Structural study of direct associations between cellular RNA polymerase and regulatory factors during the transcription cycle

总结 我们的长期研究目标是了解细胞RNA聚合酶的转录机制。我们 研究已经做出了重大贡献，提供了RNA聚合酶在不同阶段的结构， 转录周期在接下来的五年里，我们将继续研究细菌中的转录机制 和古生菌，以提供对转录的基本机制的见解，这是从细菌保守 变成人类 在过去的20年里，X射线晶体学在结构生物学中发挥了重要作用， RNA聚合酶和揭示了许多重要的结构。RNA聚合酶在细胞中的明显阶段 转录循环的主流通常是指RNA聚合酶核心酶、全酶、开放酶、 与强启动子、转录起始和延伸复合物的复合物。这些结构已经 由于其稳定的性质而被很好地表征。细胞RNA聚合酶的结构生物学挑战 是可视化RNA聚合酶与其他调节因子和配体的瞬时相互作用， 在转录主流中的每个明显阶段之间或在转录主流的分支途径处 周期由于其难以捉摸的性质，这种大分子组装体的结晶一直是瓶颈， 因此限制了X射线晶体学的方法。 近几年来，低温电子学对大分子结构的分辨 由于多种技术进步，冷冻电镜（cryo-EM）已经得到了极大的改进，使我们能够可视化 大分子复合物的非均相和大规模组装。我们将使用结构生物学方法 包括cryo-EM和X射线晶体学以及其他生物化学方法来可视化这些 瞬时相互作用，并研究其对转录过程的影响。本研究的主要目标是：1） 大肠杆菌RNA聚合酶与DksA/ppGpp之间的相互作用， 细菌RNA聚合酶与ATP依赖的马达酶之间的相互作用 用于在转录周期结束时拯救停滞的RNA聚合酶; 3）古细菌之间的相互作用 RNA聚合酶和ATP依赖性转录终止因子Eta用于破坏停滞的转录 延伸复合物启动转录偶联DNA修复。

Summary The long-term goal of our research is to understand transcription mechanisms of cellular RNA polymerase. Our research has made major contributions to provide structures of RNA polymerase at the different stage of transcription cycle. During the next five years, we will continue to study the transcription machinery in bacteria and archaea to provide insights of the fundamental mechanism of transcription, which is conserved from bacteria to human. Over the last 20 years, X-ray crystallography has been playing a major role in the structural biology of RNA polymerase and revealed many important structures. RNA polymerases at evident stages in the mainstream of the transcription cycle are often referred to RNA polymerase core enzyme, holoenzymes, open complex with a strong promoter, transcription initiation and elongation complexes. These structures have been well characterized due to their stable natures. A challenge in the structural biology of cellular RNA polymerase is to visualize transient interactions of RNA polymerase with other regulatory factors and ligands that occur in between each evident stage in the mainstream or at the branched pathways from the mainstream of transcription cycle. Due to their elusive natures, crystallization of such macromolecular assemblies has been a bottleneck and thus limited the approach by X-ray crystallography. In the last couple of years, the resolution of macromolecular structures determined by cryo-electron microscopy (cryo-EM) has been drastically improved due to multiple technical advances, allowing us to visualize heterogenous and large assembly of macromolecular complexes. We will use structural biology methods including both cryo-EM and X-ray crystallography together with other biochemical approaches to visualize these transient interactions and investigate their effects for transcription process. Major targets of our study are: 1) the interaction between the Escherichia coli RNA polymerase and DksA/ppGpp for transcription regulation during the stringent response; 2) the interaction between bacterial RNA polymerase and ATP-dependent motor enzyme for rescuing stalled RNA polymerase at the end of transcription cycle; and 3) the interaction between archaeal RNA polymerase and ATP-dependent transcription termination factor Eta for disrupting a stalled transcription elongation complex to initiate the transcription-coupled DNA repair.