REGULATION OF PYRIMIDINE GENE EXPRESSION IN BACTERIA

本建议有三个目标。一是 继续努力确定和描述监管要素， 参与控制嘧啶基因表达的机制 大肠埃希菌特别令人感兴趣的控制机制是： pyrBI启动子转录起始期间的调控， UTP诱导的逆转录RNA合成，（ii）pyrBI的衰减控制 表达，其中在pyrBI中UTP敏感的转录暂停 前导区调节Rho非依赖性转录终止， pyrB基因前的衰减子，和（iii）pyrB基因前的衰减子的翻译控制， pyrC和pyrD通过CTP/GTP敏感性选择的替代表达 导致多种合成的转录起始位点 具有不同翻译潜力的抄本。具体目标 包括通过pyrBI启动子的突变分析的表征 序列所需的重复RNA合成和物理状态 RNA聚合酶在这个过程中。其他目标包括 引起核苷酸敏感性的序列和结构的鉴定 pyrBI前导区中的转录暂停，分离和 表征改变UTP的顺式和反式作用突变- pyrBI表达的敏感调节，以及对pyrBI表达的研究。 pyrC转录物的核酸内切加工在 pyrC表达的翻译控制。第二个目标是使用 嘧啶基因作为探索基因中基本步骤的工具 表情pyrBI前导区将用于检查各种 Rho非依赖性转录终止的方面。我们将研究 RNA终止子发夹的独特性，终止子的作用， 尿苷残留的特定长期运行， 通过UTP水平的终止活性，和调节的上游序列 终止子处的转录通读。在一个相关的实验中，我们 将检查模板和非模板中DNA序列的影响 Rho-非依赖性终止子链对转录终止的影响。 第三个目标是调查一些 其它基因由细胞内核苷酸库大小调节， 控制机制类似于嘧啶基因的控制机制， 也就是说，通过UTP诱导的转录RNA合成的调节， 转录起始和翻译控制 替代转录起始位点的核苷酸敏感性选择。 这些简单但强大的机制仅仅依赖于 转录和翻译的基本要素。候选基因 含有推定的调节序列的基因包括其它嘧啶基因 和参与核苷酸代谢的基因。这个项目的总体目标是 该项目旨在提供有关控制机制的新信息， 细菌中的基因表达以及转录和 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.