Structural studies of RNA polymerase regulation by RNA

RNA 调节 RNA 聚合酶的结构研究

• 批准号: 8608542
• 负责人: Seth A. Darst
• 金额: $ 32.98万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8608542
• 关键词: 6S RNA; Bacteria; Bacterial RNA; Binding; Biochemical; Combined Modality Therapy; Complex; Couples; DNA; DNA-Directed RNA Polymerase; Data; Development; Elongation Factor; Enzymes; Escherichia coli; Event; Frequencies; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Holoenzymes; Lead; Maps; Molecular Conformation; Nutrient; Operon; Phase; Play; Process; RNA; Regulation; Resolution; Rifampin; Site; Specificity; Staging; Structure; Thermus; Transcript; Translations; Tuberculosis; Ursidae Family; X-Ray Crystallography; aminoacid biosynthesis; antimicrobial; attenuation; biophysical techniques; improved; insight; man; programs; promoter; public health relevance; research study; resistant strain; response; three dimensional structure

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.

描述（由申请人提供）：转录是从细菌到人保守的基因表达和RNA聚合酶（RNAP）的主要控制点，是转录的中心酶。我们的长期目标是了解转录的机制及其调节。确定RNAP的三维结构及其与DNA，RNA和调节因子的复合物是重要的一步。我们专注于高度特征的原核生物RNAP。为此，我们采用了一种合并的生化和生物物理方法。在这里，我们提出了转录周期不同阶段的转录复合物的结构/功能研究，旨在增加我们对转录本身RNA的乘积对RNAP调控的理解。具体而言，我们建议：1。使用X射线晶体学来确定在其暂停部位，有或没有NUSA或NUSA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与其他调节事件来调节基因表达的关键作用。转录暂停夫妻在称为衰减的过程中转录和翻译以控制许多氨基酸生物合成操纵子的表达。这些调节的停顿，例如在他的暂停部位，被RNA发夹稳定，该RNA发夹可能是通过变构机制在刚转录的RNA转录本中形成的。另外，外在因子（例如保守的伸长因子NUSA）可以进一步稳定停顿。我们已经将暂停的伸长复合物结晶，并将衍射数据收集到3.8电子分辨率。提出了进一步的实验i）提高这些晶体的分辨率限制，ii）陷阱的其他相关构象状态，iii）结晶了一个含有NUSA或NUSA结构域的复合物。 2。在结构上表征6S RNA/RNAP-HOLO酶复合物。 6S RNA是细菌转录程序对固定期营养限制的响应的关键参与者，它与C70-氢酶具有明显的特异性结合并抑制其功能。 6S RNA模仿开放式启动子复合物中的DNA，可以用作转录模板，在营养丰富时提供了一种释放6S RNA的机制。我们将：i）使用生化和生物物理方法来绘制大肠杆菌C70-糖酶与6s RNA的相互作用，ii）使用X射线晶体学来确定6S RNA/RNA/RNAP-Holoenzyme复合物的结构。