Genetic Regulation of the Threonine Operon of Escherichia Coli

遗传和生物化学研究表明， 细菌和噬菌体DNA编码转录信号 终止 这些位点位于基因的末端， 操纵子内的顺反子甚至基因编码 操纵子的区域。 很明显，转录 终止子在基因表达中起着重要的调节作用。 本研究的目的之一是了解 参与转录终止的分子事件 大肠杆菌苏氨酸操纵子中的衰减子位点 杆菌 将对该序列进行系统分析 以及衰减器的结构特征， 转录终止 本研究的第二个目的是 为了检查在THR中暂停位点的转录暂停， 调节区和衰减区。 的变化 标准足迹技术将用于探测两种RNA 和DNA在暂停的复合物中。 这项工作的第三个目标是 检测二级RNA结构的稳定性， 通过野生型和突变型衰减剂。 已经提出 二级RNA结构的稳定性对于 转录终止 RNA将被大量分离 足以测量热变性曲线。 这将 允许比较观察到的和计算的Tm值。 它将 也可以确定次级的稳定性 RNA结构与终止效率成正比。 的 该提案的最后一个目标是分离和绘制 编码参与转录的蛋白质的基因 体内终止。 突变体将被分离出来， 在Rho非依赖性终止子处的转录通读。 这些突变体的特征将提供以下信息： 哪些基因产物参与转录终止， vivo.

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.