RESUMEN
The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a "release and catch-up" mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle's resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.
Asunto(s)
Proteínas Bacterianas/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , ADN/genética , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Factores de Transcripción/genética , Terminación de la Transcripción GenéticaRESUMEN
During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced Escherichia coli RNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pNâ nm to 18.5 pN·nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Factores de Transcripción/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Teóricos , Transcripción Genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the 'elemental pause sequence' recognized from its frequent occurrence in the E. coli genome. σ70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause.
Asunto(s)
Escherichia coli/genética , Factores de Iniciación de Péptidos/metabolismo , Factor sigma/metabolismo , Transcripción Genética , Bacteriófago lambda/metabolismo , Composición de Base/genética , Secuencia de Bases , ADN Viral/metabolismo , Modelos Genéticos , Ácidos Nucleicos Heterodúplex , Regiones Promotoras Genéticas , ARN Bacteriano/metabolismo , Moldes GenéticosRESUMEN
The movement of RNA polymerase (RNAP) during transcription elongation is modulated by DNA-encoded elements that cause the elongation complex to pause. One of the best-characterized pause sequences is a binding site for the σ(70) initiation factor that induces pausing at a site near lambdoid phage late-gene promoters. An essential component of this σ(70)-dependent pause is the elemental pause site (EPS), a sequence that itself induces transcription pausing throughout the Escherichia coli genome and underlies other complex regulatory pause elements, such as the ops and his operon pauses. Here, we identify and provide a detailed kinetic analysis of a transcription cycle analogous to abortive cycling that underlies the σ(70)-dependent pause. We show that, in σ(70)-dependent pausing, the elemental pause acts primarily to modulate the rate at which complexes attempt to disengage the σ(70):DNA interaction. Our findings establish the σ(70)-dependent pause-encoding region as a multipartite element in which several pause-inducing components make distinct mechanistic contributions to the induction and maintenance of a regulatory transcription pause.
Asunto(s)
Escherichia coli/genética , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Elongación de la Transcripción Genética , Composición de Base/genética , Secuencia de Bases , ADN Bacteriano/genética , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Nucleótidos/genética , Probabilidad , ARN Bacteriano/genéticaRESUMEN
The stringent response of Escherichia coli reflects a global influence of the nucleotide ppGpp on gene expression in response to nutrient starvation. For critical elements of the response, the target of ppGpp is RNA polymerase, which can be either repressed or activated in a promoter-dependent way. A small protein, DksA, which binds in the secondary channel of RNA polymerase, possibly along with other general regulatory factors, contributes to ppGpp-dependent transcription regulation. Rutherford and colleagues (pp. 236-248) identify mutations in genes for the core subunits of RNA polymerase that circumvent the need for DksA in the regulation; the sites of these mutations suggest that DksA acts allosterically through the RNA polymerase active site to destabilize transcription initiation complexes.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Guanosina Tetrafosfato/metabolismo , Regiones Promotoras Genéticas , Coenzimas/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Proteínas de Escherichia coli/metabolismo , MutaciónRESUMEN
During initial transcription, RNA polymerase remains bound at the promoter and synthesizes RNA without movement along the DNA template, drawing downstream DNA into itself in a process called scrunching and thereby storing energy to sever the bonds that hold the enzyme at the promoter. We show that DNA scrunching also is the driving force behind the escape of RNA polymerase from a regulatory pause of the late gene operon of bacteriophage λ, and that this process is enhanced by the activity of the Q(λ) antiterminator. Furthermore, we show that failure of transcription complexes to escape the pause results in backtracking and arrest in a process analogous to abortive initiation. We identify a sequence element that modulates both abortive synthesis and the formation of arrested elongation complexes.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , ADN/química , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Elongación de la Transcripción Genética , Proteínas Virales/metabolismo , Mutación , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Bacteriophage lambda is one of the most extensively studied organisms and has been a primary model for understanding basic modes of genetic regulation. Here, we examine the progress of lambda gene expression during phage development by ribosome profiling and, thereby, provide a very-high-resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis. However, many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function.
