ABSTRACT
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.
Subject(s)
Escherichia coli Proteins/metabolism , Peptide Elongation Factors/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Amino Acid Sequence , Catalytic Domain , Cryoelectron Microscopy , DNA/chemistry , DNA/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Nucleic Acid Conformation , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Protein Binding , Protein Structure, Quaternary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , rRNA Operon/geneticsABSTRACT
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.
Subject(s)
Escherichia coli Proteins , Mycobacterium tuberculosis , Humans , Transcription Factors/genetics , Transcription Factors/chemistry , Transcription, Genetic , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Escherichia coli Proteins/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , DNA , Peptide Elongation Factors/metabolismABSTRACT
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.
Subject(s)
DNA, Bacterial , DNA-Directed RNA Polymerases , Escherichia coli , RNA, Bacterial , Transcription Termination, Genetic , Cryoelectron Microscopy , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/ultrastructure , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/ultrastructure , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Bacterial/ultrastructure , Terminator Regions, Genetic/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Bacterial/ultrastructureABSTRACT
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.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , Mutation/genetics , Amdinocillin/pharmacology , Bacterial Proteins/metabolism , Cell Death/drug effects , Chromosomes, Bacterial/genetics , Cytoprotection/drug effects , Cytoskeletal Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/drug effects , Mutagenesis, Insertional/genetics , Peptides/metabolism , Phenotype , Structure-Activity Relationship , Transcription, Genetic , Uridine Diphosphate Glucose/metabolismABSTRACT
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:
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Amino Acid Sequence , Escherichia coli/chemistry , Models, Molecular , Molecular Sequence Data , Operon , Protein Biosynthesis , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Ribosomal Proteins/metabolism , Sequence Alignment , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , RNA Folding , RNA, Bacterial/chemistry , Transcription, Genetic , Allosteric Regulation , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Protein Domains , RNA, Bacterial/genetics , RNA, Bacterial/metabolismABSTRACT
Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (RifR) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifR RNAPs, including the most prevalent clinical RifR RNAP substitution found in Mtb infected patients (S456>L of the ß subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.
Subject(s)
Aminoglycosides/pharmacology , Antibiotics, Antitubercular/pharmacology , DNA-Directed RNA Polymerases/metabolism , Drug Resistance, Bacterial/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/physiology , Promoter Regions, Genetic , Aminoglycosides/chemistry , Antibiotics, Antitubercular/chemistry , Binding Sites , Humans , Models, Molecular , Molecular Conformation , Protein Binding , Rifampin/pharmacology , Structure-Activity Relationship , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Ribosomes/metabolism , Algorithms , Chromatin Immunoprecipitation , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Gene Deletion , Open Reading Frames , RNA, Transfer/metabolism , Ribosomes/chemistry , Sigma Factor/chemistry , Transcription, Genetic , Transcriptional Elongation Factors/metabolismABSTRACT
Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , Escherichia coli/enzymology , RNA/chemistry , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Base Sequence , Cystamine/chemistry , Cystine/chemistry , Escherichia coli Proteins/chemistry , Guanosine Triphosphate/chemistry , Inverted Repeat Sequences , Models, Molecular , Oxidation-Reduction , Peptide Elongation Factors/chemistry , Protein Binding , Protein Conformation , Trans-Activators/chemistry , Transcriptional Elongation Factors/chemistryABSTRACT
In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA-DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991-1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.
