Genetic Regulation of the Threonine Operon of Escherichia Coli

遗传和生化研究表明，细菌和噬菌体 DNA 中的特定位点编码转录终止信号。 这些位点被发现位于基因末端、操纵子内的顺反子之间，甚至操纵子的基因编码区内。 转录终止子在基因表达中发挥重要的调节作用也已变得很清楚。 这项研究的一个目的是了解大肠杆菌苏氨酸 (thr) 操纵子衰减子位点转录终止所涉及的分子事件。 对转录终止所必需的衰减子的序列和结构特征进行系统分析。 本研究的第二个目标是检查 thr 调节区和衰减子的暂停位点的转录暂停。 标准足迹技术的变体将用于探测暂停复合物中的 RNA 和 DNA。 这项工作的第三个目标是检查野生型和突变型弱化子编码的二级 RNA 结构的稳定性。 有人提出，RNA 二级结构的稳定性对于转录终止很重要。 RNA 的分离量足以测量热变性曲线。 这将允许比较观察到的和计算出的 Tm 值。 还可以确定 RNA 二级结构的稳定性是否与终止效率成正比。 该提案的最后一个目标是分离和定位编码参与体内转录终止的蛋白质的基因突变。 将分离出增加 rho 独立终止子转录通读的突变体。 这些突变体的表征将提供有关哪些基因产物参与体内转录终止的信息。

Genetic and biochemical studies have shown that specific sites in bacterial and bacteriophage DNA encode signals for transcription termination. These sites have been found at the ends of genes, between cistrons within an operon and even within gene coding regions of operons. It has also become clear that transcription terminators play an important regulatory role in gene expression. One objective of this research is to gain an understanding of the molecular events involved in transcription termination at the attenuator site in the threonine (thr) operon of Escherichia coli. A systematic analysis will be undertaken of the sequence and structural features of the attenuator that are necessary for transcription termination. A second objective of this study is to examine transcriptional pausing at the pause site in the thr regulatory region and at the attenuator. Variations of the standard footprinting technique will be used to probe both RNA and DNA in paused complexes. A third objective of this work is to examine the stability of the secondary RNA structures encoded by wild-type and mutant attenuators. It has been proposed that the stability of the secondary RNA structure is important for transcription termination. RNA will be isolated in quantities sufficient to measure thermal denaturation profiles. This will permit comparison of observed and calculated Tm values. It will also be be possible to determine if the stability of secondary RNA structure is proportional to termination efficiency. The last objective of the proposal is to isolate and map mutations in genes that encode proteins that participate in transcription termination in vivo. Mutants will be isolated that increase transcriptional readthrough at rho-independent terminators. Characterization of these mutants will provide information on which gene products are involved in transcription termination in vivo.