STRUCTURAL STUDIES OF BACTERIOPHAGE N4 RNA POLYMERASE AND DNA COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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，我们希望请求X25光束线时间以原子分辨率收集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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 X25 beamline time 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.