RESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Transcrição Gênica , Proteínas de Escherichia coli/genética , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismoRESUMO
To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others. Cross-referencing the errors that we detected with various genetic and epigenetic features of the human genome revealed that the in vivo error rate in human cells changes along the length of a transcript and is further modified by genetic context, repetitive elements, epigenetic markers, and the speed of transcription. Our experiments further suggest that BRCA1, a DNA repair protein implicated in breast cancer, has a previously unknown role in the suppression of transcription errors. Finally, we analyzed the distribution of transcription errors in multiple tissues of a new mouse model and found that they occur preferentially in neurons, compared to other cell types. These observations lend additional weight to the idea that transcription errors play a key role in the progression of various neurological disorders, including Alzheimer's disease.
Assuntos
RNA , Transcrição Gênica , Animais , Camundongos , Humanos , RNA/genética , Transcriptoma , Proteínas/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.
Assuntos
Bacillus subtilis , Transcrição Gênica , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , RNA/metabolismoRESUMO
Transcription elongation is a highly processive process that is punctuated by RNA polymerase (RNAP) pausing. Long-lived pauses can provide time for diverse regulatory events to occur, which play important roles in modulating gene expression. Transcription elongation factors can dramatically affect RNAP pausing in vitro. The genome-wide role of such factors in pausing in vivo has been examined only for NusG in Bacillus subtilis. NusA is another transcription elongation factor known to stimulate pausing of B. subtilis and Escherichia coli RNAP in vitro. Here, we present the first in vivo study to identify the genome-wide role of NusA in RNAP pausing. Using native elongation transcript sequencing followed by RNase digestion (RNET-seq), we analyzed factor-dependent RNAP pausing in B. subtilis and found that NusA has a relatively minor role in RNAP pausing compared to NusG. We demonstrate that NusA has both stimulating and suppressing effects on pausing in vivo. Based on our thresholding criteria on in vivo data, NusA stimulates pausing at 129 pause peaks in 93 different genes or 5' untranslated regions (5' UTRs). Putative pause hairpins were identified for 87 (67%) of the 129 NusA-stimulated pause peaks, suggesting that RNA hairpins are a common component of NusA-stimulated pause signals. However, a consensus sequence was not identified as a NusA-stimulated pause motif. We further demonstrate that NusA stimulates pausing in vitro at some of the pause sites identified in vivo. IMPORTANCE NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing in vitro; however, the essential nature of NusA had prevented an assessment of its role in pausing in vivo. Using a NusA depletion strain and RNET-seq, we identified a similar number of NusA-stimulated and NusA-suppressed pause peaks throughout the genome. NusA-stimulated pausing was confirmed at several sites in vitro. However, NusA did not always stimulate pausing at sites identified in vivo, while in other instances NusA stimulated pausing at sites not observed in vivo. We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis.
