Structural studies of RNA polymerase regulation by RNA

描述(申请人提供)：转录是基因表达的主要控制点，RNA聚合酶(RNAP)是转录的中心酶，从细菌到人类都是保守的。我们的长期目标是了解转录的机制及其调控。确定RNAP及其与DNA、RNA和调节因子的复合体的三维结构是必不可少的一步。我们专注于高度特征化的原核生物RNAP。为此，我们采用了生化和生物物理相结合的方法。在这里，我们提出在转录周期的不同阶段对转录复合体的结构/功能进行研究，旨在加深我们对转录本身产物RNA对RNAP调控的理解。具体地说，我们建议：1.使用X射线结晶学来确定Thermus RNAP暂停转录复合体的高分辨率结构，无论是否有Nusa或Nusa结构域，在His停顿位置。转录暂停通过协调RNAP与其他调控事件在基因表达调控中发挥关键作用。转录暂停将转录和翻译结合起来，在一个称为衰减的过程中控制许多氨基酸生物合成操纵子的表达。这些调节停顿，如在His停顿位置，是由一个RNA发夹稳定的，它可能通过变构机制在刚刚转录的RNA转录本中形成。此外，外在因素，如保守的伸长因子Nusa，可以进一步稳定暂停。我们已经结晶了一个暂停的伸长复合体，并收集了3.8E分辨率的衍射数据。提出了进一步的实验，以提高这些晶体的分辨极限，ii)捕获暂停的络合物的额外相关构象状态，以及iii)结晶包含Nusa或Nusa结构域的络合物。2.6S RNA/RNAP-全酶复合体的结构表征6S RNA是细菌转录程序对稳定期营养限制做出反应的关键分子，它与C70-全酶具有显著的特异性并抑制其功能。6S RNA模拟开放启动子复合体中的DNA，可以作为转录模板，提供一种在营养变得充足时释放6S RNA的机制。我们将：i)使用生化和生物物理方法来定位E.coliC70-全酶与6S RNA的相互作用；ii)使用X射线结晶学来确定6S RNA/RNAP-全酶复合体的结构。

DESCRIPTION (provided by applicant): Transcription is the major control point of gene expression and RNA polymerase (RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. To this end, we bring to bear a combined biochemical and biophysical approach. Here we propose structure/function studies of transcription complexes in different stages of the transcription cycle, aimed towards adding to our understanding of RNAP regulation by the product of transcription itself, RNA. Specifically, we propose to: 1. Use X-ray crystallography to determine high-resolution structures of Thermus RNAP paused transcription complexes, with and without NusA or NusA domains, at the his pause site. Transcriptional pausing plays key roles in the regulation of gene expression by coordinating RNAP with other regulatory events. Transcriptional pausing couples transcription and translation to control the expression of many amino acid biosynthesis operons in a process called attenuation. These regulatory pauses, such as at the his pause site, are stabilized by an RNA hairpin that forms in the just transcribed RNA transcript, likely through an allosteric mechanism. In addition, extrinsic factors, such as the conserved elongation factor NusA, can further stabilize the pause. We've crystallized a paused elongation complex and collected diffraction data to 3.8 E-resolution. Further experiments are proposed to i) improve the resolution limit of these crystals, ii) trap additional relevant conformational states of the paused complex, and iii) crystallize a complex containing NusA or NusA domains. 2. Structurally characterize the 6S RNA/RNAP-holoenzyme complex. The 6S RNA, a key player in the response of the bacterial transcriptional program to nutrient limitation in stationary phase, binds with marked specificity to C70-holoenzyme and inhibits its function. The 6S RNA mimics the DNA in an open promoter complex, and can serve as a transcription template, providing a mechanism for releasing the 6S RNA when nutrients become plentiful. We will: i) Use biochemical and biophysical approaches to map E. coli C70-holoenzyme interactions with 6S RNA, ii) Use X-ray crystallography to determine structures of 6S RNA/RNAP-holoenzyme complexes.