Chatterjee, Jayanta MukhopadhyayAbstract:A new drug target – the ‘switch region’ – has been identified within Bacterial RNA polymerase (RNAP), the enzyme that mediates Bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit Bacterial RNA synthesis and kill bacteria. Since the new target is present in most Bacterial species, compounds that bind to the new target are active against a broad spectrum of Bacterial species. Since the new target is different from targets of other antiBacterial agents, compounds that bind to the new target are not cross-resistant with other antiBacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antiBacterial drug discovery.
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Structural Organization of Bacterial RNA Polymerase Holoenzyme and the RNA Polymerase-Promoter Open Complex
Cell, 2002Co-Authors: Vladimir Mekler, Jayanta Mukhopadhyay, Ekaterine Kortkhonjia, Jennifer L. Knight, Andrei Revyakin, Achillefs N. Kapanidis, Wei Niu, Yon W. Ebright, Ronald M. Levy, Richard H. EbrightAbstract:Abstract We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of Bacterial RNA polymerase holoenzyme and the Bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding σ 70 -(RNA polymerase core), σ 70 -DNA, and σ 70 -RNA interactions. The positions of σ 70 regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of σ 70 region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.
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RNA Polymerase: Structural Similarities Between Bacterial RNA Polymerase and Eukaryotic RNA Polymerase II☆☆☆
Journal of Molecular Biology, 2000Co-Authors: Richard H. EbrightAbstract:Abstract Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.
Katsuhiko S. Murakami - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of Bacterial RNA Polymerase Inhibitors in a Staphylococcus aureus-Based Wound Infection Model in SKH1 Mice.
ACS infectious diseases, 2020Co-Authors: Jörg Haupenthal, Vadim Molodtsov, Yannik Kautz, Walid A. M. Elgaher, Linda Pätzold, Teresa Röhrig, Matthias W. Laschke, Thomas Tschernig, Anna K. H. Hirsch, Katsuhiko S. MurakamiAbstract:Chronic wounds infected with pathogens such as Staphylococcus aureus represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the Bacterial RNA polymerase (RNAP). In this work, we describe the establishment of an in vivo wound infection model based on the pathogen S. aureus and hairless Crl:SKH1-Hrhr (SKH1) mice. The model proved to be a valuable preclinical tool to study selected RNAP inhibitors after topical application. While rifampicin showed a reduction in the loss of body weight induced by the bacteria, an acceleration of wound healing kinetics, and a reduced number of colony forming units in the wound, the ureidothiophene-2-carboxylic acid 1 was inactive under in vivo conditions, probably due to strong plasma protein binding. The cocrystal structure of compound 1 with RNAP, that we hereby also present, will be of great value for applying appropriate structural modifications to further optimize the compound, especially in terms of plasma protein binding.
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Structural basis of reiterative transcription from the pyrG and pyrBI promoters by Bacterial RNA polymerase.
Nucleic acids research, 2020Co-Authors: Yeonoh Shin, Mark Hedglin, Katsuhiko S. MurakamiAbstract: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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Structural basis of reiterative transcription from the pyrG and pyrBI promoters by Bacterial RNA polymerase
2019Co-Authors: Yeonoh Shin, Mark Hedglin, Katsuhiko S. MurakamiAbstract: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 8 poly-G RNA bases synthesized using 3 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. Structures and biochemical assays revealed how the RNA transcript from the pyrG promoter is guided toward the Rifampin-binding pocket then the main channel of RNA polymerase and provided insight into RNA slippage during reiterative transcription of the pyrG promoter. Lastly, we determined a structure of a reiterative transcription complex at the pyrBI promoter and revealed an alteRNAtive mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme. SIGNIFICANCE STATEMENT RNA polymerase synthesizes multiple bases of RNA using a single base of the template DNA due to slippage between RNA transcript and template DNA. This noncanonical RNA synthesis is called “reiterative transcription,” playing several regulatory roles cellular organisms and viruses. In this study, we determined a series of X-ray crystal structures of a Bacterial RNA polymerase engaged in reiterative transcription and characterized a role of template DNA during reiterative transcription by biochemical assays. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. We also provide insights into the regulation of gene expression using two alteRNAtive ways of reiterative transcription.
