Transcription Regulation Through RNAP Secondary Channel

通过 RNAP 二级通道进行转录调控

• 批准号: 8115215
• 负责人: DMITRY G VASSYLYEV
• 金额: $ 39.67万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115215
• 关键词: Anti-Bacterial Agents; Antibiotics; Base Sequence; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Models; C-terminal; Complex; DNA; Data; Development; Diphosphates; Escherichia coli; Event; Funding; Gene Expression; Genetic Transcription; Goals; Hot Spot; Life; Mediating; Medicine; Messenger RNA; Modeling; Molecular Weight; Nucleic acid sequencing; Organism; Pattern; Pharmacotherapy; Phase; Protein Region; Proteins; RNA; Regulation; Reporting; Resolution; Signal Transduction; Site; Source; Structure; Surface; Transcript; Transcription Elongation; Transcription Initiation; Transcriptional Regulation; antibiotic design; base; design; interest; microcin; promoter; public health relevance; small molecule; tagetitoxin; transcription factor

DESCRIPTION (provided by applicant): ABSTRACT: Transcription is the first step in gene expression and a target for numerous regulatory factors: signals in the transcribed DNA, and in the nascent mRNA, proteins, low molecular weight effectors, etc. One of the most important goals in transcription studies now is identification and "high-resolution" characterization of "hot spots" on RNAP that mediate its interactions with regulators; these sites not only determine the patterns of gene expression, but could also serve as targets for the rational design of antibiotics. Studies of bacterial RNAP complexed with antibiotics should provide short-cuts to the development of effect drug therapies, and a unique opportunity to study dynamic regulatory mechanisms using a "static" crystallographic approach. Among the identified regulatory "hot-spots", the secondary channel (SC) is of special interest. This pore is a major entrance for incoming substrates and accommodates the extruding 39-end of the RNA during transcriptional pausing and arrest. Recently, the SC has emerged as a major binding site for a rapidly expanding group of transcription regulators and catalytic co-factors. This pore apparently can accommodate several accessory proteins: DksA, transcript cleavage Gre-factors, Gfh1, DksA, as well as small RNAP effectors/ antibiotics pyrophosphate, non-complementary NTPs, tagetitoxin, microcin and ppGpp, and thus mediates control of all steps in transcription, from initiation to termination. The goal of this project is to elucidate the basic mechanisms of regulation of transcription elongation through the SC based on high-resolution structural analysis. We will study two model systems, E. coli and T. thermophilus. We propose the following specific aims: 1. We will determine the structures of the EC containing a full transcription bubble in the pre-translocated and backtracked states, and in the presence and absence of the small molecule effectors/inihibitors. 2. We will determine the EC structures with various "SC" transcription factors. The Gre-factors, Gfh1, DksA are among our major targets. Our long-term goal is to elucidate the mechanism of transcription through determining the structures of complexes that represent functional intermediates in the transcription cycle and analysis of the regulatory interactions that ultimately determine the patterns of gene expression in all organisms. We plan to integrate all the available biochemical and biophysical data to build a comprehensive model of the transcription apparatus by elucidating the continuum of structures, from initiation complexes formed at various promoters to those regulatory states that exist during processive elongation. We are particularly interested in elucidation of the structures and mechanisms of the transcription factors (in particular, antibiotics) acting at different steps in transcription. The proposed study represents an essential step in this direction. PUBLIC HEALTH RELEVANCE: Transcription is the first step in gene expression and a target for numerous regulatory factors. Elongation is a most long-lived and highly processive phase of transcription: the elongation complex (EC) is capable of uninterrupted synthesis of thousands nt-long RNA chains. High-resolution structural studies of the bacterial EC appear of central importance for understanding the basic principles of transcription and regulation of gene expression that in turn is essential for design of the new effective anti-bacterial agents. In particular, studies of bacterial RNAP complexed with antibiotics constitute a crucial subset of the structural projects which may provide a short-cut to medicine on one hand and a unique opportunity to study dynamic regulatory mechanisms using a "static" crystallographic approach on the other.

描述（由申请人提供）： 摘要：转录是基因表达的第一步，也是许多调节因素的目标：转录DNA中的信号，以及新生的mRNA中的信号，蛋白质，低分子重量效应子等。现在，转录研究中最重要的目标之一是识别和“高分辨率”对RNAP的表征的“高分辨率”表征，以介导RNAP的相互作用，以介导其与调节器的相互作用；这些位点不仅决定了基因表达的模式，而且还可以作为抗生素合理设计的靶标。与抗生素复合的细菌RNAP的研究应为效应药物疗法的发展提供短暂的介绍，以及使用“静态”晶体学方法研究动态调节机制的独特机会。 在确定的调节“热点”中，次级通道（SC）具有特殊意义。该孔是传入底物的主要入口，并在转录暂停和逮捕过程中适应了RNA的39端。最近，SC已成为快速扩展的转录调节剂和催化副因素的主要结合位点。显然，该孔可以容纳多种附件蛋白：DKSA，转录裂解GRE因子，GFH1，DKSA以及小的RNAP效应子/抗生素焦磷酸，非平衡NTPS，Tagetitoxin，Micrococin，microcoin and PPGPP，从而介导了所有在转录中的终止，从而介导了所有步骤。 该项目的目的是根据高分辨率结构分析通过SC阐明转录伸长的基本机制。我们将研究两个模型系统，即大肠杆菌和嗜热链球菌。我们提出以下特定目的：1。我们将确定在预先分配和回溯状态中包含完全转录气泡的EC结构，以及在存在和不存在小分子效应子/无限型的情况下。 2。我们将确定具有各种“ SC”转录因子的EC结构。 GRE因子GFH1，DKSA是我们的主要目标。 我们的长期目标是通过确定代表转录周期中功能性中间体的复合物的结构以及调节相互作用的分析，这些复合物的结构最终决定了所有生物体中基因表达的模式。我们计划通过阐明结构的连续性，从各种启动子形成的启动络合物到过程延长过程中存在的那些调节状态，从而整合所有可用的生化和生物物理数据，以构建转录设备的全面模型。我们对阐明转录因子的结构和机制（尤其是抗生素）作用于转录的不同步骤。拟议的研究代表了这一方向的重要步骤。 公共卫生相关性： 转录是基因表达的第一步，也是众多调节因素的目标。伸长率是最长寿，最高度的转录阶段：伸长复合物（EC）能够不间断地合成数千个NT长的RNA链。细菌EC的高分辨率结构研究对于理解基因表达的转录和调节的基本原理至关重要，而基因表达的基本原理反过来是设计新有效的抗细菌剂的必不可少的。特别是，对与抗生素复合的细菌RNAP的研究构成了结构项目的关键子集，该子集可能一方面为医学提供了短暂的速度，并且是使用另一种“静态”晶体学方法研究动态调节机制的独特机会。