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Article ; Online: Reductionism

Svetlov, Dmitri / Artsimovitch, Irina

ACS infectious diseases

2021 Volume 7, Issue 11, Page(s) 2948–2952

Abstract: The tragic consequences of the COVID-19 pandemic have led to admirable responses by the global scientific community, including a profound acceleration in the pace of research and exchange of findings. However, this has had considerable costs of its own, ... ...

Abstract	The tragic consequences of the COVID-19 pandemic have led to admirable responses by the global scientific community, including a profound acceleration in the pace of research and exchange of findings. However, this has had considerable costs of its own, as erroneous conclusions have propagated faster than researchers have been able to detect and correct them. We illustrate the specific misunderstandings that have resulted from reductionist approaches to the study of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), which are but one instance of a regrettably growing trend in structural biology. Far from merely being cautionary tales about the conduct of scientific research, these errors have had significant practical impact, by hampering a correct understanding of RdRp structure and mechanism, its inhibition by nucleoside analogues such as remdesivir, and the discovery and characterization of such analogues. After correcting these misunderstandings, we close with several recommendations for a broader correction of the course of scientific research.
MeSH term(s)	Antiviral Agents ; Biology ; COVID-19 ; Humans ; Pandemics ; RNA, Viral ; SARS-CoV-2
Chemical Substances	Antiviral Agents ; RNA, Viral
Language	English
Publishing date	2021-10-06
Publishing country	United States
Document type	Journal Article
ISSN	2373-8227
ISSN (online)	2373-8227
DOI	10.1021/acsinfecdis.1c00492
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain.

Wang, Bing / Svetlov, Dmitri / Artsimovitch, Irina

Nucleic acids research

2021 Volume 49, Issue 15, Page(s) 8822–8835

Abstract: The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved ... ...

Abstract	The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.
MeSH term(s)	Amino Acid Sequence ; Catalytic Domain ; Coenzymes/metabolism ; Coronavirus RNA-Dependent RNA Polymerase/metabolism ; Diphosphates/pharmacology ; Diphosphonates/pharmacology ; Guanosine Triphosphate/metabolism ; Manganese ; Models, Molecular ; Nidovirales/chemistry ; Nidovirales/enzymology ; Nucleotides/metabolism ; Protein Domains ; RNA-Binding Proteins/chemistry ; RNA-Binding Proteins/metabolism ; RNA-Dependent RNA Polymerase/antagonists & inhibitors ; RNA-Dependent RNA Polymerase/chemistry ; RNA-Dependent RNA Polymerase/metabolism ; SARS-CoV-2/enzymology ; Uridine Triphosphate/metabolism ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/metabolism
Chemical Substances	Coenzymes ; Diphosphates ; Diphosphonates ; NS8 protein, SARS-CoV-2 ; NSP9 protein, SARS-CoV-2 ; Nucleotides ; RNA-Binding Proteins ; Viral Nonstructural Proteins ; Manganese (42Z2K6ZL8P) ; diphosphoric acid (4E862E7GRQ) ; Guanosine Triphosphate (86-01-1) ; Coronavirus RNA-Dependent RNA Polymerase (EC 2.7.7.48) ; NSP12 protein, SARS-CoV-2 (EC 2.7.7.48) ; NSP7 protein, SARS-CoV-2 (EC 2.7.7.48) ; RNA-Dependent RNA Polymerase (EC 2.7.7.48) ; Uridine Triphosphate (UT0S826Z60)
Language	English
Publishing date	2021-08-04
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkab677
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Article ; Online: Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19.

Wang, Bing / Svetlov, Dmitri / Bartikofsky, Dylan / Wobus, Christiane E / Artsimovitch, Irina

Molecules (Basel, Switzerland)

2022 Volume 27, Issue 12

Abstract: The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost- ... ...

