RESUMO
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
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
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.
Assuntos
Doença das Coronárias , Interleucina-1beta , Mortalidade , Infarto do Miocárdio , Fator de Crescimento Transformador beta1 , beta-Caroteno 15,15'-Mono-Oxigenase , Doença das Coronárias/epidemiologia , Humanos , Incidência , Interleucina-1beta/genética , Masculino , Infarto do Miocárdio/epidemiologia , Irlanda do Norte/epidemiologia , Estudos Prospectivos , Fatores de Risco , Fator de Crescimento Transformador beta1/genética , beta-Caroteno 15,15'-Mono-Oxigenase/genéticaRESUMO
BACKGROUND: Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer's disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. METHODS: Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. RESULTS: Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. CONCLUSIONS: Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a 'Warburg like' endophenotype that is observable in young females decades prior to clinically manifest AD.
Assuntos
Aerobiose , Apolipoproteína E4/fisiologia , Glucose/metabolismo , Glicólise , Sintomas Prodrômicos , Adolescente , Adulto , Idoso , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Apolipoproteína E4/genética , Astrócitos/metabolismo , Sequência de Bases , Química Encefálica , Células Cultivadas , Diagnóstico Precoce , Metabolismo Energético , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Técnicas de Introdução de Genes , Humanos , Metabolômica , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oxirredução , Fosforilação Oxidativa , Consumo de Oxigênio/genética , Caracteres Sexuais , Análise de Célula Única , Adulto JovemRESUMO
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
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.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antituberculose/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/fisiologia , Regiões Promotoras Genéticas , Aminoglicosídeos/química , Antibióticos Antituberculose/química , Sítios de Ligação , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Rifampina/farmacologia , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
RNA polymerases (RNAPs) contain a conserved 'secondary channel' which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a 'delayed inhibition' mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.
Assuntos
Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Ribossômico/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Ribossômico/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
In bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2- to 3-nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well defined. RNAP contains a clamp domain that closes around the nucleic acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , RNA/metabolismo , Regiões Terminadoras Genéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos/metabolismo , Nucleotídeos/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.
Assuntos
Transcrição Gênica , Pareamento de Bases/genética , Sequência de Bases , Sequência Consenso/genética , DNA/genética , Cinética , Mutação/genética , Nucleotídeos/metabolismo , RNA/genética , Moldes Genéticos , Elongação da Transcrição GenéticaRESUMO
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
Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. RNA folding pathways leading to functional ON and OFF regulation involve the formation of metastable states within well-defined sequence intervals during transcription. The kinetic requirements for the formation and preservation of these metastable states in the context of transcription remain unresolved. Here, we reversibly trap the previously defined regulatory relevant metastable intermediate of the Mesoplasma florum 2'-deoxyguanosine (2'dG)-sensing riboswitch using a photocaging-ligation approach, and monitor folding to its native state by real-time NMR in both presence and absence of ligand. We further determine transcription rates for two different bacterial RNA polymerases. Our results reveal that the riboswitch functions only at transcription rates typical for bacterial polymerases (10-50 nt s-1) and that gene expression is modulated by 40-50% only, while subtle differences in folding rates guide population ratios within the structural ensemble to a specific regulatory outcome.
Assuntos
Riboswitch/genética , Transdução de Sinais/genética , Transcrição Gênica , Sequência de Bases , Simulação por Computador , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Conformação de Ácido NucleicoRESUMO
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
Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket.
Assuntos
Antibióticos Antituberculose/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Inibidores Enzimáticos/metabolismo , Fidaxomicina/metabolismo , Mycobacterium tuberculosis/enzimologia , Antibióticos Antituberculose/química , Microscopia Crioeletrônica , Inibidores Enzimáticos/química , Fidaxomicina/química , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Bacteriano/genética , Regiões Terminadoras Genéticas/genética , DNA/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Modelos Químicos , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , RNA/biossíntese , Substituição de Aminoácidos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA/química , RNA/genéticaRESUMO
In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.
Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Riboswitch , Transcrição Gênica , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Benzenossulfonatos , Sítios de Ligação , Carbocianinas , Simulação por Computador , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Corantes Fluorescentes , Modelos Genéticos , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Imagem Individual de Molécula , Fatores de Tempo , Elongação da Transcrição GenéticaRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Escherichia coli/enzimologia , Biocatálise , Domínio Catalítico , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Mutação , Nucleotídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Transcrição GênicaRESUMO
The rates of RNA synthesis and the folding of nascent RNA into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA-exit channel can either increase pausing by interacting with RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain has been proposed to mediate some effects of nascent-RNA structures. However, the connections among RNA structure formation and RNAP clamp movement and catalytic activity remain uncertain. Here, we assayed exit-channel structure formation in Escherichia coli RNAP with disulfide cross-links that favor closed- or open-clamp conformations and found that clamp position directly influences RNA structure formation and RNAP catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening through interactions with the flap that slow translocation.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , Dobramento de RNARESUMO
Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.