RESUMO
NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Óperon de RNAr/genéticaRESUMO
Transcript termination is essential for accurate gene expression and the removal of RNA polymerase (RNAP) at the ends of transcription units. In bacteria, two mechanisms are responsible for proper transcript termination: intrinsic termination and Rho-dependent termination. Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho-RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination. We describe current knowledge, discuss key outstanding questions, and highlight the importance of defining the structural rearrangements of RNAP that are involved in the two mechanisms of transcript termination.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores de Alongamento de Peptídeos/genética , Fator Rho/genética , Fatores de Transcrição/genética , Terminação da Transcrição Genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fator Rho/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.
Assuntos
Proteínas de Escherichia coli , Mycobacterium tuberculosis , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/química , Transcrição Gênica , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Escherichia coli/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , DNA , Fatores de Alongamento de Peptídeos/metabolismoRESUMO
Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.
Assuntos
Proteínas de Bactérias , Farmacorresistência Bacteriana , Evolução Molecular , Aptidão Genética , Mycobacterium tuberculosis , Rifampina , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Conservada , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genômica , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Rifampina/farmacologia , Rifampina/uso terapêutico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.
Assuntos
DNA Bacteriano , RNA Polimerases Dirigidas por DNA , Escherichia coli , RNA Bacteriano , Terminação da Transcrição Genética , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/ultraestrutura , Regiões Terminadoras Genéticas/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestruturaRESUMO
The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , SARS-CoV-2 , Humanos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/ultraestrutura , COVID-19/virologia , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , RNA Viral/biossíntese , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Especificidade por Substrato , Guanosina Trifosfato/metabolismo , Capuzes de RNARESUMO
Travis et al. (2020) reveal how Francisella tularensis uses stress-induced ppGpp to activate its virulent pathogenesis program by tethering an αCTD-DNA organizer (PigR) to a σ-organizing heterodimer (MglA-SspA), highlighting the remarkable diversity of transcriptional mechanisms in under-studied bacteria.
Assuntos
Francisella tularensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato , Fator sigma/genética , VirulênciaRESUMO
The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Mutação/genética , Andinocilina/farmacologia , Proteínas de Bactérias/metabolismo , Morte Celular/efeitos dos fármacos , Cromossomos Bacterianos/genética , Citoproteção/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mutagênese Insercional/genética , Peptídeos/metabolismo , Fenótipo , Relação Estrutura-Atividade , Transcrição Gênica , Uridina Difosfato Glucose/metabolismoRESUMO
Transcriptional pausing by multisubunit RNA polymerases (RNAPs) is a key mechanism for regulating gene expression in both prokaryotes and eukaryotes and is a prerequisite for transcription termination. Pausing and termination states are thought to arise through a common, elemental pause state that is inhibitory for nucleotide addition. We report three crystal structures of Thermus RNAP elemental paused elongation complexes (ePECs). The structures reveal the same relaxed, open-clamp RNAP conformation in the ePEC that may arise by failure to re-establish DNA contacts during translocation. A kinked bridge-helix sterically blocks the RNAP active site, explaining how this conformation inhibits RNAP catalytic activity. Our results provide a framework for understanding how RNA hairpin formation stabilizes the paused state and how the ePEC intermediate facilitates termination.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Thermus thermophilus/enzimologia , Thermus/enzimologia , Elongação da Transcrição Genética , Domínio Catalítico , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/metabolismo , Sequências Repetidas Invertidas , Nucleotídeos/química , Nucleotídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Terminação da Transcrição GenéticaRESUMO
Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile (Cdiff) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown. Cdiff infections are a leading cause of nosocomial deaths1. Fidaxomicin, which inhibits RNA polymerase, targets Cdiff with minimal effects on gut commensals, reducing recurrence of Cdiff infection2,3. Here we present the cryo-electron microscopy structure of Cdiff RNA polymerase in complex with fidaxomicin and identify a crucial fidaxomicin-binding determinant of Cdiff RNA polymerase that is absent in most gut microbiota such as Proteobacteria and Bacteroidetes. By combining structural, biochemical, genetic and bioinformatic analyses, we establish that a single residue in Cdiff RNA polymerase is a sensitizing element for fidaxomicin narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Clostridioides , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA , Fidaxomicina/química , Fidaxomicina/farmacologia , Fidaxomicina/uso terapêutico , HumanosRESUMO
Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Animais , Humanos , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Using a fluorescence method called colocalization single-molecule spectroscopy (CoSMoS), Friedman and Gelles dissect the kinetics of transcription initiation at a bacterial promoter. Ultimately, CoSMoS could greatly aid the study of the effects of DNA sequence and transcription factors on both prokaryotic and eukaryotic promoters.
