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，也是一个主要的调控检查点，由dna依赖性RNA聚合酶（rnap）完成。水热菌（Thermus aquaticus, Taq） RNAP核心酶的晶体结构已被确定。将大肠杆菌RNAP的功能数据与Taq RNAP的结构数据进行叠加，可以提出转录复合体的假设结构-功能模型。这些模型将特定的生化功能分配给几个RNAP结构元素。为了验证这些模型的预测，将对RNAP进行合理的诱变。作为模型系统，我们将使用来自Taq的RNAP，这是唯一已知具有高分辨率结构的细菌酶，以及E. co/i RNAP，这是在功能上研究得最好的。对Taq RNAP的研究将充分利用结构信息，有望对RNAP的机制和结构提供新的认识。对大肠杆菌酶的研究有望为RNAP的机制和调控提供新的见解。建立了研究E. co/i RNAP的遗传系统和生化分析方法；没有研究Taq RNAP的遗传系统。我们开发了一个E. co/i表达系统，该系统允许过量产生带有工程突变的重组Taq RNAP，并且我们证明了许多用于研究E. co/i转录的技术适用于研究Taq RNAP。以下是我们的具体目标。1)对Tag和E. co/i RNAP β和β -prime亚基的潜在功能重要结构元件进行系统诱变。以下RNAP元件将首先被靶向：β“舵”，涉及转录泡的建立和维持RNA-DNA杂交的适当长度；β“皮瓣”，涉及新生RNA结合和转录终止的控制；二级通道，被认为将NTP引导到催化中心，并以转录复合物的非生产性反向构象接受新生RNA的3'端。2)克隆。过表达和纯化与RNAP相互作用的标签转录因子。编码Taq RNAP sigma因子、转录终止因子和转录物切割因子的基因将被克隆，重组Taq因子将用于扩展转录检测库，以测试RNAP突变体，获得与RNAP共晶，并与结构组合作，用于结构确定。这项工作将直接有助于我们了解细菌转录的分子基础，并将为开发针对细菌RNAP的新药提供必要的信息。细菌和真核生物之间惊人的RNAP保护程度确保了我们的结果将直接与真核生物转录相关，而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.