Assuntos
Bacillus subtilis , Proteínas de Escherichia coli , Regiões 5' não Traduzidas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , TranscriptomaRESUMO
NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, ΔnusG, and NusA depletion ΔnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Expressão Gênica , Terminação da Transcrição Genética , Fatores de Elongação da Transcrição/genética , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Fatores de Elongação da Transcrição/metabolismoRESUMO
Promoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10-20-bp distance from many promoters. The pauses in the 10-15-bp register of the promoter are dictated by the canonical -10 element, 6-7 nt spacer and "YR+1Y" motif centered at the transcription start site. The promoters for the pauses in the 16-20-bp register contain an additional -10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Bacteriano/genética , Fator sigma/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
Although transcription by RNA polymerase (RNAP) is highly processive, elongation can be transiently halted by RNAP pausing. Pausing provides time for diverse regulatory events to occur such as RNA folding and regulatory factor binding. The transcription elongation factors NusA and NusG dramatically affect the frequency and duration of RNAP pausing, and hence regulation of transcription. NusG is the only transcription factor conserved in all three domains of life; its homolog in archaea and eukaryotes is Spt5. This review focuses on NusG-dependent pausing, which is a common occurrence in Bacillus subtilis. B. NusG induces pausing about once per 3 kb at a consensus TTNTTT motif in the non-template DNA strand within the paused transcription bubble. A conserved region of NusG contacts the TTNTTT motif to stabilize the paused transcription elongation complex (TEC) in multiple catalytically inactive RNAP conformations. The density of NusG-dependent pause sites is 3-fold higher in untranslated regions, suggesting that pausing could regulate the expression of hundreds of genes in B. subtilis. We describe how pausing in 5' leader regions contributes to regulating the expression of B. subtilis genes by transcription attenuation and translation control mechanisms. As opposed to the broadly accepted view that NusG is an anti-pausing factor, phylogenetic analyses suggest that NusG-dependent pausing is a widespread mechanism in bacteria. This function of NusG is consistent with the well-established role of its eukaryotic homolog Spt5 in promoter-proximal pausing. Since NusG is present in all domains of life, NusG-dependent pausing could be a conserved mechanism in all organisms.
Assuntos
Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica , Filogenia , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
Transcription is punctuated by RNA polymerase (RNAP) pausing. These pauses provide time for diverse regulatory events that can modulate gene expression. Transcription elongation factors dramatically affect RNAP pausing in vitro, but the genome-wide role of such factors on pausing has not been examined. Using native elongating transcript sequencing followed by RNase digestion (RNET-seq), we analyzed RNAP pausing in Bacillus subtilis genome-wide and identified an extensive role of NusG in pausing. This universally conserved transcription elongation factor is known as Spt5 in archaeal and eukaryotic organisms. B. subtilis NusG shifts RNAP to the posttranslocation register and induces pausing at 1,600 sites containing a consensus TTNTTT motif in the nontemplate DNA strand within the paused transcription bubble. The TTNTTT motif is necessary but not sufficient for NusG-dependent pausing. Approximately one-fourth of these pause sites were localized to untranslated regions and could participate in posttranscription initiation control of gene expression as was previously shown for tlrB and the trpEDCFBA operon. Most of the remaining pause sites were identified in protein-coding sequences. NusG-dependent pausing was confirmed for all 10 pause sites that we tested in vitro. Putative pause hairpins were identified for 225 of the 342 strongest NusG-dependent pause sites, and some of these hairpins were shown to function in vitro. NusG-dependent pausing in the ribD riboswitch provides time for cotranscriptional binding of flavin mononucleotide, which decreases the concentration required for termination upstream of the ribD coding sequence. Our phylogenetic analysis implicates NusG-dependent pausing as a widespread mechanism in bacteria.
Assuntos
Bacillus subtilis/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Óperon/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Translocação Genética/genéticaRESUMO
