GROWTH RATE-DEPENDENT CONTROL OF GENE EXPRESSION

长期目标是阐明一种基本的 基因调控过程，生长速率依赖性基因调控 表情 两个大肠杆菌戊糖酶基因 将研究磷酸途径：gnd，编码6-磷酸葡萄糖酸 脱氢酶和编码葡萄糖6-磷酸脱氢酶的ZWF。 生长速率依赖性的gnd表达调控是在 翻译效率并涉及“内部互补序列” (ICS)“，编码序列内的阴性对照位点， 与核糖体结合位点（RBS）互补。 提供依据 由ICS组成的长距离mRNA二级结构的形成 并制备RBS、gnd-kan蛋白融合株， 突变体将被选择和表征;此外，一个专门的 核糖体系统将被用来证明形成的二级 结构和核糖体浓度在调节中的作用。 效果 生长率和调节突变对单链的影响 RBS和ICS将通过体内RNA的甲基化和随后的 通过转录扩增引物延伸（TAPE）进行分析， 为此目的而开发的。 噬菌体T7 RNA聚合酶的表达 系统将用于确定转录-翻译的作用 耦合在规则中。 gnd mRNA前导序列的其他突变 将被准备和表征，以确定为什么它的次要 结构似乎是正常表达水平所必需的， 增长率依赖性调节。 此外，遗传选择将进行 确定领导者的功能是否取决于一个约束因素， TAPE将对领导者进行结构-功能研究 法 zwf的生长速率依赖性调节机制 表达将通过选择和表征来确定， 调节突变体与zwf-kan蛋白融合菌株， 制备一组缺失和碱基置换突变， 上游调控区。 这些突变也将用于识别 超氧阴离子自由基诱导zwf表达顺式作用位点 根的 ppGpp在zwf生长速率调节中的潜在作用 将用允许表达水平的质粒菌株测定。 在一些实施方案中，所述突变体具有待改变的核苷酸和缺乏所述核苷酸的突变体。

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.