Abstract	The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site (i.e., RNA synthesis) of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise.
MeSH term(s)	Antiviral Agents/chemistry ; Antiviral Agents/pharmacology ; Antiviral Agents/therapeutic use ; COVID-19/drug therapy ; Drug Discovery ; Humans ; Molecular Docking Simulation ; Pandemics ; RNA ; RNA-Dependent RNA Polymerase ; SARS-CoV-2
Chemical Substances	Antiviral Agents ; RNA (63231-63-0) ; RNA-Dependent RNA Polymerase (EC 2.7.7.48)
Language	English
Publishing date	2022-06-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules27123815
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Article ; Online: Going Retro, Going Viral

Bing Wang / Dmitri Svetlov / Dylan Bartikofsky / Christiane E. Wobus / Irina Artsimovitch

Molecules, Vol 27, Iss 3815, p

Experiences and Lessons in Drug Discovery from COVID-19

2022 Volume 3815

Abstract	The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site (i.e., RNA synthesis) of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise.
Keywords	SARS-CoV-2 RdRp ; NiRAN ; nucleotide analogs ; fidaxomicin ; rifamycins ; Organic chemistry ; QD241-441
Subject code	540
Language	English
Publishing date	2022-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: NMPylation and de-NMPylation of SARS-CoV-2 Nsp9 by the NiRAN domain

Wang, Bing / Svetlov, Dmitri / Artsimovitch, Irina

bioRxiv

Abstract: Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all ... ...

Abstract	Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all RNA viruses and has been a focus of mechanistic studies and drug discovery. The second active site resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. Both catalytic reactions are essential for viral replication, but the mechanism and targets of NiRAN are poorly characterized. One recent study showed that NiRAN transfers NMP to the first residue of RNA-binding protein Nsp9. Another study reported a structure of SARS-CoV-2 replicase with an extended Nsp9 in the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 Nsp12 efficiently and reversibly NMPylates the native but not the extended Nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas a substitution of a catalytic RdRp Asp residue does not. NMPylation is inhibited by nucleotide analogs, pyrophosphate, and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We hypothesize that Nsp9 remodels both active sites of Nsp12 to support initiation of RNA synthesis by RdRp and subsequent capping of the product RNA by the NiRAN domain.
Keywords	covid19
Language	English
Publishing date	2021-06-14
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2021.06.13.448258
Database	COVID19

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Article ; Online: Flipping states: a few key residues decide the winning conformation of the only universally conserved transcription factor.

Shi, Da / Svetlov, Dmitri / Abagyan, Ruben / Artsimovitch, Irina

Nucleic acids research

2017 Volume 45, Issue 15, Page(s) 8835–8843

Abstract: Transcription factors from the NusG family bind to the elongating RNA polymerase to enable synthesis of long RNAs in all domains of life. In bacteria, NusG frequently co-exists with specialized paralogs that regulate expression of a small set of targets, ...

Abstract	Transcription factors from the NusG family bind to the elongating RNA polymerase to enable synthesis of long RNAs in all domains of life. In bacteria, NusG frequently co-exists with specialized paralogs that regulate expression of a small set of targets, many of which encode virulence factors. Escherichia coli RfaH is the exemplar of this regulatory mechanism. In contrast to NusG, which freely binds to RNA polymerase, RfaH exists in a structurally distinct autoinhibitory state in which the RNA polymerase-binding site is buried at the interface between two RfaH domains. Binding to an ops DNA sequence triggers structural transformation wherein the domains dissociate and RfaH refolds into a NusG-like structure. Formation of the autoinhibitory state, and thus sequence-specific recruitment, represents the decisive step in the evolutionary history of the RfaH subfamily. We used computational and experimental approaches to identify the residues that confer the unique regulatory properties of RfaH. Our analysis highlighted highly conserved Ile and Phe residues at the RfaH interdomain interface. Replacement of these residues with equally conserved Glu and Val counterpart residues in NusG destabilized interactions between the RfaH domains and allowed sequence-independent recruitment to RNA polymerase, suggesting a plausible pathway for diversification of NusG paralogs.
MeSH term(s)	Amino Acid Sequence ; Amino Acid Substitution ; Binding Sites ; DNA, Bacterial/chemistry ; DNA, Bacterial/genetics ; DNA, Bacterial/metabolism ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/genetics ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Evolution, Molecular ; Gene Expression Regulation, Bacterial ; Glutamic Acid/chemistry ; Glutamic Acid/metabolism ; Isoleucine/chemistry ; Isoleucine/metabolism ; Models, Molecular ; Peptide Elongation Factors/chemistry ; Peptide Elongation Factors/genetics ; Peptide Elongation Factors/metabolism ; Phenylalanine/chemistry ; Phenylalanine/metabolism ; Protein Binding ; Protein Folding ; Protein Interaction Domains and Motifs ; Sequence Alignment ; Sequence Homology, Amino Acid ; Trans-Activators/chemistry ; Trans-Activators/genetics ; Trans-Activators/metabolism ; Transcription Factors/chemistry ; Transcription Factors/genetics ; Transcription Factors/metabolism ; Transcription, Genetic ; Valine/chemistry ; Valine/metabolism
Chemical Substances	DNA, Bacterial ; Escherichia coli Proteins ; NusG protein, E coli ; Peptide Elongation Factors ; RfaH protein, E coli ; Trans-Activators ; Transcription Factors ; Isoleucine (04Y7590D77) ; Glutamic Acid (3KX376GY7L) ; Phenylalanine (47E5O17Y3R) ; DNA-Directed RNA Polymerases (EC 2.7.7.6) ; Valine (HG18B9YRS7)
Language	English
Publishing date	2017-09-06
Publishing country	England
Document type	Journal Article
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkx523
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Article ; Online: Locking the nontemplate DNA to control transcription.