RESUMO
NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a ß barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-ß conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Escherichia coli/química , Modelos Moleculares , Dados de Sequência Molecular , Óperon , Biossíntese de Proteínas , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Ribossômicas/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Cellular life depends on transcription of DNA by RNA polymerase to express genetic information. RNA polymerase has evolved not just to read information from DNA and write it to RNA but also to sense and process information from the cellular and extracellular environments. Much of this information processing occurs during transcript elongation, when transcriptional pausing enables regulatory decisions. Transcriptional pauses halt RNA polymerase in response to DNA and RNA sequences and structures at locations and times that help coordinate interactions with small molecules and transcription factors important for regulation. Four classes of transcriptional pause signals are now evident after decades of study: elemental pauses, backtrack pauses, hairpin-stabilized pauses, and regulator-stabilized pauses. In this review, I describe current understanding of the molecular mechanisms of these four classes of pause signals, remaining questions about how RNA polymerase responds to pause signals, and the many exciting directions now open to understand pausing and the regulation of transcript elongation on a genome-wide scale.
Assuntos
Genes Bacterianos , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sequence-specific pausing by RNA polymerase (RNAP) during transcription plays crucial and diverse roles in gene expression. In bacteria, RNA structures are thought to fold within the RNA exit channel of the RNAP and can increase pause lifetimes significantly. The biophysical mechanism of pausing is uncertain. We used single-particle cryo-EM to determine structures of paused complexes, including a 3.8-Å structure of an RNA hairpin-stabilized, paused RNAP that coordinates RNA folding in the his operon attenuation control region of E. coli. The structures revealed a half-translocated pause state (RNA post-translocated, DNA pre-translocated) that can explain transcriptional pausing and a global conformational change of RNAP that allosterically inhibits trigger loop folding and can explain pause hairpin action. Pause hairpin interactions with the RNAP RNA exit channel suggest how RNAP guides the formation of nascent RNA structures.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Dobramento de RNA , RNA Bacteriano/química , Transcrição Gênica , Regulação Alostérica , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Domínios Proteicos , RNA Bacteriano/genética , RNA Bacteriano/metabolismoRESUMO
NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Escherichia coli/fisiologia , Fatores de Alongamento de Peptídeos/fisiologia , Fatores de Transcrição/fisiologia , Terminação da Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Proteínas de Bactérias , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Fatores de Alongamento de Peptídeos/metabolismo , Conformação Proteica , RNA Bacteriano , Fator Rho/metabolismo , Fator Rho/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). A surface-exposed domain inserted into the catalytic trigger loop (TL) of Escherichia coli RNAP, called SI3, modulates pausing and is essential for growth. Here we describe a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution (ß'Ala941âThr; ΔSI3*). ΔSI3* increased transcription rate in vitro relative to ΔSI3, possibly explaining its viability, but retained both positive and negative effects of ΔSI3 on pausing. ΔSI3* inhibited pauses stabilized by nascent RNA structures (pause hairpins; PHs) but enhanced other pauses. Using NET-seq, we found that ΔSI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at ΔSI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. ΔSI3*-suppressed pauses also were associated with apparent pausing one nucleotide upstream from the consensus sequence, often generating tandem pause sites. These '-2 pauses' were stimulated by pyrophosphate in vitro and by addition of apyrase to degrade residual NTPs during NET-seq sample processing. We propose that some pauses are readily reversible by pyrophosphorolysis or single-nucleotide cleavage. Our results document multiple ways that SI3 modulates pausing in vivo and may explain discrepancies in consensus pause sequences in some NET-seq studies.
Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , Escherichia coli , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Domínios ProteicosRESUMO
Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.
Assuntos
RNA Polimerases Dirigidas por DNA , RNA , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , RNA/genética , DNA , Nucleotídeos/química , Transcrição GênicaRESUMO
DNA in bacterial chromosomes is organized into higher-order structures by DNA-binding proteins called nucleoid-associated proteins (NAPs) or bacterial chromatin proteins (BCPs). BCPs often bind to or near DNA loci transcribed by RNA polymerase (RNAP) and can either increase or decrease gene expression. To understand the mechanisms by which BCPs alter transcription, one must consider both steric effects and the topological forces that arise when DNA deviates from its fully relaxed double-helical structure. Transcribing RNAP creates DNA negative (-) supercoils upstream and positive (+) supercoils downstream whenever RNAP and DNA are unable to rotate freely. This (-) and (+) supercoiling generates topological forces that resist forward translocation of DNA through RNAP unless the supercoiling is constrained by BCPs or relieved by topoisomerases. BCPs also may enhance topological stress and overall can either inhibit or aid transcription. Here, we review current understanding of how RNAP, BCPs, and DNA topology interplay to control gene expression.
Assuntos
Proteínas de Bactérias , Cromatina , DNA Bacteriano , RNA Polimerases Dirigidas por DNA , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cromatina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Bactérias/metabolismo , Bactérias/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/genéticaRESUMO
The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.