GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

基因表达的生长速率依赖性控制

• 批准号: 3274531
• 负责人: Richard E Wolf
• 金额: $ 19.89万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274531
• 关键词: Escherichia coli; Salmonella typhimurium; bacterial DNA; bacterial genetics; bacterial proteins; bacterial virus; binding proteins; cell growth regulation; chemical binding; enzyme induction /repression; enzyme substrate complex; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic promoter element; genetic recombination; genetic strain; genetic translation; glucose 6 phosphate dehydrogenase; messenger RNA; microorganism culture; microorganism growth; microorganism metabolism; molecular cloning; mutant; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phosphogluconate dehydrogenase; plasmids; protein biosynthesis; regulatory gene; structural genes

The long term objective is to elucidate the mechanisms of a fundamental genetic regulatory process, growth rate dependent regulation of gene expression. Two Escherichia coli genes for enzymes of the hexose monophosphate shunt will be studied: gnd, which encodes 6 phosphogluconate dehydrogenase (6PGD) and zwf, which encodes glucose 6-phosphate dehydrogenase (G6PD). Growth rate dependent regulation of gnd expression occurs at the posttranscriptional level and involves a site of negative control which lies within the coding sequence for 6PGD and which is highly complementary to the ribosome binding site (RBS). A model is proposed which suggests that the regulation involves complementary base pairing in the mRNA between the internal homology sequence (IHS) and the RBS. To test the model a series of RBS and IHS deoxyoligonucleotides containing one or more mutations at specific sites will be synthesized, cloned by a new method, and the effect of the mutations singly and in combinations will be determined for the growth rate dependent regulation of gnd-lacZ protein fusions. The level of 6PGD is growth rate uninducible in hisT mutants of Salmonella typhimurium which are defective in the tRNA modifying enzyme pseudouridine synthase I (PSUI). Genetic analysis and in vitro experiments with positive regulatory role of hisT by binding to a putative PSUI recognition site which is within the RBS-IHS hairpin and destabilizing the structure. The growth rate dependence of the amount, synthesis rate, functional and chemical half-life and utilization of gnd mRNA will be determined and the hypothesis tested that the growth rate dependent regulation of 6PGD level is effected by endonucleolytic cleavage of the MRNA at the IHS. The molecular basis for the overproduction and altered growth rate dependence of G6PD level in mutants with cis-dominant zwf mutations will be determined by cloning and DNA sequence analysis. These data and the properties of zwf-lac operon and protein fusions should show whether zwf and gnd are regulated by similar mechanisms. Achieving the project's specific aims should provide a detailed understanding of the unusual mechanism that regulates gnd expression and in knowledge of growth rate dependent regulation of non-ribosomal genes and thus bacterial growth.

长期目标是阐明 基本遗传调节过程，增长率依赖性 基因表达的调节。 两个大肠杆菌基因 将研究己糖单磷酸分流的酶：GND， 编码6个磷酸葡萄糖酸脱氢酶（6pgd）和 ZWF，编码6-磷酸葡萄糖脱氢酶（G6PD）。 GND表达的增长率依赖性调节发生在 转录后级别，涉及一个负面的站点 控制在6pgd和6pgd的编码顺序中 这是与核糖体结合位点高度互补的 （RB）。 提出了一个模型，该模型表明该法规 涉及在mrna中的互补碱基配对 内部同源序列（IHS）和RB。 测试 模拟一系列含有含有的RB和IHS脱氧乙烯核苷酸 在特定地点的一个或多个突变将合成， 由新方法克隆，突变的效果单独克隆 并将确定增长率的组合 GND-LACZ蛋白融合的依赖调节。 水平 6PGD是沙门氏菌的HAST突变体中不可诱导的生长速率 在tRNA修饰酶中有缺陷的鼠伤寒 假氨酸合酶I（PSUI）。 遗传分析和体外 通过与A结合，具有阳性调节作用的实验 推定的PSUI识别网站，该网站位于RBS-IHS中 发夹和破坏结构的稳定。 增长率 量的依赖性，合成率，功能和 化学半衰期和GND mRNA的利用率将是 确定并假设测试了增长率 6pgd水平的依赖调节受 IHS mRNA的内核裂解。 这 分子基础过度生产和生长速率改变 G6PD水平在顺式ZWF的突变体中的依赖性 突变将通过克隆和DNA序列确定 分析。 这些数据以及ZWF-LAC操纵子和 蛋白质融合应显示ZWF和GND是否受调节 通过类似的机制。 实现该项目的具体目标 应该对异常的详细理解 调节GND表达和了解的机制 非核糖体基因的增长率依赖性调节 因此细菌生长。