REGULATION OF PYRIMIDINE GENE EXPRESSION IN BACTERIA

本提案有三个目标。第一个是 继续努力确定和确定监管要素和特征 细胞中嘧啶基因表达调控机制的研究进展 大肠埃希菌。特别感兴趣的控制机制是(I) PyrBI启动子转录启动过程中的调控 UTP诱导的重复RNA合成，(II)PyrBI的衰减控制 对UTP敏感的转录暂停在pyrBI中的表达 前导区调控Rho非依赖性转录终止 在pyrB基因之前的衰减器，和(Iii)翻译控制 用CTP/GTP敏感的突变体筛选PYRC和PYRD的表达 转录起始点导致合成多个 不同翻译潜力的成绩单。具体目标 包括PYRBI启动子突变分析的特征 重复合成RNA所需的序列及其物理状态 RNA聚合酶在这一过程中。其他目标包括 识别引起核苷酸敏感的序列和结构 在pyrBI前导区转录暂停，分离和 改变UTP的顺式和反式作用突变的特征 对pyrBI基因表达的敏感调控，以及对该基因表达的研究 PyrC转录本的内切加工可能在肿瘤中的作用 PyrC基因表达的翻译调控。第二个目标是使用 作为探索基因基本步骤的工具的嘧啶基因 表情。PyrBI前导区域将用于检查各种 Rho非依赖性转录终止的几个方面。我们将研究 RNA终止子发夹的独特性，终止子的作用- 规定的长程尿苷残留量，可能的调节 UTP水平的终止活动，以及调节 终止符上的转录通读。在一个相关的实验中，我们 将检测DNA序列在模板和非模板中的影响 转录终止时Rho非依赖性终止子的链。 第三个目标是调查一些 其他基因由细胞内核苷酸池大小调节，使用 控制机制类似于对嘧啶基因所描述的机制， 也就是说，UTP诱导的重复RNA合成在 转录启动和翻译控制由 核苷酸敏感的选择性转录起始点的选择。 这些简单但强大的机制仅依赖于对 转录和翻译的基本要素。候选基因 包含可能的调控序列的还有其他的嘧啶基因 以及与核苷酸代谢有关的基因。这个项目的总体目标是 项目是提供关于控制机制的新信息 基因在细菌中的表达以及转录和转录的关键步骤 翻译。

There are three objectives in the present proposal. The first is to continue efforts to identify and characterize regulatory elements and mechanisms involved in controlling pyrimidine gene expression in Escherichia coli. Control mechanisms of particular interest are (i) regulation during transcriptional initiation at the pyrBI promoter through UTP-induced reiterative RNA synthesis, (ii) attenuation control of pyrBI expression in which UTP-sensitive transcriptional pausing in the pyrBI leader region regulates Rho-independent transcriptional termination at an attenuator preceding the pyrB gene, and (iii) translational control of pyrC and pyrD expression by CTP/GTP-sensitive selection of alternative transcriptional start sites that results in the synthesis of multiple transcripts with different potentials for translation. Specific aims include the characterization by mutational analysis of pyrBI promoter sequences required for reiterative RNA synthesis and the physical state of RNA polymerase during this process. Additional aims include the identification of sequences and structures that cause nucleotide-sensitive transcriptional pausing in the pyrBI leader region, the isolation and characterization of cis- and trans-acting mutations that alter UTP- sensitive regulation of pyrBI expression, and the investigation of the possible role of endonucleolytic processing of pyrC transcripts in translational control of pyrC expression. The second objective is to use the pyrimidine genes as tools to probe fundamental steps in gene expression. The pyrBI leader region will be used to examine various aspects of Rho-independent transcriptional termination. We will examine the uniqueness of RNA terminator hairpins, the role of terminator- specified long runs of uridine residues, possible regulation of termination activity by UTP levels, and upstream sequences that modulate transcriptional readthrough at terminators. In a related experiment, we will examine the effects of DNA sequence in the template and nontemplate strands of a Rho-independent terminator on transcriptional termination. The third objective is to investigate the possibility that a number of other genes are regulated by intracellular nucleotide pool sizes using control mechanisms similar to those described for the pyrimidine genes, namely, regulation by UTP-induced reiterative RNA synthesis during transcriptional initiation and translational control mediated by nucleotide-sensitive selection of alternative transcriptional start sites. These simple but powerful mechanisms rely only on the subtle modulation of the basic elements of transcription and translation. Candidate genes containing putative regulatory sequences include other pyrimidine genes and genes involved in nucleotide metabolism. The overall goal of this project is to provide new information about the mechanisms controlling gene expression in bacteria and about key steps in transcription and translation.