Nedialkov, Yuri / Svetlov, Dmitri / Belogurov, Georgiy A / Artsimovitch, Irina

Molecular microbiology

2018 Volume 109, Issue 4, Page(s) 445–457

Abstract: Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause-free RNA ... ...

Abstract	Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause-free RNA synthesis. However, recent structures of paused transcription elongation complexes show that the clamp does not open and suggest alternative mechanisms of antipausing. Among these mechanisms, direct contacts of NusG/Spt5 proteins with the nontemplate DNA in the transcription bubble have been proposed to prevent unproductive DNA conformations and thus inhibit arrest. We used Escherichia coli RfaH, whose interactions with DNA are best characterized, to test this idea. We report that RfaH stabilizes the upstream edge of the transcription bubble, favoring forward translocation, and protects the upstream duplex DNA from exonuclease cleavage. Modeling suggests that RfaH loops the nontemplate DNA around its surface and restricts the upstream DNA duplex mobility. Strikingly, we show that RfaH-induced DNA protection and antipausing activity can be mimicked by shortening the nontemplate strand in elongation complexes assembled on synthetic scaffolds. We propose that remodeling of the nontemplate DNA controls recruitment of regulatory factors and R-loop formation during transcription elongation across all life.
MeSH term(s)	Chromosomal Proteins, Non-Histone/metabolism ; DNA, Bacterial/genetics ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/genetics ; Escherichia coli Proteins/metabolism ; Nucleic Acid Conformation ; Peptide Elongation Factors/metabolism ; RNA, Bacterial/biosynthesis ; Trans-Activators/metabolism ; Transcription Factors/metabolism ; Transcription, Genetic/genetics ; Transcription, Genetic/physiology ; Transcriptional Elongation Factors/metabolism
Chemical Substances	Chromosomal Proteins, Non-Histone ; DNA, Bacterial ; Escherichia coli Proteins ; NusG protein, E coli ; Peptide Elongation Factors ; RNA, Bacterial ; RfaH protein, E coli ; Trans-Activators ; Transcription Factors ; Transcriptional Elongation Factors ; SPT5 transcriptional elongation factor (138673-72-0) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2018-04-25
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	619315-8
ISSN	1365-2958 ; 0950-382X
ISSN (online)	1365-2958
ISSN	0950-382X
DOI	10.1111/mmi.13983
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Article ; Online: RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

NandyMazumdar, Monali / Nedialkov, Yuri / Svetlov, Dmitri / Sevostyanova, Anastasia / Belogurov, Georgiy A / Artsimovitch, Irina

Proceedings of the National Academy of Sciences of the United States of America

2016 Volume 113, Issue 52, Page(s) 14994–14999

Abstract: Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize ... ...