Asunto(s)
Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Viral de la Expresión Génica/genética , Genoma Viral/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Mutación del Sistema de Lectura/genética , ARN Mensajero/genéticaRESUMEN
RNA polymerase of both bacteria and eukaryotes can stall or pause within tens of base pairs of its initiation site at the promoter, a state that may reflect important regulatory events in early transcription. In the bacterial model system, the σ(70) initiation factor stabilizes such pauses by binding a downstream repeat of a promoter segment, especially the '-10' promoter element. We first show here that the '-35' promoter element also can stabilize promoter-proximal pausing, through interaction with σ(70) region 4. We further show that an essential element of either type of pause is a sequence just upstream of the site of pausing that stabilizes RNA polymerase backtracking. Although the pause is not intrinsically backtracked, we suggest that the same sequence element is required both to stabilize the paused state and to potentiate backtracking.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Factor sigma/genética , Transcripción GenéticaRESUMEN
Bacterial transcription termination, described mostly for Escherichia coli, occurs in three recognized ways: intrinsic termination, an activity only of the core RNAP enzyme and transcript sequences that encode an RNA hairpin and terminal uridine-rich segment; termination by the enzyme Rho, an ATP-dependent RNA translocase that releases RNA by forcing uncharacterized structural changes in the elongating complex; and Mfd-dependent termination, the activity of an ATP-dependent DNA translocase that is thought to dissociate the elongation complex by exerting torque on a stalled RNAP. Intrinsic termination can be described in terms of the nucleic acid movements in the process, whereas the enzymatic mechanisms have been illuminated importantly by definitive structural and biochemical analysis of their activity.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Terminación de la Transcripción Genética , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , ARN Bacteriano/metabolismo , Factor Rho/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The DNA recognition sequence for the transcriptional activator, CII protein, which is critical for lysogenization by bacteriophage lambda, overlaps the -35 region of the P(RE) promoter. Data presented here show that activation by CII does not change the pattern of cleavage of the -35 region of P(RE) by iron (S)-1-(p-bromoacetamidobenzyl)-EDTA (Fe-BABE) conjugated to the sigma subunit of RNA polymerase (RNAP). Thus, the overall interaction between sigma and the -35 region of P(RE) is not significantly altered by CII. Therefore, the effects of the activator on RNAP binding to the promoter and formation of open complexes do not reflect a large-scale qualitative change in the nature of the interaction between RNAP and promoter DNA. The ability of CII to stimulate lysogenization is reduced in the presence of plasmid-borne rpoA variants encoding alanine substitutions at several positions in the C-terminal domain of the alpha subunit. However, it has not been possible to identify residues that directly affect the interaction between the activator and RNA polymerase.
Asunto(s)
Bacteriófago lambda/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Aminoácidos/análisis , Secuencia de Bases , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Factor sigma/metabolismo , Activación Transcripcional , Proteínas ViralesRESUMEN
Intrinsic transcription terminators of Escherichia coli and other bacteria, consisting primarily of an RNA hairpin preceding a terminal uridine-rich RNA segment, suffice to dissociate the otherwise stable elongation complex of core RNA polymerase. The essential functions of the hairpin and U-rich segments have been established, although the precise mechanism of termination is unknown. We identify another element of the terminator, namely the non-template DNA strand in the region of the terminal transcription bubble. Failure of the terminal bubble to rewind through complementary base-pairing strongly reduces the efficiency of terminator function, suggesting that the natural pathway of termination consists of coupled rewinding of the DNA template and unwinding of the RNA/DNA hybrid at the site of release.
