Bacterial gene regulation by the NTP substrates of transcription initiation

描述（由申请人提供）：自从发现抑制蛋白和激活蛋白以来，对细菌基因调控的认识发生了巨大变化。我们现在知道，基因调控还涉及许多不同类型的转录和翻译控制机制，这些机制在没有调节蛋白参与的情况下感知小分子细胞效应物的水平。在许多情况下，效应物是转录的三磷酸核苷（NTP）底物，它们的浓度在转录起始和延伸期间被RNA聚合酶直接感知。本研究的主要目的是阐明新的和进一步表征已知的细菌基因调控机制，包括起始和/或转录起始位点转换过程中ntp敏感的重复转录。这些反应产生具有不同5′端序列和不同翻译潜力的替代转录本。重复转录是由于新生转录物与其模板之间的滑动而导致核苷酸的重复添加。起始位点切换是在同一启动子内选择转录起始的替代位点。拟议的研究将集中在大肠杆菌操纵子编码参与嘧啶生物合成或回收的蛋白质。我们的第一个目标是确定嘧啶介导的pyrG表达调节的机制。pyrG基因编码CTP合成酶，该合成酶催化嘧啶核苷酸生物合成途径的最后一步。初步研究表明，起始核苷酸CTP水平通过限制CTP依赖的重复转录和转录起始位点切换的机制控制pyrG的表达。第二个目标是继续我们对重复转录或起始位点切换所需的启动子序列的表征，这些启动子序列决定了所产生的转录本的各种命运。获得的结果将使我们能够预测和操纵重复转录和起始位点切换的不同机制。我们的第三个目标是表征突变RNA聚合酶选择有缺陷的重复转录在体内。分析将包括含有非活性位点rpoC突变的RNA聚合酶，该突变在起始（不一定直接）抑制非生产性重复转录。此外，该分析将包括含有活性位点突变的RNA聚合酶突变体，这些突变体因其在伸长过程中促进或抑制重复转录的能力而被选中。最终目的是在RNA聚合酶中绘制由重复转录产生的转录本的路径。我们将检查转录本，不能有效地延长以及转录本，可以切换到正常的延伸模式。最后两个目标的结果应该为RNA聚合酶的机制提供新的信息。从我们的研究中获得的知识应该可以更好地预测基因组序列的基因调节机制，为生物技术的目的操纵细菌，设计治疗细菌性疾病的新疗法，以及更好地了解所有细胞的内部工作。

DESCRIPTION (provided by applicant): The perception of bacterial gene regulation has changed dramatically since the discoveries of repressor and activator proteins. We now know that gene regulation also involves many different types of transcriptional and translational control mechanisms that sense the level of small-molecule cellular effectors without the involvement of regulatory proteins. In many cases, the effectors are the nucleoside triphosphate (NTP) substrates of transcription, and their concentrations are sensed directly by RNA polymerase during transcription initiation and elongation. The principal objective of this study is to elucidate new and further characterize known mechanisms of bacterial gene regulation involving NTP-sensitive reiterative transcription during initiation and/or transcription start-site switching. These reactions produce alternative transcripts with different 5'-end sequences and different potentials for translation. Reiterative transcription is the repetitive addition of a nucleotide due to slippage between a nascent transcript and its template. Start-site switching is the selection of alternate sites of transcription initiation within the same promoter. The proposed studies will focus on Escherichia coli operons that encode proteins involved in pyrimidine biosynthesis or salvage. Our first aim is to define the mechanism of pyrimidine-mediated regulation of pyrG expression. The pyrG gene encodes CTP synthetase, which catalyzes the final step of the pyrimidine nucleotide biosynthetic pathway. Preliminary studies indicate that the levels of the initiating nucleotide CTP control pyrG expression through a mechanism involving limited CTP-dependent reiterative transcription and transcription start-site switching. The second aim is to continue our characterization of promoter sequences required for reiterative transcription or start-site switching and that dictate the various fates of the resulting transcripts. The results obtained will allow us to predict and manipulate the distinct mechanisms of reiterative transcription and start-site switching. Our third aim is to characterize mutant RNA polymerases selected for defective reiterative transcription in vivo. The analysis will include RNA polymerases containing non-active site rpoC mutations that inhibit non- productive reiterative transcription during initiation (not necessarily directly). Additionally, this analysis will include RNA polymerase mutants containing active site mutations selected for their abilities to either promote or inhibit reiterative transcription during elongation. The final aim is to map within RNA polymerase the path followed by transcripts produced by reiterative transcription. We will examine transcripts that cannot be productively elongated as well as transcripts that can switch to the normal elongation mode. The results from the last two aims should provide new information regarding the mechanics of RNA polymerase. The knowledge gained from our research should allow better predictions of mechanisms of gene regulation from genomic sequences, manipulation of bacteria for the purposes of biotechnology, the design of new therapies for the treatment of bacterial diseases, and a better understanding of the inner workings of all cells. PUBLIC HEALTH RELEVANCE: Bacterial gene regulation involves many different types of transcriptional and translational control mechanisms, including those in which small-molecule cellular effectors are sensed without the involvement of regulatory proteins. In this study, we will elucidate and characterize regulatory mechanisms in which effectors are the nucleoside triphosphate substrates of transcription, and their concentrations are sensed directly by RNA polymerase. Defining these mechanisms in molecular detail enhances our understanding of the inner workings of bacteria and increases our ability to control bacterial pathogens and to create new tools for biotechnology.