Subject(s)
Bacteria/genetics , DNA-Directed RNA Polymerases/genetics , RNA, Bacterial/genetics , Terminator Regions, Genetic/genetics , DNA/genetics , Nucleic Acid Conformation , Protein Structure, Secondary , Transcription, Genetic/geneticsABSTRACT
The active site of multisubunit RNA polymerases (RNAPs) is highly conserved from humans to bacteria. This single site catalyzes both nucleotide addition required for RNA transcript synthesis and excision of incorrect nucleotides after misincorporation as a proofreading mechanism. Phosphoryl transfer and proofreading hydrolysis are controlled in part by a dynamic RNAP component called the trigger loop (TL), which cycles between an unfolded loop and an α-helical hairpin [trigger helices (TH)] required for rapid nucleotide addition. The precise roles of the TL/TH in RNA synthesis and hydrolysis remain unclear. An invariant histidine residue has been proposed to function in the TH form as a general acid in RNA synthesis and as a general base in RNA hydrolysis. The effects of conservative, nonionizable substitutions of the TL histidine (or a neighboring TL arginine conserved in bacteria) have not yet been rigorously tested. Here, we report that glutamine substitutions of these residues, which preserve polar interactions but are incapable of acid-base chemistry, had little effect on either phosphoryl transfer or proofreading hydrolysis by Escherichia coli RNAP. The TL substitutions did, however, affect the backtracking of RNAP necessary for proofreading and potentially the reactivity of the backtracked nucleotide. We describe a unifying model for the function of the RNAP TL, which reconciles available data and our results for representative RNAPs. This model explains diverse effects of the TL basic residues on catalysis through their effects on positioning reactants for phosphoryl transfer and easing barriers to transcript backtracking, rather than as acid-base catalysts.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Models, Chemical , Mutation, Missense , Nucleic Acid Conformation , RNA/biosynthesis , Amino Acid Substitution , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , RNA/chemistry , RNA/geneticsABSTRACT
The secondary channel (SC) of multisubunit RNA polymerases (RNAPs) allows access to the active site and is a nexus for the regulation of transcription. Multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but the dynamics of these factors' interactions with RNAP and how they function without cross-interference are unclear. In Escherichia coli, GreB is an SC protein that promotes proofreading by transcript cleavage in elongation complexes backtracked by nucleotide misincorporation. Using multiwavelength single-molecule fluorescence microscopy, we observed the dynamics of GreB interactions with elongation complexes. GreB binds to actively elongating complexes at nearly diffusion-limited rates but remains bound for only 0.3-0.5 s, longer than the duration of the nucleotide addition cycle but far shorter than the time needed to synthesize a complete mRNA. Bound GreB inhibits transcript elongation only partially. To test whether GreB preferentially binds backtracked complexes, we reconstituted complexes stabilized in backtracked and nonbacktracked configurations. By verifying the functional state of each molecular complex studied, we could exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, GreB binds rapidly and randomly to elongation complexes, patrolling for those requiring nucleolytic rescue, and its short residence time minimizes RNAP inhibition. The results suggest a general mechanism by which SC factors may cooperate to regulate RNAP while minimizing mutual interference.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Transcription, Genetic , Transcriptional Elongation Factors/metabolism , Benzenesulfonates , Binding Sites , Carbocyanines , Computer Simulation , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/metabolism , Fluorescent Dyes , Models, Genetic , Models, Molecular , Monte Carlo Method , Protein Binding , Single Molecule Imaging , Time Factors , Transcription Elongation, GeneticABSTRACT
Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Peptide Elongation Factors/metabolism , Transcription Factors/metabolism , Base Sequence , Escherichia coli Proteins/genetics , Gene Deletion , Genome, Bacterial , Peptide Elongation Factors/genetics , Protein Binding , Transcription Factors/genetics , Transcription, GeneticABSTRACT
In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the ß gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the ß' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.