Abstract	Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.
MeSH term(s)	DNA, Bacterial/chemistry ; DNA-Directed RNA Polymerases/chemistry ; Escherichia coli/chemistry ; Escherichia coli Proteins/chemistry ; Gene Deletion ; Nucleic Acid Conformation ; Oligonucleotides/genetics ; Peptide Elongation Factors/chemistry ; Promoter Regions, Genetic ; Protein Binding ; Protein Conformation ; Sigma Factor/chemistry ; Thermus/chemistry ; Transcription, Genetic
Chemical Substances	DNA, Bacterial ; Escherichia coli Proteins ; Oligonucleotides ; Peptide Elongation Factors ; Sigma Factor ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2016-12-12
Publishing country	United States
Document type	Journal Article
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1613673114
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Article ; Online: In silico discovery of small molecules that inhibit RfaH recruitment to RNA polymerase.

Svetlov, Dmitri / Shi, Da / Twentyman, Joy / Nedialkov, Yuri / Rosen, David A / Abagyan, Ruben / Artsimovitch, Irina

Molecular microbiology

2018 Volume 110, Issue 1, Page(s) 128–142

Abstract: RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus ... ...

Abstract	RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP β subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP β' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.
MeSH term(s)	Bacterial Proteins/antagonists & inhibitors ; Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/genetics ; DNA-Directed RNA Polymerases/metabolism ; Drug Design ; Escherichia coli/drug effects ; Escherichia coli/genetics ; Escherichia coli/pathogenicity ; Escherichia coli Proteins/antagonists & inhibitors ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Klebsiella pneumoniae/drug effects ; Klebsiella pneumoniae/genetics ; Klebsiella pneumoniae/pathogenicity ; Ligands ; Molecular Docking Simulation ; Peptide Elongation Factors/antagonists & inhibitors ; Peptide Elongation Factors/chemistry ; Peptide Elongation Factors/genetics ; Ribosomes/chemistry ; Ribosomes/genetics ; Ribosomes/metabolism ; Small Molecule Libraries/chemistry ; Small Molecule Libraries/pharmacology ; Trans-Activators/antagonists & inhibitors ; Trans-Activators/chemistry ; Trans-Activators/genetics ; Virulence/drug effects
Chemical Substances	Bacterial Proteins ; Escherichia coli Proteins ; Ligands ; Peptide Elongation Factors ; RfaH protein, E coli ; Small Molecule Libraries ; Trans-Activators ; DNA-Directed RNA Polymerases (EC 2.7.7.6) ; RNA polymerase beta subunit (EC 2.7.7.6)
Language	English
Publishing date	2018-10-02
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	619315-8
ISSN	1365-2958 ; 0950-382X
ISSN (online)	1365-2958
ISSN	0950-382X
DOI	10.1111/mmi.14093
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Article: Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.

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

Cell

2005 Volume 122, Issue 3, Page(s) 351–363

Abstract: Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations ... ...

Abstract	Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.
MeSH term(s)	Allosteric Regulation ; Anti-Bacterial Agents/antagonists & inhibitors ; Anti-Bacterial Agents/chemistry ; Anti-Bacterial Agents/pharmacology ; Catalysis ; Crystallography, X-Ray ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/drug effects ; DNA-Directed RNA Polymerases/metabolism ; Magnesium/pharmacology ; Models, Molecular ; Molecular Structure ; Protein Conformation ; Rifamycins/antagonists & inhibitors ; Rifamycins/chemistry ; Rifamycins/pharmacology ; Signal Transduction/drug effects ; Signal Transduction/physiology ; Structure-Activity Relationship ; Thermus thermophilus/drug effects ; Thermus thermophilus/enzymology ; Transcription Factors/chemistry ; Transcription Factors/drug effects ; Transcription Factors/metabolism
Chemical Substances	Anti-Bacterial Agents ; Rifamycins ; Transcription Factors ; DNA-Directed RNA Polymerases (EC 2.7.7.6) ; Magnesium (I38ZP9992A)
Language	English
Publishing date	2005-08-12
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2005.07.014
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In stock of ZB MED Cologne/Königswinter

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Inter-library loan at ZB MED

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Inter-library loan at ZB MED

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In stock of ZB MED Cologne/Königswinter

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Full text online

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In stock of ZB MED Cologne/Königswinter

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In stock of ZB MED Cologne/Königswinter

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Full text online

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In stock of ZB MED Cologne/Königswinter

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In stock of ZB MED Cologne/Königswinter

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