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Structural biology of Bacterial RNA polymerase.
Biomolecules, 2015Co-Authors: Katsuhiko S. MurakamiAbstract:Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477–42485), an enormous amount of biochemical, biophysical and genetic data has been collected on Bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to Bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to Bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to Bacterial transcription research and discuss the role that small molecules play in inhibiting Bacterial RNA transcription.
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Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme
The Journal of biological chemistry, 2014Co-Authors: Ritwika Basu, Danil Pupov, Daria Esyunina, Andrey Kulbachinskiy, Brittany A. Warner, Vadim Molodtsov, Carlos Fernández-tornero, Katsuhiko S. MurakamiAbstract:The Bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5′-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.
Andrey Kulbachinskiy - One of the best experts on this subject based on the ideXlab platform.
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Dual role of the σ factor in primer RNA synthesis by Bacterial RNA polymerase
FEBS letters, 2018Co-Authors: Daria Esyunina, Danil Pupov, Andrey KulbachinskiyAbstract:Bacterial RNA polymerase (RNAP) serves as a primase during replication of single-stranded plasmids and filamentous phages. Primer RNA (prRNA) synthesis from the origin regions of these replicons depends on the σ factor that normally participates in promoter recognition. However, it was proposed that σ may not be required for origin recognition but is rather involved in RNA extension by RNAP. Here, by analyzing the natural replication origin of bacteriophage M13 and synthetic ssDNA templates, we show that interactions of σ with promoter-like motifs stabilize priming complexes and can control prRNA synthesis by trapping RNAP on the template. Thus, the σ factor is involved in both DNA recognition and RNA priming, unifying its functions in transcription initiation from double- and single-stranded templates.
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Interplay between σ region 3.2 and secondary channel factors during promoter escape by Bacterial RNA polymerase.
The Biochemical journal, 2017Co-Authors: Ivan Petushkov, Danil Pupov, Daria Esyunina, Vladimir Mekler, Konstantin Severinov, Andrey KulbachinskiyAbstract:In Bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5′-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ 70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5′-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.
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σ38-dependent promoter-proximal pausing by Bacterial RNA polymerase.
Nucleic acids research, 2016Co-Authors: Ivan Petushkov, Daria Esyunina, Andrey KulbachinskiyAbstract:Transcription initiation by Bacterial RNA polymerase (RNAP) requires a variable σ subunit that directs it to promoters for site-specific priming of RNA synthesis. The principal σ subunit responsible for expression of house-keeping genes can bind the transcription elongation complex after initiation and induce RNAP pausing through specific interactions with promoter-like motifs in transcribed DNA. We show that the stationary phase and stress response σ38 subunit can also induce pausing by Escherichia coli RNAP on DNA templates containing promoter-like motifs in the transcribed regions. The pausing depends on σ38 contacts with the DNA template and RNAP core enzyme and results in formation of backtracked transcription elongation complexes, which can be reactivated by Gre factors that induce RNA cleavage by RNAP. Our data suggest that σ38 can bind the transcription elongation complex in trans but likely acts in cis during transcription initiation, by staying bound to RNAP and recognizing promoter-proximal pause signals. Analysis of σ38-dependent promoters reveals that a substantial fraction of them contain potential pause-inducing motifs, suggesting that σ38-depended pausing may be a common phenomenon in Bacterial transcription.
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Lineage-specific variations in the trigger loop modulate RNA proofreading by Bacterial RNA polymerases
Nucleic Acids Research, 2016Co-Authors: Daria Esyunina, Danil Pupov, Matti Turtola, I. A. Bass, Georgiy A. Belogurov, Saulius Klimasauskas, Andrey KulbachinskiyAbstract:RNA cleavage by Bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in Bacterial stress adaptation.
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Aptamers to the sigma factor mimic promoter recognition and inhibit transcription initiation by Bacterial RNA polymerase.
