Kinetic Studies of Transcription Elongation

描述（由申请人提供）：拟议研究的主要目标是阐明调控转录延伸的详细动力学机制。转录，即DNA指导的RNA合成，是导致基因表达的级联事件中的第一步。RNA聚合酶（RNAP）在转录中的核心作用是以高保真度和合理的速率合成新生RNA链。RNAP似乎已经发展到催化多种反应，并显示出前所未有的动态灵活性。在过去的十年中，我们的理解转录的调节在伸长水平上有了很大的发展，然而，我们的理解大多来自静态伸长复合物或在调节事件的位置，如暂停和终止位点的研究。因此，这些研究与RNA合成机制的相关性问题仍不清楚。为了了解一种具有这种构象和功能多样性的酶，必须确定每个途径中的所有步骤，并确定哪些步骤可能是限速的，从而受到调控。值得注意的是，只有瞬态动力学方法可以识别单个限速步骤。coliRNAP除了催化位点外，还含有一个变构结合位点。模板化的核苷三磷酸（NTP）而非模板化的NTP与该位点的结合增加了核苷酸掺入的速率。这些数据表明，RNA聚合酶可以存在于缓慢催化合成的状态（未活化）和快速催化合成的状态（活化）中，从缓慢状态到快速状态的转变是由模板化的NTP与变构位点的结合诱导的。我们假设这种构象转换对转录延伸和终止的调节至关重要，在下一个资助期，我们将测试这个模型的许多预测，并进一步研究NTP结合在调节转录延伸中的作用。此外，为了开发一个完整的延伸模型，我们还将表征转录物切割，这对维持准确和进行性合成很重要。我们将利用T. Thermophilus RNAP用于这些后面的研究。最后，最近发表的酵母RNAP II和T. aquaticus RNAP核心酶将我们带入了转录研究的新时代，为在原子水平上理解RNA合成机制提供了前所未有的机会。因此，我们将利用这些信息开始了解，在氨基酸水平上，在延长的调节中的NTP结合和构象转换的作用。

DESCRIPTION (provided by applicant): The main goal of the proposed studies is to elucidate the detailed kinetic mechanisms that regulate and control transcription elongation. Transcription, the DNA directed synthesis of RNA, is the first step in the cascade of events that leads to gene expression. The central role of RNA polymerase (RNAP) in transcription is to synthesize the nascent RNA chain with high fidelity and at a reasonable rate. RNAP appears to have evolved such that it catalyzes multiple reactions and displays an unprecedented level of dynamic flexibility. In the past ten years, our understanding of the regulation of transcription at the level of elongation has evolved considerably; however, most of our understanding comes from studies either of static elongation complexes or at positions of regulatory events such as pause and termination sites. As such, the question as to the relevance of these studies to the mechanism of RNA synthesis remains unclear. To understand an enzyme that exhibits such conformational and functional diversity, it is essential to identify all steps in each of the pathways and to determine which step(s) might be rate-limiting and thus subject to regulation. Significantly, only transient-state kinetic methods can identify individual rate-limiting steps.In the previous grant period, using kinetics, we demonstrated that E. coli RNAP contains an allosteric binding site in addition to the catalytic site. Binding of the templated nucleoside triphosphate (NTP), but not non-templated NTPs, to this site increases the rate of nucleotide incorporation. The data suggest that RNA polymerase can exist in a state that catalyzes synthesis slowly (unactivated) and one that catalyzes synthesis rapidly (activated), with the transition from the slow to the fast state being induced by binding of the templated NTP to the allosteric site. We hypothesize that this conformational switch is paramount to the regulation of transcription elongation and termination.In the next grant period, we will test many predictions of this model and further investigate the role of NTP binding in regulating transcription elongation. In addition, to develop an integrated model of elongation, we also will characterize transcript cleavage which is important for maintaining accurate and processive synthesis. We will take advantage of the thermal stability and high cleavage activity of T. thermophilus RNAP for these latter studies. Finally, the recent publication of crystal structures of yeast RNAP Il and T. aquaticus RNAP core enzymes bring us into a new era in the study of transcription, providing an unprecedented opportunity to understand the mechanism of RNA synthesis at the atomic level. Accordingly, we will use this information to begin to understand, at the amino acid level, the role of NTP binding and conformational transitions in the regulation of elongation.