Regulation of transcription elongation

项目摘要 RNA聚合酶(RNAP)的暂停和终止是ALL基因表达的重要组成部分 有机体。NusA和NusG是两种常见的转录延伸因子，能够刺激 细菌中的停顿和终止。暂停允许RNAP位置与RNA折叠和/或同步 调节因子结合。固有终止和Rho依赖终止是两种转录终止 在细菌中发现的机制。规范的内在终止子由不间断的RNA发夹组成 然后是U形曲线。尽管努沙已知在体外刺激内源性终止，因为努沙是 对于存活至关重要，直到最近才知道它在体内终止过程中的作用。RNA-Seq研究与B。 枯草杆菌Nusa枯竭菌株鉴定出一类需要NusA的内在终止子。努萨依赖 终结者具有较弱的RNA发夹和/或较差的U-束。也有证据表明，NusG刺激 分枝杆菌RNAP在体外不理想的内在终止子上的终止。在依赖于RHO终止中， 当Rho赶上暂停的RNAP时，它会促进文字记录的发布。E.coliNusG参与了一些Rho- 通过充当RNAP和RHO之间的桥梁来依赖终止事件。 NusA和NusG协同刺激枯草杆菌色氨酸操纵子5‘非编码区的两个位点的暂停。 NusG与暂停的DNA中的非模板DNA(NtDNA)链进行序列特异性接触 转录泡泡。因为RNAP和模板DNA必须彼此相对移动才能延伸到 恢复时，NusG与这两个组分的相互作用抑制伸长。两端富含T的ntDNA序列 暂停位点构成了一个保守的NusG识别基序。Net-seq将用于标识暂停站点 在对NusA和/或NusG有反应的整个枯草杆菌基因组中。耗尽努萨和删除的能力 没有生长缺陷的nusG使枯草芽孢杆菌成为这些研究的理想生物。通过结合RNase 使用net-seq(rnet-seq)，NusA和NusG对RNAP易位状态的影响将是 在每个停顿地点确定。同样，对NusA、NusG和NusG影响的全面基因组分析 在枯草杆菌中终止时的Rho将使用包含NusA耗竭的所有组合的菌株来执行， nusG和Rho等位基因。然后，将在体外对调节暂停位点和终止子的子集进行表征。 转录衰减机制的一个特点是存在重叠的反终止子和 5‘非编码区中形成的终止子结构。枯草杆菌yxjB的5‘非编码区包含两个这样的集合 重叠的结构。将测试一个模型，其中YxjB通过与其绑定来自动调节其表达 5‘非编码区，并通过防止两个反终止子的形成来促进在两个终止子的终止。这个 模型还假设下游的终止子发夹隔离了yxjB核糖体结合位点。因此，这一点 发夹将抑制未能终止的文字记录的翻译。体内表达的组合，在 体外转录和体外结合研究将用于阐明这些复杂的调控机制。

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.