Kinetic Studies of Transcription Elongation

描述(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引导的RNA合成，是导致基因表达的一连串事件的第一步。RNA聚合酶(RNAP)在转录过程中的核心作用是以高保真和合理的速率合成新生的RNA链。RNAP似乎已经进化到可以催化多种反应，并显示出前所未有的动态灵活性。在过去的十年中，我们对转录在伸长水平上的调控的理解已经有了很大的发展；然而，我们的大部分理解要么来自对静态伸长复合体的研究，要么来自于调控事件的位置，如暂停和终止位点。因此，关于这些研究与RNA合成机制的相关性的问题仍然不清楚。要了解一种表现出如此构象和功能多样性的酶，必须确定每个途径中的所有步骤，并确定哪个步骤(S)可能是限速步骤，从而受到调控。值得注意的是，只有暂态动力学方法才能识别单个限速步骤。在之前的授权期，我们使用动力学方法证明了E.coliRNAP除了催化部位外还含有变构结合部位。与模板核苷三磷酸(NTP)结合，但不与非模板NTP结合，可提高核苷酸掺入速率。这些数据表明，RNA聚合酶可以存在于缓慢(未激活)催化合成和快速(激活)催化合成的状态，从慢状态到快状态的转变是由模板NTP与变构位点结合引起的。我们假设这种构象转换对转录延长和终止的调控至关重要。在下一个授权期，我们将检验这个模型的许多预测，并进一步研究NTP结合在调控转录延长中的作用。此外，为了开发一个完整的伸长模型，我们还将表征转录本的切割，这对于保持准确和过程的合成是重要的。我们将利用T.thermophilus RNAP的热稳定性和高切割活性进行后续研究。最后，最近发表的酵母RNAP Il和T.Aquaticus RNAP核心酶的晶体结构将我们带入了转录研究的新纪元，为从原子水平上理解RNA合成的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在氨基酸水平上了解NTP结合和构象转变在伸长调节中的作用。

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.