Macrolide antibiotics bind to 23S rRNA within the peptide exit tunnel of the ribosome, causing the translating ribosome to stall when an appropriately positioned macrolide arrest motif is encountered in the nascent polypeptide. Tylosin is a macrolide antibiotic produced by Streptomyces fradiae Resistance to tylosin in S. fradiae is conferred by methylation of 23S rRNA by TlrD and RlmAII Here, we demonstrate that yxjB encodes RlmAII in Bacillus subtilis and that YxjB-specific methylation of 23S rRNA in the peptide exit tunnel confers tylosin resistance. Growth in the presence of subinhibitory concentrations of tylosin results in increased rRNA methylation and increased resistance. In the absence of tylosin, yxjB expression is repressed by transcription attenuation and translation attenuation mechanisms. Tylosin-dependent induction of yxjB expression relieves these two repression mechanisms. Induction requires tylosin-dependent ribosome stalling at an RYR arrest motif at the C terminus of a leader peptide encoded upstream of yxjB Furthermore, NusG-dependent RNA polymerase pausing between the leader peptide and yxjB coding sequences is essential for tylosin-dependent induction. Pausing synchronizes the position of RNA polymerase with ribosome position such that the stalled ribosome prevents transcription termination and formation of an RNA structure that sequesters the yxjB ribosome binding site. On the basis of our results, we are renaming yxjB as tlrBIMPORTANCE Antibiotic resistance is a growing health concern. Resistance mechanisms have evolved that provide bacteria with a growth advantage in their natural habitat such as the soil. We determined that B. subtilis, a Gram-positive soil organism, has a mechanism of resistance to tylosin, a macrolide antibiotic commonly used in the meat industry. Tylosin induces expression of yxjB, which encodes an enzyme that methylates 23S rRNA. YxjB-dependent methylation of 23S rRNA confers tylosin resistance. NusG-dependent RNA polymerase pausing and tylosin-dependent ribosome stalling induce yxjB expression, and hence tylosin resistance, by preventing transcription termination upstream of the yxjB coding sequence and by preventing repression of yxjB translation.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Fatores de Alongamento de Peptídeos/metabolismo , RNA Ribossômico 23S/genética , Ribossomos/metabolismo , Tilosina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Metilação , Regiões Promotoras Genéticas , Ligação Proteica , RNA Ribossômico 23S/química , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Difosfatos/metabolismo , Escherichia coli/enzimologia , RNA Bacteriano/genética , Fator sigma/metabolismo , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/genética , Fosforilação , Regiões Promotoras Genéticas , Fator sigma/genéticaRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/química , RNA/química , Elongação da Transcrição Genética , Pareamento de Bases , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/enzimologia , Movimento , Domínios Proteicos , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismoRESUMO
Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3'-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.
Assuntos
Dano ao DNA , DNA/química , DNA/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , DNA/metabolismo , Reparo do DNA , Replicação do DNA , Mutação , RNA Polimerase II/química , RNA Polimerase II/genéticaRESUMO
RNA nanoparticles consisting of multiple RNA strands of different sequences forming various three-dimensional structures emerge as promising carriers of siRNAs, RNA aptamers, and ribozymes. In vitro transcription of a mixture of dsDNA templates encoding all the subunits of the RNA nanoparticle may result in cotranscriptional self-assembly of the nanoparticle. Based on our experience with production of RNA nanorings, RNA nanocubes, and RNA three-way junctions, we propose a strategy for optimization of the cotranscriptional production of chemically modified ribonuclease-resistant RNA nanoparticles.
Assuntos
Nanopartículas , RNA/química , RNA/genética , Transcrição Gênica , Regiões 5' não Traduzidas , Sequência de Bases , Halogenação , MutaçãoRESUMO
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.
Assuntos
Proteínas de Bactérias , RNA Polimerases Dirigidas por DNA , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Holoenzimas/biossíntese , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.
Assuntos
Proteínas de Escherichia coli/genética , Genes Reporter/genética , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos , Regiões Promotoras Genéticas , RNA/biossíntese , RNA/genéticaRESUMO
Cells and viruses possess several known 'restart' pathways to overcome lesions during DNA replication. However, these 'bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.
Assuntos
Bacteriófago T7/genética , Dano ao DNA/genética , DNA Helicases/metabolismo , Replicação do DNA/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Dímeros de Pirimidina/metabolismo , Bacteriófago T7/metabolismo , Linhagem Celular , Escherichia coliRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Alostérica , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/enzimologia , Conformação Proteica , Espalhamento a Baixo Ângulo , Transcrição Gênica , Difração de Raios XRESUMO
BACKGROUND: Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. RESULTS: We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5' end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5' G-dC base pair and the 3' end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5' untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. CONCLUSIONS: In E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Translocação Genética , Ilhas de CpG , Replicação do DNA , DNA Antissenso/genética , DNA Antissenso/metabolismo , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biblioteca Gênica , Estudos de Associação Genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
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.