MECHANISM, ACTIVATION AND CONTROL OF RRNA TRANSCRIPTION

负责制造蛋白质（如核糖体RNA、核糖体蛋白、翻译因子和trna）的机制对所有生物体的生长和发育至关重要。根据细胞营养状态对翻译机制合成的调节一直是微生物生理学研究的中心问题，至少已有40年的历史。最近，对大肠杆菌中负责rRNA转录的机制的理解也变得清晰起来，可以为细菌转录提供基本的见解。我们现在准备解决的核心问题不仅是我们对一般转录机制的理解，而且是不同的调控系统如何协同工作。提案中解决的问题按任意顺序分为三个具体目标：在第一个目标中，我们建议确定细菌启动子中的识别元件UP元件如何与RNA聚合酶(RNAP) α亚基相互作用，以及它是否影响酶向复合物募集后的步骤，可能是通过α -sigma亚基相互作用。在第二个目标中，我们建议确定rrn转录因子FIS如何增加许多启动子的转录，以及它如何影响它们对营养条件的反应。我们建议在两个rRNA启动子rrnB P1和rrnE P1上定义FIS和α之间的相互作用。在第三个目标中，我们建议通过NTP感知和其他负责rRNA转录的机制来确定rRNA合成调控的分子细节。然而，孤立地确定这些机制是不够的。我们现在建议开始确定在稳态生长、上升和固定阶段影响翻译装置合成调节的多种机制之间的关系。

The machinery responsible for making proteins (e.g. ribosomal RNA, ribosomal proteins, translation factors, and tRNAs) is central to growth and development of all organisms. The regulation of the synthesis of the translation machinery in response to the nutritional state of the cell has been a central issue in the study of microbial physiology for at least forty years. More recently, it has also become clear that an understanding of the mechanisms responsible for rRNA transcription in Escherichia coli can provide fundamental insights into bacterial transcription in general. We are now poised to address questions central not only to our understanding of general transcription mechanisms, but also to how different regulatory systems work together. The questions addressed in the proposal are divided into three specific aims in arbitrary order: In the first aim, we propose to determine how a recognition element in bacterial promoters, the UP element, interacts with the RNA polymerase (RNAP) alpha subunit and whether it affects steps after recruitment of the enzyme to the complex, perhaps through alpha-sigma subunit interactions. In the second aim, we propose to determine how the rrn transcription factor FIS increases transcription of many promoters and how it can affect their responses to nutritional conditions. We propose to define the interactions between FIS and alpha at two rRNA promoters, rrnB P1 and at rrnE P1. In the third aim, we propose to determine the molecular details of regulation of rRNA synthesis by NTP sensing and other mechanisms responsible for rRNA transcription. However, it is not sufficient to determine these mechanisms in isolation. We now propose to begin to determine the relationship(s) between the multiple mechanisms affecting regulation of the synthesis of the translation apparatus during steady state growth, upshifts, and in stationary phase.