GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

长期的目标是阐明一个 基本遗传调节过程，生长速度依赖 基因表达的调控。 两个大肠杆菌基因， 将研究单磷酸己糖支路的酶：GND， 其编码6磷酸葡萄糖酸脱氢酶（6PGD）， zwf，其编码葡萄糖6-磷酸脱氢酶（G6 PD）。 gnd表达的生长速率依赖性调节发生在 转录后水平，并涉及一个网站的负 位于6PGD的编码序列内的控制， 与核糖体结合位点高度互补 （RBS）. 提出了一个模型，该模型表明， 在mRNA中的互补碱基配对， 内部同源序列（IHS）和RBS。 测试 模拟一系列RBS和IHS脱氧寡核苷酸， 将合成特定位点的一个或多个突变， 用一种新的方法克隆， 并在组合中将被确定为生长速率 gnd-lacZ蛋白融合的依赖性调节。 水平 6PGD在沙门氏菌hisT突变体中是生长速率不可诱导的 缺乏tRNA修饰酶的鼠伤寒杆菌 假尿苷合酶I（PSUI）。 遗传分析和体外 hisT通过与一种 RBS-IHS内的推定PSUI识别位点 发夹和结构不稳定。 增速 量、合成速率、功能和 gnd mRNA的化学半衰期和利用率将 确定和假设检验，增长率 6PGD水平的依赖性调节受到以下因素的影响： 在IHS处的mRNA的内切核酸裂解。 的 生产过剩和增长率改变的分子基础 顺式显性zwf突变体中G6 PD水平的依赖性 突变将通过克隆和DNA测序来确定 分析. 这些数据与zwf-lac操纵子和 蛋白质融合应显示zwf和gnd是否受到调节 类似的机制。 实现项目的具体目标 应该能详细了解 调节gnd表达的机制， 非核糖体基因的生长速率依赖性调节， 从而细菌生长。

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.