RESUMEN
The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component-the histone tails, the specific histone-DNA contacts, and the DNA sequence-contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we show that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by affecting distinctly the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by chromatin remodeling and transcription factors.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Nucleosomas , Transcripción Genética , Levaduras/genética , ADN/metabolismo , Histonas/química , ARN Polimerasa II/metabolismo , Levaduras/metabolismoRESUMEN
Transcription elongation by multi-subunit RNA polymerases (RNAPs) is regulated by auxiliary factors in all organisms. NusG/Spt5 is the only universally conserved transcription elongation factor shared by all domains of life. NusG is a component of antitermination complexes controlling ribosomal RNA operons, an essential antipausing factor, and a transcription-translation coupling factor in Escherichia coli. We employed RNET-seq for genome-wide mapping of RNAP pause sites in wild-type and NusG-depleted cells. We demonstrate that NusG is a major antipausing factor that suppresses thousands of backtracked and nonbacktracked pauses across the E. coli genome. The NusG-suppressed pauses were enriched immediately downstream from the translation start codon but were also abundant elsewhere in open reading frames, small RNA genes, and antisense transcription units. This finding revealed a strong similarity of NusG to Spt5, which stimulates the elongation rate of many eukaryotic genes. We propose a model in which promoting forward translocation and/or stabilization of RNAP in the posttranslocation register by NusG results in suppression of pausing in E. coli.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Proteínas de Escherichia coli/genética , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Factores de Transcripción/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismoRESUMEN
UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.
Asunto(s)
Daño del ADN/efectos de la radiación , Dímeros de Pirimidina/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , ADN de Hongos/biosíntesis , ADN de Hongos/genética , Genoma Fúngico/fisiología , Dímeros de Pirimidina/genética , ARN Polimerasa II/genética , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genéticaRESUMEN
Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA-DNA base pair at the upstream edge of the transcription bubble. DNA-DNA base pairing immediately upstream from the RNA-DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA-DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , ADN/química , ARN/química , Elongación de la Transcripción Genética , Emparejamiento Base , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/enzimología , Movimiento , Dominios Proteicos , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismoRESUMEN
In the process of transcription elongation, RNA polymerase (RNAP) pauses at highly nonrandom positions across genomic DNA, broadly regulating transcription; however, molecular mechanisms responsible for the recognition of such pausing positions remain poorly understood. Here, using a combination of statistical mechanical modeling and high-throughput sequencing and biochemical data, we evaluate the effect of thermal fluctuations on the regulation of RNAP pausing. We demonstrate that diffusive backtracking of RNAP, which is biased by repetitive DNA sequence elements, causes transcriptional pausing. This effect stems from the increased microscopic heterogeneity of an elongation complex, and thus is entropy-dominated. This report shows a linkage between repetitive sequence elements encoded in the genome and regulation of RNAP pausing driven by thermal fluctuations.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Entropía , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , ARN Bacteriano/metabolismo , Análisis de Secuencia de ARN , TemperaturaRESUMEN
In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5'-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.
Asunto(s)
Daño del ADN , Purinas/metabolismo , ARN Polimerasa II/metabolismo , Secuencia de Bases , ADN/química , Oxidación-Reducción , Transcripción GenéticaRESUMEN
Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.
Asunto(s)
Conformación de Ácido Nucleico , ARN Mensajero/química , Regiones Terminadoras Genéticas , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Chloroflexi/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Inversión de SecuenciaRESUMEN
Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α2ßß'ω·σA can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD.
Asunto(s)
Proteínas Bacterianas , ARN Polimerasas Dirigidas por ADN , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Regiones Promotoras Genéticas , Transcripción Genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/biosíntesis , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Holoenzimas/biosíntesis , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP ß' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , ADN Viral/química , ADN Viral/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Difosfatos/metabolismo , Modelos Genéticos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Nucleótidos/genética , Nucleótidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Viral/química , ARN Viral/genética , Moldes Genéticos , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica/fisiología , ARN Mensajero/biosíntesis , Saccharomyces cerevisiae/fisiología , Elongación de la Transcripción Genética/fisiología , Factores de Transcripción TFII/metabolismo , Factores de Elongación Transcripcional/metabolismo , Escherichia coli , Regulación de la Expresión Génica/genética , Cinética , Método de Montecarlo , Pinzas Ópticas , Saccharomyces cerevisiae/genéticaRESUMEN
Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN in the absence or presence of RNAP and the solution structures of RapA and RapAΔN either ligand-free or in complex with RNAP. Determined by small-angle x-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ(70) on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Alostérica , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/enzimología , Conformación Proteica , Dispersión del Ángulo Pequeño , Transcripción Genética , Difracción de Rayos XRESUMEN
To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Mutación/genética , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Dominio Catalítico , Isomerismo , Datos de Secuencia Molecular , Nucleótidos/metabolismo , ARN Polimerasa II/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5' end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination.
