GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

我们的长期目标是阐明一种基本的 基因调控过程，依赖生长速率的基因调控 表情。大肠杆菌戊糖酶的两个基因 将研究磷酸途径：编码6-磷酸葡萄糖酸的GND 脱氢酶和编码葡萄糖6-磷酸脱氢酶的zwf。 生长速率依赖的GND表达调控处于 翻译效率与内部互补序列的关系 (ICS)“，编码序列中的一个负控制位点，高度 与核糖体结合位点(RBS)互补。为…提供证据 由ICS组成的长程mRNA二级结构的形成 并将制备RBS、GND-Kan融合蛋白菌株并进行调控 将选择和鉴定突变体；此外，还将有一个专门的 将使用核糖体系统来演示次生核糖体的形成 结构和核糖体浓度在调节中的作用。其效果 生长速度和调控突变对细胞单链的影响 RBS和ICS将通过体内RNA的甲基化和随后的 用转录扩增引物延伸(TAPE)进行分析，方法是 为此目的而开发的。噬菌体T7 RNA聚合酶的表达 系统将用来确定转录-翻译的作用 调整中的耦合。GND信使核糖核酸前导序列的额外突变 将被准备和表征，以确定为什么它的次要 结构似乎对于正常的表达水平和 依赖增长率的调控。此外，还将进行遗传选择 Out以确定前导函数是否取决于绑定因子，以及 带子的结构-功能研究将通过带子进行 方法。ZWF生长速率依赖性调控机制的研究 表达将由以下各项的选择和特性决定 ZWF-Kan蛋白融合菌株的调控突变体 一组缺失突变和碱基替换突变的制备 上游监管区域。突变也将被用来识别 超氧阴离子自由基诱导zwf表达的顺式作用部位 激进分子。PpGpp在ZWF生长速度调控中的潜在作用 将由一种质粒菌株来确定，该质粒菌株允许 核苷酸是不同的，并且与没有核苷酸的突变体在一起。

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.