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，也是一个主要的调节检查点，由DNA依赖的RNA聚合酶(RNAP)执行。水热菌(Thermus Aquaticus，Taq)RNAP核心酶的晶体结构最近被确定。来自E.coliRNAP的功能数据和来自Taq RNAP的结构数据的叠加使得能够提出假设的转录复合体的结构-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的合理突变。作为模型系统，我们将使用来自Taq的RNAP和E.co/I RNAP，Taq是唯一已知其高分辨率结构的细菌酶，而E.co/I RNAP是功能研究最好的。Taq RNAP的研究将最大限度地利用结构信息，有望为RNAP的机制和结构提供新的见解。对大肠杆菌酶的研究有望为RNAP的机制和调控提供深入的认识。已经建立了研究E.co/I RNAP的遗传系统和生化分析方法，但还没有研究Taq RNAP的遗传系统。我们开发了一种E.co/I表达系统，允许用工程突变来过量生产重组Taq RNAP，并且我们证明了许多研究E.co/I转录的技术都适用于Taq RNAP的研究。以下是我们的具体目标。1)对Tag和E.co/I RNAP Beta和Beta-Prime亚基的潜在功能重要结构元件进行系统的突变。最初将针对以下RNAP元件：Beta‘’“舵”，参与转录气泡的建立和RNA-DNA杂交的适当长度的维持；Beta‘Flat’，参与新生RNA的结合和转录终止的控制；二级通道，被认为引导NTP到催化中心，并接受新生RNA的3‘端的非生产性回溯构象的转录复合体2)克隆。高效表达和纯化与RNAP相互作用的Tag转录因子。编码Taq RNAP sigma因子、转录终止因子和转录本切割因子的基因将被克隆，重组Taq因子将被用于扩展转录检测的谱系，用于测试RNAP突变体，获得与RNAP的共晶，并与结构基团合作，用于结构测定。所提出的工作将直接有助于我们了解细菌转录的分子基础，并将产生针对细菌RNAP的新药开发所需的信息。细菌和真核生物之间令人惊讶的RNAP保守程度确保了我们的结果将直接与真核转录相关，而拟议的分析是不可能的。

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.