Post-transcriptional regulation of RpoS synthesis

RpoS 合成的转录后调控

• 批准号: 6572041
• 负责人: THOMAS A. ELLIOTT
• 金额: $ 22.48万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6572041
• 关键词: Escherichia coli; RNA binding protein; antisense nucleic acid; bacterial RNA; bacterial proteins; cell cycle; gene mutation; genetic regulation; microorganism growth; microorganism metabolism; molecular genetics; nucleic acid structure; posttranscriptional RNA processing; protein biosynthesis; site directed mutagenesis; transcription factor

DESCRIPTION (provided by applicant): RpoS is a sigma factor, discovered and best studied in the enteric bacteria, that is important in orchestrating responses to many stresses. RpoS activity is greatly increased during stationary phase after growth in rich medium, by limitation for individual nutrients (e.g. carbon or nitrogen), by high osmolarity medium, and after entry into the eukaryotic host cell, among other stimuli. Expression of more than 50 genes responds to RpoS, and the cognate gene products act to mitigate the adverse consequences of stress for the cell. RpoS matters in the real world, where "feast and famine" is the norm. Our goal is to understand the mechanisms regulating RpoS abundance, which are poorly understood. The principal control occurs by post-transcriptional regulation of RpoS synthesis, and by regulated protein turnover. We focus here on the control of RpoS synthesis. Escherichia coil is our model organism, but the results should be broadly applicable, in two senses. First, they should illuminate the important role of RpoS in pathogenic genera such as Salmonella and Yersinia, and they will also advance our understanding of post-transcriptional gene regulation.Genetic analysis has suggested that one known RNA-binding protein, Hfq, and another possible RNA binding protein, DksA, are likely to interact with rpoS mRNA to control its expression. The small molecule "alarmone" ppGpp also has a role. The target mRNA has an antisense element that pairs with the ribosome binding site to limit translation. The function of the antisense element is counteracted in a way that requires the RNA-binding proteins and under at least some conditions, a trans-acting anti-antisense RNA. Experiments described in the specific aims utilize mainly genetic but also physical approaches: to verify the secondary structure of the rpoS mRNA, to identify the important proximal factors and their sites of action, and to determine exactly what happens to this mRNA to increase its expression.

描述(由申请人提供)：rpos是一种西格玛因子，在肠道细菌中被发现并得到最好的研究，它在协调对许多压力的反应中非常重要。在富营养液中生长后的稳定期，受个别营养物质(如碳或氮)的限制，在高渗透压下，以及在进入真核宿主细胞后等刺激下，rpos的活性在稳定期显著增加。超过50个基因的表达对rpos有反应，同源基因产物起到减轻细胞应激不良后果的作用。RPOS在现实世界中很重要，在现实世界中，“盛宴和饥荒”是常态。我们的目标是了解调节RPO丰度的机制，这些机制还知之甚少。主要的控制是通过转录后调节rpos的合成和调节蛋白质的周转。我们在这里重点介绍RPOS合成的控制。大肠杆菌是我们的模式生物，但在两种意义上，结果应该是广泛适用的。首先，他们应该阐明rpos在沙门氏菌和耶尔森氏菌等致病属中的重要作用，他们也将促进我们对转录后基因调控的理解。遗传分析表明，一个已知的RNA结合蛋白Hfq和另一个可能的RNA结合蛋白DksA可能与rpos mRNA相互作用来控制其表达。小分子“警报器”ppGpp也有作用。靶基因具有与核糖体结合部位配对的反义元件，以限制翻译。反义元件的功能以一种需要RNA结合蛋白和至少在某些条件下需要反义RNA的方式被抵消。在特定目的中描述的实验主要使用遗传方法，但也使用物理方法：验证rpos mRNA的二级结构，确定重要的近端因子及其作用部位，并准确地确定该mRNA发生了什么以增加其表达。