Biochemical and biophysical research communications, 2015Co-Authors: Nataliya Miropolskaya, Andrey KulbachinskiyAbstract:Promoter recognition by Bacterial RNA polymerase (RNAP) is a multi-step process involving multiple protein-DNA interactions and several structural and kinetic intermediates which remain only partially characterized. We used single-stranded DNA aptamers containing specific promoter motifs to probe the interactions of the Thermus aquaticus RNAP σ(A) subunit with the -10 promoter element in the absence of other parts of the promoter complex. The aptamer binding decreased intrinsic fluorescence of the σ subunit, likely as a result of interactions between the -10 element and conserved tryptophan residues of the σ DNA-binding region 2. By monitoring these changes, we demonstrated that DNA binding proceeds through a single rate-limiting step resulting in formation of very stable complexes. Deletion of the N-terminal domain of the σ(A) subunit increased the rate of aptamer binding while replacement of this domain with an unrelated N-terminal region 1.1 from the Escherichia coli σ(70) subunit restored the original kinetics of σ-aptamer interactions. The results demonstrate that the key step in promoter recognition can be modelled in a simple σ-aptamer system and reveal that highly divergent N-terminal domains similarly modulate the DNA-binding properties of the σ subunit. The aptamers efficiently suppressed promoter-dependent transcription initiation by the holoenzyme of RNA polymerase, suggesting that they may be used for development of novel transcription inhibitors.
Seth A. Darst - One of the best experts on this subject based on the ideXlab platform.
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Utilization of variably spaced promoter-like elements by the Bacterial RNA polymerase holoenzyme during early elongation
Molecular microbiology, 2010Co-Authors: Pukhrambam Grihanjali Devi, Seth A. Darst, Elizabeth A Campbell, Bryce E. NickelsAbstract:The Bacterial RNA polymeras holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. During initiation, members of the sigma(70) family of sigma factors contact two conserved promoter elements, the -10 and -35 elements, which are separated by approximately 17 base pairs (bp). sigma(70) family members contain four flexibly linked domains. Two of these domains, sigma(2) and sigma(4), contain determinants for interactions with the promoter -10 and -35 elements respectively. sigma(2) and sigma(4) also contain core-binding determinants. When bound to core the inter-domain distance between sigma(2) and sigma(4) matches the distance between promoter elements separated by approximately 17 bp. Prior work indicates that during early elongation the nascent RNA-assisted displacement of sigma(4) from core can enable the holoenzyme to adopt a configuration in which sigma(2) and sigma(4) are bound to 'promoter-like' DNA elements separated by a single base pair. Here we demonstrate that holoenzyme can also adopt configurations in which sigma(2) and sigma(4) are bound to 'promoter-like' DNA elements separated by 0, 2 or 3 bp. Thus, our findings suggest that displacement of sigma(4) from core enables the RNA polymerase holoenzyme to adopt a broad range of 'elongation-specific' configurations.
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Structural, functional, and genetic analysis of sorangicin inhibition of Bacterial RNA polymerase
The EMBO journal, 2005Co-Authors: Elizabeth A Campbell, Herbert Irschik, Konstantin Severinov, Olga Pavlova, Nikolay Zenkin, Fred Leon, Rolf Jansen, Seth A. DarstAbstract:A combined structural, functional, and genetic approach was used to investigate inhibition of Bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP β subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2–3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.
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New inhibitors targeting Bacterial RNA polymerase
Trends in biochemical sciences, 2004Co-Authors: Seth A. DarstAbstract:The Bacterial RNA polymerase is a proven target for antimicrobials because it is inhibited by rifampicin, a potent antibiotic that is a key component of tuberculosis therapy. The Bacterial RNA polymerase is also a molecular machine that presents numerous targets for small-molecule inhibitors that have not yet been exploited. Recent work has uncovered new classes of inhibitors. Further applications of high-throughput screening, medicinal chemistry and high-resolution structural studies hold great promise for the development of antimicrobials.
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Bacterial RNA Polymerases: The Wholo Story
Current opinion in structural biology, 2003Co-Authors: Katsuhiko S. Murakami, Seth A. DarstAbstract:Recent structural and biophysical results have provided unprecedented insights into the structure and function of the Bacterial RNA polymerase holoenzyme as it goes through the steps of transcription initiation. Comparisons with structural analyses of evolutionarily unrelated RNA polymerases reveal unexpected general features of the initiation process.
