CRYSTALLOGRAPHIC STUDIES OF DNA AND RNA POLYMERASES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 本项目的总体目标是为理解DNA和RNA聚合酶的起始和延伸机制、它们从起始阶段到延伸阶段的转变、因子对聚合酶的调节以及这些聚合酶确保正确核苷酸插入的机制提供结构基础。这一目标将通过确定与功能相关蛋白质复合并结合到适当的DNA或RNA底物上的聚合酶的晶体结构，以及通过适当的生化实验来实现。此外，我们的目标是建立复制体的组件组装的结构。其中包括DNA聚合酶III、tau和分叉DNA底物之间的复制叉状复合体，以及包括与其DNA底物结合的水热杆菌Dna B解旋酶的原始体的结构。此外，DNAB解旋酶的结构与其DNA和核苷酸底物以及DNAG和解旋酶加载器蛋白的解旋酶结合域结合。蛋白质引发的phi29 DNA聚合酶启动DNA合成的结构基础及其向延伸期转变的机制将通过这一转变中的一系列中间态结构来确定。我们将从其与半乳糖和乳糖操纵子启动子DNA以及分解代谢基因激活蛋白调节转录因子的复合体结构中，来研究来自大肠杆菌的多亚单位RNA聚合酶对转录启动的调控。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of this project is to provide a structural basis for understanding the mechanisms of initiation and elongation by DNA and RNA polymerases, their transitions from initiation to elongation phases, the regulation of polymerases by factors and the mechanisms by which these polymerases assure that the correct nucleotide is inserted. This objective will be achieved by determining the crystal structures of polymerases complexed with functionally associated proteins and bound to appropriate DNA or RNA substrates, as well as by appropriate biochemical experiments. Further, we aim to establish the structures of component assemblies of the replisome. These include a replication fork complex between DNA polymerase III, tau and a forked-DNA substrate, as well as the structure of the primasome that includes Thermus aquaticus DnaB helicase complexed with the DnaG primase bound to their DNA substrate. Additionally, the structure of the DnaB helicase bound to its DNA and nucleotide substrates as well as the helicase binding domain of DnaG and helicase loader protein. The structural basis of the initiation of DNA synthesis by the protein primed phi29 DNA polymerase and the mechanism of its transition to the elongation phase will be determined through a series of structures of intermediate states in this transition. The regulation of transcription initiation by the multi-subunit RNA polymerase from E. coli will be examined from its structures complexed with the gal and lac operon promoter DNAs and the catabolite gene activator protein regulator transcription factor.