REGULATION OF PYRIMIDINE GENE EXPRESSION IN BACTERIA

描述：此应用程序的目标是阐明新的 细菌中基因调控的机制及其分子特征 监管过程中涉及的互动。在拟议的研究中 研究人员将试图识别大肠杆菌中的操纵子 在转录起始过程中受重复转录的调节。 这个反应涉及到特定核苷酸的重复加成。 (通常是U或A)到新成绩单的3‘端，因为滑动 在转录本中的同源多聚体序列和互补序列之间 DNA模板中的序列。由此产生的成绩单不太可能是 有效地扩展到包括下游序列，因此它们的合成 导致基因表达减少。此前，特恩博博士发现 几个受UTP依赖的重复序列调控的嘧啶操纵子 转录，即pyrBI、carAB、codBA和UPP操纵子，它们编码 嘧啶代谢酶。在这些示例中，用于 调节重复转录被发现有很大的不同。他 确定了一些具有启动子的其他操纵子，其序列包含 可能存在依赖于UTP的重复转录的位点。在《目标1》中，他将 确定重复转录是否发生在这些启动子和 它是否参与监管，这可能是由 细胞内UTP水平的变化。这些操纵子包括CyA (腺苷环化酶)、metY-NusA-InfB(tRNAfMet、NusA和IF2)、GAL (半乳糖分解代谢)、尿苷激酶(UDK)、三磷酸腺苷合成酶亚基(ATP)、 苯丙氨酰-tRNA合成酶(PheST)、嘌呤生物合成(PurMN)和PROS (脯氨酰-tRNA合成酶)。在目标2中，他将继续分析 依赖UTP的重复转录(及其相关基因调控) 使用pyrBI、carAB、codBA和UPP操纵子的启动子。他会的 确定所需的启动子序列，检查转录的影响 起始位置(相对于-10区域)，探索RNA的作用 转录本-DNA模板杂交的稳定性，并检测其参与 转录裂解(GRE)因子。此外，他还将决定 拴系新生幼体5‘端对重复转录的影响 转录到RNA聚合酶(RNAP)的活性部位，试图测量 重复转录之前和期间的RNAP足迹，以及 决定Sigma因子在重复转录过程中的命运。在AIM 3，他将研究PurHD(嘌呤生物合成)和glnLG(NtrB和NTRC 氮调节剂)操纵子参与涉及ATP依赖的调控 重复转录，因为它们的启动子被预测包含 适合该反应的序列。调查员将确定 重复转录是否发生在这些启动子上，并用于 嘌呤(ATP和/或GTP)介导的操纵子表达调控。在目标4中， 他将继续分离和鉴定改变 对PYRBI表达的UTP敏感调控。在顺式基因中起作用的突变可能 影响UTP依赖的重复转录所需的序列， 转录延长/暂停，或内在转录 终止。反式作用的突变可能会影响影响 上述活动、RNAP、翻译机构的组件、 以及控制翻译速度的要素。每个突变(和 受影响的基因产物)将被鉴定，并在pyrBI表达中的步骤 受突变的影响将被确定。结果应该会提供 关于转录周期中每个步骤的重要新信息和 也谈到了控制翻译的因素。在目标5中，他将决定 每种检测到的细胞内UTP浓度范围 PyrBI和carAB操纵子的控制机制和明显的 CodBA和UPP的单一、反复转录调控机制 歌剧。在目标6中，他将继续他们对内在的研究 通过检测突变的体内效应来终止转录 位于PyrBI衰减器所需的胸苷束中。这个 研究人员还将检查DNA序列(模板或 非模板链)和对特定类型的RNA的要求 发夹在固有终止中。总而言之，这些研究应该 提供一般类型的基因调控的新实例，并进一步定义 基因表达的关键步骤。尽管这些研究是与E. 这项工作很可能适用于基因表达的研究。 以及在所有细菌中的调控，可能在真核生物中也是如此。

DESCRIPTION: The objective of this application is to elucidate new mechanisms of gene regulation in bacteria and to characterize the molecular interactions involved in the regulatory process. In the proposed research the investigator will attempt to identify operons in E. coli that are regulated by reiterative transcription during transcriptional initiation. This reaction involves the repetitive addition of a particular nucleotide (usually U or A) to the 3' end of the nascent transcript due to slippage between a homopolymeric sequence in the transcript and a complementary sequence in the DNA template. The resulting transcripts are unlikely to be productively extended to include downstream sequences, thus their synthesis results in reduced gene expression. Previously, Dr. Turnbough identified several pyrimidine operons that are regulated by UTP-dependent reiterative transcription, namely the pyrBI, carAB, codBA and upp operons, which encode pyrimidine metabolic enzymes. In these examples the mechanism employed to modulate reiterative transcription were found to be quite different. He identified a number of other operons with promoters whose sequences contain likely sites for UTP-dependent reiterative transcription. In Aim 1, he will determine whether reiterative transcription occurs at these promoters and whether it is involved in regulation, which presumably is mediated by changes in the intracellular levels of UTP. These operons include cya (adenylate cyclase), metY-nusA-infB (tRNAfMet, NusA, and IF2), gal (galactose catabolism), udk (uridine kinase), atp (ATP synthetase subunits), pheST (phenylalanyl-tRNA synthetase), purMN (purine biosynthesis), and proS (prolyl-tRNA synthetase). In Aim 2, he will continue the analysis of UTP-dependent reiterative transcription (and associated gene regulation) using the promoters of the pyrBI, carAB, codBA, and upp operons. He will identify required promoter sequences, examine the effects of transcriptional start site position (relative to the -10 region), explore the role of RNA transcript-DNA template hybrid stability, and examine the involvement of transcript cleavage (Gre) factors. In addition, he will determine the effect on reiterative transcription of tethering the 5' end of the nascent transcript to the active site of RNA polymerase (RNAP), attempt to measure the RNAP footprint before and during reiterative transcription, and determine the fate of sigma factor during reiterative transcription. In Aim 3, he will examine the purHD (purine biosynthesis) and glnLG (NtrB and NtrC nitrogen regulators) operons for regulation involving ATP-dependent reiterative transcription since their promoters are predicted to contain sequences appropriate for this reaction. The investigator will determine whether reiterative transcription occurs at these promoters and is used for purine (ATP and/or GTP)-mediated regulation of operon expression. In Aim 4, he will continue to isolate and characterize mutations that alter UTP-sensitive regulation of pyrBI expression. Mutations that act in cis may affect sequences required for UTP-dependent reiterative transcription, transcriptional elongation/pausing, or intrinsic transcriptional termination. Mutations that act in trans may affect factors that influence the foregoing activities, RNAP, components of the translational machinery, and elements that control the rate of translation. Each mutation (and affected gene product) will be identified, and the step in pyrBI expression affected by the mutation will be determined. The results should provide important new information about each step in the transcription cycle and also about factors controlling translation. In Aim 5, he will determine the ranges of intracellular UTP concentrations detected by each of the multiple control mechanisms of the pyrBI and carAB operons and by the apparently single, reiterative transcription control mechanisms of the codBA and upp operons. In Aim 6, he will continue their studies on intrinsic transcriptional termination by examining the in vivo effects of mutations located in the required thymidine tract of the pyrBI attenuator. The investigator will also examine the role of DNA sequence (template or nontemplate strand) and the requirements for particular types of RNA hairpins in intrinsic termination. Taken together, these studies should provide new examples of general types of gene regulation and further define critical steps in gene expression. Although these studies are done with E. coli, the work is likely to be applicable to the study of gene expression and regulation in all bacteria and probably in eukaryotes, as well.