Structural studies of RNA polymerase regulation by RNA

描述（由申请人提供）：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及其调控的机制。确定RNAP及其与DNA、RNA和调控因子的复合物的三维结构是必不可少的一步。我们专注于高度表征的原核rnap。为此目的，我们采用生物化学和生物物理相结合的方法。在这里，我们提出了转录复合体在转录周期的不同阶段的结构/功能研究，旨在增加我们对转录本身的产物RNA对RNAP的调控的理解。具体而言，我们建议：1。使用x射线晶体学确定在他的暂停位点有和没有NusA或NusA结构域的Thermus RNAP暂停转录复合物的高分辨率结构。转录暂停通过协调RNAP与其他调控事件在基因表达调控中起关键作用。转录暂停偶联转录和翻译来控制许多氨基酸生物合成操纵子的表达，这一过程被称为衰减。这些调控停顿，比如在his停顿位点，被刚转录的RNA转录物中形成的RNA发夹所稳定，可能是通过一种变构机制。此外，外部因素，如保守伸长因子NusA，可以进一步稳定暂停。我们已经结晶了一个暂停延伸复合物，并收集了3.8 e分辨率的衍射数据。提出了进一步的实验来i)提高这些晶体的分辨率限制，ii)捕获暂停配合物的其他相关构象态，以及iii)结晶包含NusA或NusA结构域的配合物。2. 6S RNA/ rnap -全酶复合物的结构特征。6S RNA是细菌在固定期对营养限制的转录反应中的一个关键角色，它与c70全酶具有明显的特异性结合并抑制其功能。6S RNA模仿开放启动子复合体中的DNA，可以作为转录模板，在营养丰富时提供一种释放6S RNA的机制。我们将：i)使用生化和生物物理方法绘制大肠杆菌c70全酶与6S RNA的相互作用图，ii)使用x射线晶体学确定6S RNA/ rnap -全酶复合物的结构。

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.