Asunto(s)
Bacteriófago lambda , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Bases , Escherichia coli/virología , Genes Reporteros , Transcripción Genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through â¼10(3) nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity.
Asunto(s)
Análisis de Secuencia de ARN/métodos , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/metabolismoRESUMEN
The fidelity of RNA synthesis depends on both accurate template-mediated nucleotide selection and proper maintenance of register between template and RNA. Loss of register, or transcriptional slippage, is particularly likely on homopolymeric runs in the template. Transcriptional slippage can alter the coding capacity of mRNAs and is used as a regulatory mechanism. Here we describe mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II that substantially increase the level of transcriptional slippage. Alleles of RPB1 (RPO21) with elevated slippage rates were identified among 6-azauracil-sensitive mutants and were also isolated using a slippage-dependent reporter gene. Biochemical characterization of polymerase II isolated from these mutants confirms elevated levels of transcriptional slippage.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Mutación , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Cromosomas/ultraestructura , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Oligonucleótidos/genética , Unión Proteica , ARN/metabolismo , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the ß subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Mutación , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas/ultraestructura , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/enzimología , Operón Lac , Modelos Genéticos , Datos de Secuencia Molecular , Fenotipo , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 +/- 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t(-3/2) power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 +/- 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.
Asunto(s)
ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética , Fenómenos Biomecánicos , Escherichia coli/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Moldes Genéticos , Factores de Elongación Transcripcional/metabolismoRESUMEN
Intrinsic transcription termination signal in DNA consists of a short inverted repeat followed by a T-rich stretch. Transcription of this sequence by RNA polymerase (RNAP) results in formation of a "termination hairpin" (TH) in the nascent RNA and in rapid dissociation of the transcription elongation complex (EC) at termination points located 7-8 nt downstream of the base of TH stem. RNAP envelops 15 nt of the RNA following RNA growing 3'-end, suggesting that folding of the TH is impeded by a tight protein environment when RNAP reaches the termination points. To monitor TH folding under this constraint, we halted Escherichia coli ECs at various distances downstream from a TH and treated them with single-strand specific RNase T1. The EC interfered with TH formation when halted at 6, 7, and 8, but not 9, nt downstream from the base of the potential stem. Thus, immediately before termination, the downstream arm of the TH is protected from complementary interactions with the upstream arm. This protection makes TH folding extremely sensitive to the sequence context, because the upstream arm easily engages in competing interactions with the rest of the nascent RNA. We demonstrate that by de-synchronizing TH formation and transcription of the termination points, this subtle competition significantly affects the efficiency of transcription termination. This finding can explain previous puzzling observations that sequences far upstream of the TH or point mutations in the terminator that preserve TH stability affect termination. These results can help understand other time sensitive co-transcriptional processes in pro- and eukaryotes.
Asunto(s)
Escherichia coli/genética , Pliegue del ARN , ARN/química , Regiones Terminadoras Genéticas , Transcripción Genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/metabolismo , Conformación de Ácido Nucleico , ARN/metabolismoRESUMEN
RNA polymerase can cleave a phosphodiester bond at the 3' end of a nascent RNA in the presence of pyrophosphate producing NTP. Pyrophosphorolysis has been characterized during elongation steps of transcription where its rate is significantly slower than the forward rate of NMP addition. In contrast, we report here that pyrophosphorolysis can occur in a millisecond time scale during the transition of Escherichia coli RNA polymerase from initiation to elongation at the psbA2 promoter. This rapid pyrophosphorolysis occurs during productive RNA synthesis as opposed to abortive RNA synthesis. Dissociation of σ70 or RNA extension beyond nine nucleotides dramatically reduces the rate of pyrophosphorolysis. We argue that the rapid pyrophosphorolysis allows iterative cycles of cleavage and re-synthesis of the 3' phosphodiester bond by the productive complexes in the early stage of transcription. This iterative process may provide an opportunity for the σ70 to dissociate from the RNA exit channel of the enzyme, enabling RNA to extend through the channel.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Difosfatos/metabolismo , Escherichia coli/enzimología , ARN Bacteriano/genética , Factor sigma/metabolismo , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/genética , Fosforilación , Regiones Promotoras Genéticas , Factor sigma/genéticaRESUMEN
RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the "lid" and "rudder," two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.