Asunto(s)
Modelos Biológicos , Transcripción Genética/fisiología , Secuencia de Bases , Escherichia coli/fisiología , Datos de Secuencia Molecular , MutaciónRESUMEN
After promoter escape in Escherichia coli, the initiating σ(70) factor is retained by core RNA polymerase (RNAP) for at least tens of nucleotides. While it is bound, σ(70) can engage a repeat of a promoter DNA element located downstream of the promoter and thereby induce a transcription pause. The σ(70)-dependent promoter-proximal pause that occurs at all lambdoid phage late gene promoters is essential to regulation of the late gene operons. Several, and possibly many, E. coli promoters have associated σ(70)-dependent pauses. Clearly characterized σ(70)-dependent pauses occur within 25 nucleotides of the start site, but σ(70)-dependent pausing might occur farther downstream as well. In this review, we summarize evidence for σ(70)-dependent promoter-proximal and promoter-distal pausing, and we discuss its potential regulatory function and mechanistic basis.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Bacteriano/biosíntesis , Factor sigma/metabolismo , Transcripción Genética , ADN Bacteriano/metabolismo , Modelos Biológicos , Modelos Químicos , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/biosíntesisAsunto(s)
Codón Iniciador/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Iniciación de la Cadena Peptídica Traduccional/genética , Elementos Reguladores de la Transcripción , Elongación de la Transcripción Genética , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción Genética , Transcripción GenéticaRESUMEN
The elongation phase of transcription by RNA polymerase is highly regulated and modulated. Both general and operon-specific elongation factors determine the local rate and extent of transcription to coordinate the appearance of transcript with its use as a messenger or functional ribonucleoprotein or regulatory element, as well as to provide operon-specific gene regulation.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Factores de Elongación Transcripcional/metabolismo , Bacterias/genética , Bacteriófago lambda/metabolismo , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
The universal bacterial transcription elongation factor NusA mediates elongation activities of RNA polymerase. By itself, NusA induces transcription pausing and facilitates intrinsic termination, but NusA also is a cofactor of antiterminators that antagonize pausing and prevent termination. We show that NusA is required for lambda-related phage 82 antiterminator Q(82) to construct a stable complex in which RNA-based termination mechanisms have restricted access to the emerging transcript; this result suggests a locale for both Q(82) and NusA near the beta flap domain of RNA polymerase. Furthermore, as NusA is not required for the antipausing activity of Q(82) in vitro, we distinguish two distinct activities of antiterminators, namely antipausing and RNA occlusion, and discuss their roles in Q(82) function.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Factores de Elongación de Péptidos/metabolismo , ARN Bacteriano/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Modelos Genéticos , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Factor Rho/metabolismo , Ribonucleasa H/metabolismo , Factores de Elongación TranscripcionalRESUMEN
By using DNA heteroduplexes that inhibit rewinding of the upstream part of the transcription bubble, we show that transcript release in termination by the enzymes Mfd and Rho is facilitated by reannealing of DNA in the upstream region of the transcription bubble, as is also true for termination by intrinsic terminators. We also show that, like Mfd, the Rho termination factor promotes forward translocation of RNA polymerase. These results support termination models in which external forces imposed on nucleic acids induce concerted rewinding of DNA and unwinding of the DNA/RNA hybrid, possibly accompanied by forward translocation of RNA polymerase, leading to transcription complex dissociation.
Asunto(s)
ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Regiones Terminadoras Genéticas/genética , Transcripción Genética/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , ARN/genética , ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Cinética , Datos de Secuencia Molecular , Nucleótidos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation, the stability of interactions between sigma(70) (the primary sigma factor in Escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent RNA. Here we demonstrate that the nascent RNA-mediated destabilization of an interaction between sigma(70) region 4 and the flap domain of the beta subunit is required for the bacteriophage lambda Q antiterminator protein to contact holoenzyme during early elongation. We demonstrate further that the requirement for nascent RNA in the process by which Q engages RNAP can be bypassed if sigma(70) region 4 is removed. Our findings illustrate how a regulator can exploit the nascent RNA-mediated reconfiguration of the holoenzyme to gain access to the enzyme during early elongation.