STRUCTURAL STUDIES OF BACTERIOPHAGE N4 RNA POLYMERASE AND DNA COMPLEX

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们使用噬菌体N4 RNA聚合酶（N4 RNAP）作为模型系统来了解转录机制。我们利用SeMet-protein MAD方法和在X25/NSLS获得的数据集，在2.3A分辨率下解析了载脂蛋白酶的结构。最近，我们用分子置换的方法解决了N4 RNAP-DNA复合体的结构。数据集采用主源x射线采集，分辨率为2.4A。转录起始阶段rnap - dna -底物复合体（转录起始复合体）的结构尚未得到解决。通过共晶法制备了起爆复合晶体，并在主源x射线下进行了2.4A分辨率的衍射。为了了解转录模仿反应的分子细节，我们希望利用X25光束线时间在原子分辨率上收集转录起始复杂晶体数据集。该结构将是转录起始阶段的第一个快照，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.