Mechanism, Activation, and Control of rRNA Transcription

项目总结/摘要： 翻译器的合成是所有生物体生长和发育的核心， 并且其调节已经是分子微生物学的中心问题超过50年。最近， 很明显，对rRNA和tRNA的机制的理解 转录可以提供对一般转录机制的基本了解。 我们在前一个项目期间确定，核苷酸衍生物ppGpp不仅 直接调节rRNA启动子，但它也直接调节转录更广泛 超过700份成绩单出现了负面或正面的变化， 在ppGpp诱导的5分钟内。在提案的主要目的中，我们将分析 通过ppGpp及其辅因子DksA调节转录。我们发现有两种不同的 ppGpp在E. coli RNA聚合酶（RNAP）。我们将决定 ppGpp对每个结合位点的亲和力，确定结合是否是协同的， 在每个部位检测ppGpp作用的机制模型，包括阳性对照的机制， 确定不同启动子上的每个ppGpp结合位点在体外和体内的调节作用， 体内，并解决的作用，这两个网站在转录延伸的调节。最后我们 将使用代谢组学和蛋白质组学方法来鉴定蛋白质上的ppGpp靶点， RNAP并探讨其意义。 在第二个目标中，我们将研究转录因子的机制，如DksA，结合 直接到RNAP。这些包括TraR及其同源物，远距离DksA样蛋白携带在 广泛分布的染色体外分子; R.类球蛋白DksA样和CardD样蛋白 这似乎是重要的调节对光的反应;和E. coli Crl，a Sigma S 全酶组装因子 我们最近发现E.大肠杆菌rRNA操纵子形成细菌核仁样结构， 不依赖于转录，但仍然需要rRNA启动子区。第三 目的，我们将继续确定该结构的顺式和反式作用决定因素及其 细胞生理学的后果。

PROJECT SUMMARY / ABSTRACT: The synthesis of the translation apparatus is central to growth and development of all organisms, and its regulation has been a central issue in molecular microbioogy for over 50 years. Recently, it has become clear that an understanding of the mechanisms responsible for rRNA and tRNA transcription can provide fundamental insights into the mechanism of transcription in general. We determined in the previous project period that not only does the nucleotide derivative ppGpp directly regulate rRNA promoters, but it also directly regulates transcription much more widely than previously expected, with more than 700 transcripts changing either negatively or positively within 5 minutes of ppGpp induction. In the major aim of the proposal, we will analyze the regulation of transcription by ppGpp and its cofactor DksA. We found that there are two distinct binding sites for ppGpp on E. coli RNA polymerase (RNAP). We will determine the binding affinities of ppGpp for each binding site, determine whether binding is cooperative, develop and test mechanistic models for ppGpp action at each site including the mechanism of positive control, determine the regulatory roles of each ppGpp binding site on different promoters in vitro and in vivo, and address the roles of the two sites in regulation of transcription elongation. Finally, we will use metabolomic and proteomic approaches to identify ppGpp targets on proteins other than RNAP and investigate their significance. In the second aim, we will investigate the mechanisms of transcription factors that, like DksA, bind directly to RNAP. These include TraR and its homologs, distant DksA-like proteins carried on widely-distributed extrachromosomal elements; R. sphaeroides DksA-like and CarD-like proteins that appear to be important for regulation for responses to light; and E. coli Crl, a Sigma S holoenzyme assembly factor. We recently discovered that E. coli rRNA operons form a bacterial nucleolus-like structure that is not dependent on transcription but nevertheless requires the rRNA promoter region. In the third aim, we will continue to determine the cis- and trans-acting determnants for this structure and its consequences for cellular physiology.