GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

基因表达的生长速率依赖性控制

• 批准号: 3274528
• 负责人: Richard E Wolf
• 金额: $ 23.31万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274528
• 关键词: DNA footprinting; Escherichia coli; RNA biosynthesis; RNA methylation; Salmonella typhimurium; bacterial DNA; bacterial genetics; bacterial proteins; bacteriophage T7; binding proteins; cell growth regulation; chimeric proteins; enzyme induction /repression; gene expression; genetic manipulation; genetic mapping; genetic promoter element; genetic translation; glucose 6 phosphate dehydrogenase; messenger RNA; microorganism culture; microorganism growth; molecular cloning; mutant; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phosphogluconate dehydrogenase; posttranscriptional RNA processing; protein biosynthesis; protein structure function; regulatory gene; ribosomes; structural genes; superoxides

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 pentose phosphate pathway will be studied: gnd, which encodes 6-phosphogluconate dehydrogenase, and zwf, which encodes glucose 6-phosphate dehydrogenase. Growth rate dependent regulation of gnd expression is at the level of translational efficiency and involves the "internal complementary sequence (ICS)", a negative control site within the coding sequence that is highly complementary to the ribosome binding site (RBS). To provide evidence for the formation of a long-range mRNA secondary structure composed of the ICS and the RBS, gnd-kan protein fusion strains will be prepared and regulatory mutants will be selected and characterized; in addition, a dedicated ribosome system will be used to demonstrate formation of the secondary structure and the role of ribosome concentration in regulation. The effect of growth rate and regulatory mutations on the single-strandedness of the RBS and ICS will be determined by methylation of in vivo RNA and subsequent analysis by transcription-amplified primer extension (TAPE), a method to be developed for this for this purpose. A phage T7 RNA polymerase expression system will be used to determine the role of transcription-translation coupling in the regulation. Additional mutations of the gnd mRNA leader will be prepared and characterized, in order to determine why its secondary structure appears to be required for the normal level of expression and for growth rate dependent regulation. Also, genetic selections will be carried out to determine whether leader function depends on a binding factor, and structure-function studies of the leader will be conducted by the TAPE method. The mechanism for growth rate dependent regulation of zwf expression will be determined by the selection and characterization of regulatory mutants with zwf-kan protein fusion strains and by the preparation of a set of deletion and base-substitution mutations in the upstream regulatory region. The mutations will also be used to identify the cis-acting site for induction of zwf expression by superoxide free radicals. The potential role of ppGpp in growth rate regulation of zwf will be determined with a plasmid strain that allows the level of the nucleotide to be varied and with a mutant that is devoid of the nucleotide.

长期目标是阐明基本的机制 遗传调节过程，基因的增长率依赖性调节 表达。 两个用于戊糖酶的大肠杆菌基因 将研究磷酸盐途径：GND，编码6-磷酸葡萄糖酸盐 脱氢酶和ZWF，编码6-磷酸葡萄糖脱氢酶。 GND表达的增长率依赖性调节处于 翻译效率，并涉及“内部互补序列 （ICS）”，编码顺序中的负面对照站点 补充核糖体结合位点（RBS）。 提供证据 由ICS组成的远程mRNA二级结构的形成 RBS，将准备GND-KAN蛋白融合菌株并进行调节 突变体将被选择和表征；此外，一个专门的 核糖体系统将用于演示次级的形成 核糖体浓度在调节中的结构和作用。 效果 关于单链的生长速率和调节突变 RB和ICS将通过体内RNA的甲基化和随后的甲基化确定 通过转录放大引物扩展（磁带）分析，一种方法 为此目的为此开发。 噬菌体T7 RNA聚合酶表达 系统将用于确定转录翻译的作用 该法规中的耦合。 GND mRNA领导者的其他突变 将被准备和表征，以确定为什么其次要 正常表达水平和 增长率依赖性调节。 另外，将携带遗传选择 确定领导者功能是否取决于结合因子，并且 领导者的结构功能研究将由磁带进行 方法。 ZWF的增长率依赖性调节机制 表达将取决于选择和表征 具有ZWF-KAN蛋白融合菌株的调节突变体以及 准备一组删除和基础替代突变 上游监管区域。 该突变也将用于识别 通过超氧化物诱导ZWF表达的顺式作用位点 激进分子。 PPGPP在ZWF增长率调节中的潜在作用 将用质粒菌株确定 核苷酸要变化，并带有缺乏核苷酸的突变体。