Post-transcriptional regulation of RpoS synthesis

描述（由申请人提供）：RpoS是一种sigma因子，在肠道细菌中发现并得到最好的研究，在协调对许多应激的反应中很重要。RpoS活性在富培养基中生长后的稳定期期间，通过限制单独的营养物（例如碳或氮），通过高渗透压培养基，以及在进入真核宿主细胞后，以及其他刺激而大大增加。超过50个基因的表达响应RpoS，并且同源基因产物起到减轻应激对细胞的不利后果的作用。在真实的世界中，“盛宴和饥荒”是常态。 我们的目标是了解调节RpoS丰度的机制，这是知之甚少。主要控制发生的转录后调节RpoS合成，并通过调节蛋白质周转。我们在这里集中在RpoS合成的控制。大肠杆菌是我们的模式生物，但结果应该是广泛适用的，在两个意义上。首先，他们应该阐明的重要作用，RpoS在致病菌属，如沙门氏菌和耶尔森氏菌，他们也将推进我们的理解转录后gene regulation.Genetic分析表明，一个已知的RNA结合蛋白，Hfq，和另一种可能的RNA结合蛋白，DksA，可能与rpoS mRNA相互作用，以控制其表达。小分子“报警素”ppGpp也有作用。靶mRNA具有与核糖体结合位点配对以限制翻译的反义元件。反义元件的功能以需要RNA结合蛋白的方式被抵消，并且在至少一些条件下，需要反式作用的抗反义RNA。具体目标中描述的实验主要利用遗传方法，但也采用物理方法：验证rpoS mRNA的二级结构，识别重要的近端因子及其作用位点，并确定该mRNA发生了什么以增加其表达。

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.