GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

长期目标是阐明其机制 基本的遗传调控过程，生长速率依赖 基因表达的调控。 两个大肠杆菌基因 将研究己糖单磷酸分流器的酶：gnd， 编码 6 个磷酸葡萄糖酸脱氢酶 (6PGD) 和 zwf，编码葡萄糖 6-磷酸脱氢酶 (G6PD)。 gnd 表达的生长速率依赖性调节发生在 转录后水平并涉及负位点 控制位于 6PGD 的编码序列内 与核糖体结合位点高度互补 （苏格兰皇家银行）。 提出了一个模型，表明监管 涉及 mRNA 之间的互补碱基配对 内部同源序列 (IHS) 和 RBS。 测试 建立一系列 RBS 和 IHS 脱氧寡核苷酸模型，其中包含 将合成特定位点的一个或多个突变， 通过新方法克隆，以及单独突变的效果 并结合起来确定增长率 gnd-lacZ 蛋白融合的依赖性调节。 水平 6PGD 是沙门氏菌 hisT 突变体不可诱导的生长速率 tRNA 修饰酶有缺陷的鼠伤寒杆菌 假尿苷合酶 I (PSUI)。 遗传分析和体外 通过结合 hisT 来发挥积极调节作用的实验 假定的 PSUI 识别位点位于 RBS-IHS 内 发夹并破坏结构的稳定性。 增长率 数量、合成速率、功能和功能的依赖性 gnd mRNA 的化学半衰期和利用将是 确定并检验了假设的增长率 6PGD水平的依赖性调节受以下因素影响 IHS 处 mRNA 的核酸内切裂解。 的 生产过剩和生长速度改变的分子基础 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序列确定 分析。 这些数据和 zwf-lac 操纵子的特性和 蛋白质融合应显示 zwf 和 gnd 是否受到调节 通过类似的机制。 实现项目的具体目标 应提供对异常情况的详细了解 调节 gnd 表达的机制和知识 非核糖体基因的生长速率依赖性调节和 因此细菌生长。

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.