Regulation of transcription elongation

转录延伸的调控

• 批准号: 9236602
• 负责人: PAUL L BABITZKE
• 金额: $ 33.94万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9236602
• 关键词: 5' Untranslated Regions; Affect; Alleles; Bacillus subtilis; Bacteria; Binding; Binding Sites; Biological Models; Cell physiology; Complex; DNA; DNA Sequence; DNA-Directed RNA Polymerase; Defect; Escherichia coli; Event; Exhibits; Gene Components; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Growth; HIV; Health; Human; In Vitro; Length; Life; Malignant Neoplasms; Methods; Methyltransferase; Modeling; Oncogenes; Operon; Organism; Positioning Attribute; Process; Proteins; Publishing; Quality Control; RNA; RNA Folding; Regulation; Reporting; Repression; Ribonucleases; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Sampling; Signal Transduction; Site; Structure; Testing; Transcript; Transcription Elongation; Transcriptional Elongation Factors; Transcriptional Regulation; Translations; attenuation; flexibility; genetic regulatory protein; genome-wide; human disease; improved; in vivo; insight; mycobacterial; prevent; response; rho; transcription factor; transcription termination; transcriptome sequencing; transcriptomics

Project Summary RNA polymerase (RNAP) pausing and termination are important components of gene expression in all organisms. NusA and NusG are two general transcription elongation factors that are capable of stimulating pausing and termination in bacteria. Pausing allows synchronization RNAP position with RNA folding and/or regulatory factor binding. Intrinsic and Rho-dependent termination are two transcription termination mechanisms identified in bacteria. Canonical intrinsic terminators consist of an uninterrupted RNA hairpin followed by a U-tract. Although NusA was known to stimulate intrinsic termination in vitro, since NusA is essential for viability its role on termination in vivo was not known until recently. RNA-seq studies with a B. subtilis NusA depletion strain identified a class of intrinsic terminator that requires NusA. NusA-dependent terminators have weak RNA hairpins and/or poor U-tracts. There is also evidence that NusG stimulates termination of mycobacterial RNAP at suboptimal intrinsic terminators in vitro. In Rho-dependent termination, Rho promotes transcript release when it catches up to paused RNAP. E. coli NusG participates in some Rho- dependent termination events by serving as a bridge between RNAP and Rho. NusA and NusG cooperatively stimulate pausing at two sites in the 5'UTR of the B. subtilis trp operon. NusG makes sequence-specific contacts with the non-template DNA (ntDNA) strand within the paused transcription bubble. As RNAP and template DNA must move with respect to one another for elongation to resume, interaction of NusG with both components inhibits elongation. The T-rich ntDNA sequence at the two pause sites constitutes a conserved NusG recognition motif. NET-seq will be used to identify pause sites throughout the B. subtilis genome that respond to NusA and/or NusG. The ability to deplete NusA and delete nusG without growth defects makes B. subtilis the ideal organism for these studies. By combining RNase footprinting with NET-seq (RNET-seq), the effect of NusA and NusG on the translocation state of RNAP will be determined at each pause site. Similarly, a comprehensive genomic analysis of the effects of NusA, NusG and Rho on termination in B. subtilis will be performed using strains containing all combinations of NusA depletion, nusG and rho alleles. A subset of regulatory pause sites and terminators will then be characterized in vitro. A hallmark of transcription attenuation mechanisms is the presence of overlapping antiterminator and terminator structures that form in the 5'UTR. The 5'UTR of B. subtilis yxjB contains two such sets of overlapping structures. A model will be tested in which YxjB autoregulates its expression by binding to its 5'UTR and promoting termination at both terminators by preventing formation of the two antiterminators. The model also posits that the downstream terminator hairpin sequesters the yxjB ribosome binding site. Thus, this hairpin would repress translation of transcripts that fail to terminate. A combination of in vivo expression, in vitro transcription and in vitro binding studies will be used to elucidate these complex regulatory mechanisms.

项目摘要 RNA聚合酶（RNAP）的暂停和终止是所有基因表达的重要组成部分。 有机体NusA和NusG是两种通用的转录延伸因子，其能够刺激转录因子的转录。 在细菌中暂停和终止。暂停允许RNAP位置与RNA折叠和/或 调节因子结合。内源性终止和Rho依赖性终止是两种转录终止 在细菌中发现的机制。典型的内在终止子由不间断的RNA发夹组成 然后是U形管尽管已知NusA在体外刺激内在终止，但由于NusA是 对于生存力至关重要，直到最近才知道其在体内终止中的作用。RNA-seq研究使用B。 枯草杆菌NusA耗竭菌株鉴定了一类需要NusA的内在终止子。NusA依赖性 终止子具有弱的RNA发夹和/或差的U-束。也有证据表明，NusG刺激 在体外，分枝杆菌RNAP在次优的内在终止子处终止。在Rho依赖性终止中， Rho在赶上暂停的RNAP时促进转录本释放。e. coli NusG参与了一些Rho- 通过充当RNAP和Rho之间的桥梁来实现依赖终止事件。 NusA和NusG协同刺激B的5 'UTR中两个位点的暂停。枯草杆菌色氨酸操纵子 NusG与暂停序列内的非模板DNA（ntDNA）链进行序列特异性接触， 转录气泡。由于RNAP和模板DNA必须相对于彼此移动以进行延伸， 重新开始，NusG与这两种组分的相互作用抑制伸长。富含T的ntDNA序列在两个 暂停位点构成保守的NusG识别基序。NET-seq将用于识别暂停位点 整个B。枯草芽孢杆菌基因组的NusA和/或NusG。能够耗尽NusA并删除 无生长缺陷的nusG形成B。枯草芽孢杆菌是这些研究的理想微生物。通过结合RNase 使用NET-seq足迹法（RNET-seq），NusA和NusG对RNAP的易位状态的影响将是 在每个暂停点确定。类似地，对NusA、NusG和NusB的作用的全面基因组分析显示， 在B中终止时的Rho。将使用含有NusA耗竭的所有组合的菌株进行枯草芽孢杆菌的筛选， nusG和rho等位基因。然后将在体外表征调节暂停位点和终止子的子集。 转录减弱机制的标志是存在重叠的抗终止子和 在5'UTR中形成的终止子结构。B的5 'UTR。subtilis yxjB包含两组这样的 重叠结构。将测试一种模型，其中YxjB通过结合其受体而自动调节其表达。 5'UTR，并通过阻止两个反终止子的形成来促进两个终止子处的终止。的 模型还假定下游终止子发夹螯合yxjB核糖体结合位点。因此，这 发夹会抑制不能终止转录本的翻译。体内表达、体内表达和体内表达的组合， 体外转录和体外结合研究将用于阐明这些复杂的调节机制。