STRUCT STUDY OF BACTERIOPHAGE N4 RNA POLYMERASE TRANSCRIPTION INITIATION COMPLEX

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。 噬菌体N4 RNA聚合酶转录起始复合体的结构研究 我们正在使用噬菌体N4 RNA聚合酶(N4 RNAP)作为一个模型系统来了解转录机制。我们用Semet-Protein MAD方法和在X25/NSLS上获得的数据集，在2.3A分辨率下求解了apo酶的结构。最近，我们用分子置换的方法解决了N4 RNAP-DNA的复杂结构。数据集是用主源X射线采集的，分辨率为2.4A。在转录起始阶段，没有人解决RNAP-DNA-底物复合体(转录起始复合体)的结构问题。我们用共结晶法制备了引发络合物晶体，并用本源X射线衍射仪对其进行了2.4A分辨率的衍射。为了了解转录模拟反应的分子细节，我们想要请求F1/CHESS的光束线时间来收集原子分辨率的转录起始复合体晶体数据集。该结构将是转录起始阶段的第一张快照，它告诉我们为什么RNAP能够进行从头反应。该项目由美国国立卫生研究院资助。

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. Structural study of bacteriophage N4 RNA polymerase transcription initiation complex We are using bacteriophage N4 RNA polymerase (N4 RNAP) as a model system to understand transcription mechanism. We have solved apo enzyme structure at 2.3A resolution by using SeMet-protein MAD method and dataset obtained at X25/NSLS. Recently, we have solved N4 RNAP-DNA complex structure by molecular replacement. The dataset was collected with home source X-ray and the resolution is 2.4A. Nobody solved the structure of RNAP-DNA-substrate complex at transcription initiation stage (transcription initiation complex). We prepared initiation complex crystal by co-crystallization and found it diffracts at 2.4A resolution with home source X-ray. To understand molecular detail of transcription imitation reaction, we want to request beamline time of F1/CHESS to collect transcription initiation complex crystal dataset at atomic resolution. The structure will be the first snap shot of transcription initiation stage and it tells us why RNAP is able to carry out de novo reaction. This project is funded by NIH.