Subject(s)
Bacterial Proteins/chemistry , DNA-Directed RNA Polymerases/chemistry , Escherichia coli Proteins/chemistry , Peptide Elongation Factors/chemistry , Trans-Activators/chemistry , Transcription Factors/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Operon , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Protein Conformation , Thermus thermophilus/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The trafficking patterns of the bacterial regulators of transcript elongation sigma(70), rho, NusA, and NusG on genes in vivo and the explanation for promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters (rho first, then NusA, then NusG). However, the interactions of elongating complexes with these regulators did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from sigma(70) peaks in the direction of transcription and co-occurred with NusA and rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of rho did not increase RNAP levels within genes downstream from the RNAP peaks, suggesting the peaks are caused by a mechanism other than rho-dependent attenuation.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Transcription, Genetic , Chromatin Immunoprecipitation , DNA-Directed RNA Polymerases/metabolism , Genes, Bacterial , Models, Genetic , Promoter Regions, Genetic/genetics , Protein Binding , Protein TransportABSTRACT
The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH-NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , Escherichia coli/enzymology , Biocatalysis , Catalytic Domain , DNA/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Models, Molecular , Mutation , Nucleotides/metabolism , Protein Folding , Protein Structure, Tertiary , Protein Unfolding , Transcription, GeneticABSTRACT
Elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase (RNAP) to modulate transcription. However, although NusG associates with RNAP transcribing most Escherichia coli genes, RfaH regulates just a few operons containing ops, a DNA sequence that mediates RfaH recruitment. Here, we describe the mechanism by which this specificity is maintained. We observe that RfaH action is indeed restricted to those several operons that are devoid of NusG in vivo. We also show that RfaH and NusG compete for their effects on transcript elongation and termination in vitro. Our data argue that RfaH recognizes its DNA target even in the presence of NusG. Once recruited, RfaH remains stably associated with RNAP, thereby precluding NusG binding. We envision a pathway by which a specialized regulator has evolved in the background of its ubiquitous paralogue. We propose that RfaH and NusG may have opposite regulatory functions: although NusG appears to function in concert with Rho, RfaH inhibits Rho action and activates the expression of poorly translated, frequently foreign genes.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins/metabolism , Peptide Elongation Factors/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Escherichia coli Proteins/genetics , Models, Molecular , Nucleic Acid Conformation , Operon , Peptide Elongation Factors/genetics , Phylogeny , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Rho Factor/genetics , Rho Factor/metabolism , Trans-Activators/genetics , Transcription Factors/geneticsABSTRACT
We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N(2)-furfuryl-dG (N(2)-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the N(2)-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.
Subject(s)
Escherichia coli Proteins/physiology , Escherichia coli/physiology , Peptide Elongation Factors/physiology , Signal Transduction/physiology , Transcription Factors/physiology , 4-Nitroquinoline-1-oxide/pharmacology , Anti-Infective Agents/pharmacology , Blotting, Far-Western , DNA Damage , DNA Repair , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Dose-Response Relationship, Drug , Drug Resistance, Bacterial , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microbial Viability/drug effects , Microscopy, Fluorescence , Mutation , Nitrofurazone/pharmacology , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Quinolones/pharmacology , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Signal Transduction/drug effects , Temperature , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transcriptional Elongation FactorsABSTRACT
The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found approximately 200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a previously uncharacterized set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a role of noncoding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , RNA/biosynthesis , Transcription, Genetic/physiology , rho GTP-Binding Proteins/metabolism , Bridged Bicyclo Compounds, Heterocyclic , Chromatin Immunoprecipitation , Genes, Bacterial/genetics , Microarray Analysis , rho GTP-Binding Proteins/antagonists & inhibitorsABSTRACT
BACKGROUND: Historically, high levels of morbidity and mortality have been associated with cardiovascular disease in the Northern Ireland population. Previously reported associations between single nucleotide polymorphisms (SNPs) and cardiovascular disease within other populations have not always been consistent. OBJECTIVE: To investigate associations between 33 SNPs with fatal or non-fatal incident coronary heart disease (CHD) events and all-cause mortality in the Northern Irish participants of the Prospective Epidemiological Study of Myocardial Infarction (PRIME). METHOD: Phase 2 of the PRIME study prospectively evaluated 2,010 men aged 58-74 years in Northern Ireland for more than 10 years for incident CHD events (myocardial infarction, percutaneous coronary intervention, coronary artery bypass, and cardiac death) and more than 15 years for all-cause mortality. SNPs previously reported in association with cardiovascular outcomes were evaluated against incident CHD events and all-cause mortality using Cox's proportional hazards models adjusted for established cardiovascular disease risk factors. RESULTS: During the follow-up period, 177 incident CHD events were recorded, and 821 men died. Both BCMO1 rs6564851 (Hazard ratio [HR] = 0.76; 95% confidence intervals [CI]: 0.60-0.96; P = 0.02) and TGFB1 rs1800469 (HR = 1.30; CI: 1.02-1.65; P = 0.04) were significantly associated with incident CHD events in adjusted models. Only IL1B rs16944 was significantly associated with all-cause mortality (HR = 1.18; CI: 1.05-1.33; P = 0.005). No associations remained significant following Bonferonni correction for multiple testing. CONCLUSION: We report a novel association between BCMO1 rs6564851 and risk of incident CHD events. In addition, TGFB1 rs1800469 and IL1B rs16944 were associated with the risk of incident CHD events and all-cause mortality outcomes respectively, supporting previously reported associations.