Structure-based mutational analysis of RNA polymerase

描述（由申请人提供）：我们的长期目标是了解转录的分子细节，并揭示潜在的结构决定因素。转录是基因表达的第一步，也是主要的调控检查点，由DNA依赖性RNA聚合酶（RNAP）完成。水生栖热菌（Taq）RNAP核心酶的晶体结构最近已被确定。将E. coli RNAP和Taq RNAP的结构数据，提出了转录复合体的结构-功能模型。这些模型将特定的生化功能分配给几个RNAP结构元件。为了测试这些模型的预测，将进行RNAP的合理诱变。作为模型系统，我们将使用来自Taq的RNAP，这是唯一已知高分辨率结构的细菌酶，以及E。co/i RNAP，这是功能上研究得最好的。对Taq RNAP的研究将充分利用结构信息，并有望为RNAP的机制和结构提供新的见解。研究E.大肠杆菌酶的研究有望为RNAP机制和调控提供新的见解。遗传系统和生化测定来研究E. co/i RNAP已经建立，没有遗传系统来研究Taq RNAP。我们开发了一个E。co/i表达系统，使过量生产重组Taq RNAP与工程突变，我们表明，许多技术开发的研究E. co/i转录适用于研究Taq RNAP。以下是我们的具体目标。1)对Tag和E. co/i RNAP β和β-初级亚基。最初将靶向以下RNAP元件：β“舵”，其涉及转录泡的建立和RNA-DNA杂交体的适当长度的维持; β“瓣”，其涉及新生RNA结合和转录终止的控制;第二信道，被认为将NTP引导至催化中心并接受处于转录的非生产性回溯构象的新生RNA的3'末端2）克隆。过表达和纯化与RNAP相互作用的Tag转录因子。本研究将克隆Taq RNAP sigma因子、转录终止因子和转录物切割因子的编码基因，并将重组Taq因子用于扩展检测RNAP突变体的转录测定方法，获得与RNAP共晶体，并与结构基团合作进行结构测定。并将产生开发靶向细菌RNAP的新药所需的信息。细菌和真核生物之间惊人的RNAP保守程度确保了我们的结果将直接与真核转录相关，而所提出的分析是不可能的。

DESCRIPTION (provided by applicant): Our long-term goal is to understand transcription in molecular detail and to uncover the underlying structural determinants. Transcription, the first step of gene expression and a major regulatory checkpoint, is performed by DNA-dependent RNA polymerases (RNAPs). A crystal structure of Thermus aquaticus (Taq) RNAP core enzyme has recently been determined. The superposition of functional data from E. coli RNAP and structural data from Taq RNAP allowed to put forward hypothetical structure-function models of transcription complex. The models assign specific biochemical functions to several RNAP structural elements. To test predictions of these models, rational mutagenesis of RNAP will be performed. As model systems we will use RNAP from Taq, the only bacterial enzyme for which high-resolution structure is known, and E. co/i RNAP, which is best-studied functionally. Studies of Taq RNAP will make the best use of structural information and are expected to provide new insights in RNAP mechanism and structure. Studies of E. coli enzyme are expected to provide insights in RNAP mechanism and regulation. Genetic systems and biochemical assays to study E. co/i RNAP have been established; there is no genetic system to study Taq RNAP. We developed an E. co/i expression system that allows to overproduce recombinant Taq RNAP with engineered mutations, and we showed that many techniques developed to study E. co/i transcription are applicable to study Taq RNAP. The following are our specific aims. 1) To perform systematic mutagenesis of potentially functionally important structural elements of Tag and E. co/i RNAP Beta and Beta-prime subunits. The following RNAP elements will be targeted initially: the Beta' "rudder", implicated in the establishment of transcription bubble and the maintenance of the proper length of RNA-DNA hybrid; the Beta "flap", implicated in the nascent RNA binding and control of transcription termination; the secondary channel, thought to direct NTP to the catalytic center and to accept the 3' end of the nascent RNA in unproductive backtracked conformation of transcription complex.2) To clone. overexpress and purify Tag transcription factors interacting with RNAP. Genes coding for Taq RNAP sigma factors, transcription termination factors, and transcript cleavage factors will be cloned and recombinant Taq factors will be used to extend the repertoire of transcription assays for testing RNAP mutants, to obtain co-crystals with RNAP, and in collaboration with a structural group, for structure determination.The proposed work will contribute directly to our understanding of molecular basis of bacterial transcription, and will generate information necessary for development of new drugs that target bacterial RNAP. The amazing degree of RNAP conservation between bacteria and eukaryotes ensures that our results will be directly relevant to eukaryotic transcription, where the proposed analyses are not possible.