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Bacterial RNA polymerase.
Current opinion in structural biology, 2001Co-Authors: Seth A. DarstAbstract:The recently determined crystal structure of a Bacterial core RNA polymerase (RNAP) provides the first glimpse of this family of evolutionarily conserved cellular RNAPs. Using the structure as a framework, a consistent picture of protein-nucleic acid interactions in transcription complexes has been accumulated from cross-linking experiments. The molecule can be viewed as a molecular machine, with distinct structural features hypothesized to perform specific functions. Comparison with the alpha-carbon backbone of a eukaryotic RNAP reveals close structural similarity.
Konstantin Severinov - One of the best experts on this subject based on the ideXlab platform.
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Interplay between σ region 3.2 and secondary channel factors during promoter escape by Bacterial RNA polymerase.
The Biochemical journal, 2017Co-Authors: Ivan Petushkov, Danil Pupov, Daria Esyunina, Vladimir Mekler, Konstantin Severinov, Andrey KulbachinskiyAbstract:In Bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5′-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ 70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5′-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.
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Interplay between the β′ Clamp and the β′ Jaw Domains during DNA Opening by the Bacterial RNA Polymerase at σ54-dependent Promoters
Journal of molecular biology, 2006Co-Authors: Sivaramesh Wigneshweraraj, Konstantin Severinov, Dhruti Savalia, Martin BuckAbstract:The Bacterial RNA polymerase (RNAP) is a multi-subunit, structurally flexible, complex molecular machine, in which activities associated with DNA opening for transcription-competent open promoter complex (OC) formation reside in the catalytic beta and beta' subunits and the dissociable sigma subunit. OC formation is a multi-step process that involves several structurally conserved mobile modules of beta, beta', and sigma. Here, we present evidence that two flexible modules of beta', the beta' jaw and the beta' clamp and a conserved regulatory Region I domain of sigma(54), jointly contribute to the maintenance of stable DNA strand separation around the trancription start site in OCs formed at sigma(54)-dependent promoters. Clearly, regulated interplay between the mobile modules of the beta' and the sigma subunits of the RNAP appears to be necessary for stable OC formation.
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Structure-activity analysis of microcinJ25: distinct parts of the threaded lasso molecule are responsible for interaction with Bacterial RNA polymerase.
Journal of Bacteriology, 2005Co-Authors: Ekaterina Semenova, Yulia Yuzenkova, Jean Peduzzi, Sylvie Rebuffat, Konstantin SeverinovAbstract:Peptide microcin J25 (MccJ25) inhibits Bacterial RNA polymerase. We show that thermolysin-cleaved MccJ25 and MccJ25 lacking amino acids 13 to 17 also inhibit transcription. Our data and structural analysis of intact and thermolysin-digested MccJ25 suggest that distinct regions of MccJ25 are involved in transcription inhibition and cell entry.
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Structure-Activity Analysis of Microcin J25: Distinct Parts of the Threaded Lasso Molecule Are Responsible for Interaction with Bacterial RNA Polymerase
Journal of bacteriology, 2005Co-Authors: Ekaterina Semenova, Yulia Yuzenkova, Jean Peduzzi, Sylvie Rebuffat, Konstantin SeverinovAbstract:Peptide microcin J25 (MccJ25) inhibits Bacterial RNA polymerase. We show that thermolysin-cleaved MccJ25 and MccJ25 lacking amino acids 13 to 17 also inhibit transcription. Our data and structural analysis of intact and thermolysin-digested MccJ25 suggest that distinct regions of MccJ25 are involved in transcription inhibition and cell entry.
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Structural, functional, and genetic analysis of sorangicin inhibition of Bacterial RNA polymerase
The EMBO journal, 2005Co-Authors: Elizabeth A Campbell, Herbert Irschik, Konstantin Severinov, Olga Pavlova, Nikolay Zenkin, Fred Leon, Rolf Jansen, Seth A. DarstAbstract:A combined structural, functional, and genetic approach was used to investigate inhibition of Bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP β subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2–3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.