GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

长期的目标是阐明一种 基本的基因调控过程，取决于增长率 基因表达的调控。大肠杆菌的两个基因 将研究己糖一磷酸分流的酶：GND， 编码6-磷酸葡萄糖酸脱氢酶(6PGD)和 ZWF，编码葡萄糖6-磷酸脱氢酶(G6PD)。 生长速率依赖的GND表达调控发生在 转录后水平，并涉及一个阴性部位 位于6PGD编码序列和 它与核糖体结合部位高度互补 (苏格兰皇家银行)。提出了一个模型，该模型表明，监管 涉及在mRNA中的互补碱基配对 内部同源序列(IHS)和RBS。要测试 一系列RBS和IHS脱氧寡核苷酸的模型 特定位置的一个或多个突变将被合成， 用一种新的方法克隆，并对单独突变的效果进行了研究 并将以组合的形式确定增长率 GND-LacZ蛋白融合的依赖调节。的水平 6PGD在沙门氏菌HIST突变株中不能诱导生长 TRNA修饰酶缺陷的小鼠 假尿苷合成酶I(PSUI)。遗传分析和体外实验 HIST结合A蛋白正向调节作用的实验研究 RBS-IHS内的推定PSUI识别站点 发夹和破坏结构的稳定。增长速度 与量、合成速率、泛函和 GND基因的化学半衰期及其利用 确定并检验了假设增长率 6PGD水平的依赖性调节受以下因素影响 在IHS的mRNA的内切核裂解。这个 生产过剩和生长速度变化的分子基础 顺式显性Zwf突变体中G6PD水平的依赖性 突变将通过克隆和DNA序列来确定 分析。这些数据和zwf-lac操纵子和 蛋白质融合应显示zwf和GND是否受调控 通过类似的机制。实现项目的具体目标 应该提供对不寻常现象的详细理解 调节GND表达的机制和已知的 非核糖体基因与生长速率相关的调控 这样细菌就会生长。

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.