Structure-based mutational analysis of RNA polymerase

描述(由申请人提供)：我们的长期目标是在分子细节上了解转录机制和调控，并揭示潜在的结构决定因素。我们专注于细菌RNA聚合酶(RNAP)，这是一种转录酶，也是一个被证实的抗生素靶标。在之前的资助期间，获得了来自水华的结晶型重组RNAP。这一进展首次允许将遗传和结构方法的力量结合起来，以了解RNAP的功能和调控。在本提案的有效期内，我们将使用现有的结构信息以及我们计划从Thermus和其他嗜热细菌获得的可结晶的野生型和突变型重组RNAP，以实现以下具体目标。1.研究转录起始过程中RNAP与启动子DNA的非规范相互作用。塞莫斯RNAP？将鉴定与识别-10共识启动子元件下游的一个新元件有关的亚基残基，并确定该元件在启动子复合体形成中的功能和结构作用。RNAP的作用？将确定启动子复合体与启动子间隔区相互作用形成启动子复合体中的亚基拉链结构元素，并建立调控RNAP对单链M13噬菌体或ORI DNA的特异性识别的规则和引物RNA合成的机制。2.研究RNAP与抗生素的相互作用。利用定点抑制和Microcin J25(MccJ25)突变体，MccJ25分子与其结合位点RNAP二级通道的相互作用将被模拟。将描述MccJ25抑制革兰氏阳性细菌和靶向这些生物的RNAP的结构同系物。对携带位点特异性突变体的嗜热RNAP的功能和结构分析将用于：1)了解结合位点突变对RNAP与Sorangicin和Rifampicin相互作用的不同影响；2)了解链霉菌素抑制转录的分子机制。将对RNAP校对中心进行突变分析。这些研究结果将对真细菌RNAP的作用机制和调控以及真核细胞RNAP的功能机制(该酶与真细菌的RNAP高度相似)有新的重要认识。从公共卫生的角度来看，拟议的工作具有重要意义，因为将获得开发针对细菌转录的新药和/或改进药物所需的信息。

DESCRIPTION (provided by applicant): Our long-term goal is to understand transcription mechanism and regulation in molecular detail and to uncover underlying structural determinants. We focus on bacterial RNA polymerase (RNAP), the enzyme of transcription and a validated antibiotic target. During the previous funding period, crystallizable recombinant RNAP from Thermus aquaticus was obtained. This advance allows, for the first time, to combine the powers of genetic and structural approaches to understand RNAP function and regulation. During the tenure of this proposal, we will use available structural information and crystallizable wild-type and mutant recombinant RNAPs from Thermus and other thermophilic bacteria that we plan to obtain to address the following specific aims. 1. Study non-canonical RNAP-promoter DNA interactions during transcription initiation. The Thermus RNAP ? subunit residues involved in recognition of a novel element downstream of the -10 consensus promoter element will be identified and the functional and structural role of this element in promoter complex formation will be established, the role of the RNAP ?' subunit zipper structural element in promoter complex formation through interactions with promoter spacer will be determined, and the rules that govern the specific recognition of single-stranded M13 phage ori DNA by RNAP and the mechanism of primer RNA synthesis will be established. 2. Study RNAP interactions with antibiotics. Using site-specific suppression and Microcin J25 (MccJ25) mutants, the interaction of MccJ25 molecule with its binding site, the RNAP secondary channel, will be modeled. Structural homologues of MccJ25 that inhibit Gram-positive bacteria and target RNAPs from these organisms will be characterized. Functional and structural analyses of thermophilic RNAPs carrying site-specific mutants will be used to i) understand the differential effects of binding site mutations on RNAP interactions with Sorangicin and Rifampicin and ii) to understand the molecular mechanism of transcription inhibition by Streptolydigin. Mutational analysis of the RNAP proofreading center will be undertaken. The results of proposed studies will lead to new important insights into eubacterial RNAP mechanism and regulation and also into the mechanism of eukarytic RNAP function (this enzyme is highly similar to the eubacterial one). The proposed work is significant from the public health standpoint since information necessary for development of new and/or improved drugs that target bacterial transcription will be obtained.