Mechanisms of biomolecular condensation in bacterial ribosomal RNA transcription

通过生物分子凝聚的原核亚细胞组织的研究最近获得了动力，证明核糖体RNA（rRNA）转录的基本过程发生在凝聚物中，类似于真核细胞的核仁。NusA是一种重要的转录抗终止因子，在大肠杆菌中能发生液-液相分离。大肠杆菌中，并形成快速生长细胞中的转录浓缩物的基础。然而，NusA如何驱动LLPS以及缩合物如何与转录效率相关还有待描述。我们自己的工作最近也表明，NusA位于基因组减少的病原体肺炎支原体中的转录-翻译偶联的连接处。分枝pneumoniae NusA进化出一个高度无序的C-末端结构域，它取代了E.大肠杆菌中表达，而在B中完全不表达。枯草杆菌。我们表明，M。pneumoniae NusA在体外和在E. coli细胞。在本提案中，我们的目标是利用三种不同细菌中NusA同源物之间的序列超变异性，在核糖体RNA转录介导的情况下剖析相分离原理、凝聚物成核机制、生长动力学、超分子结构和功能。含有NusA的抗终止复合物。我们将结合联合收割机生化和功能测定，结构研究跨尺度使用核磁共振光谱和单分子荧光显微镜和冷冻电子断层扫描的新组合，提供一个机制和定量的理解转录缩合物。总的来说，我们将提供社区在凝聚体的分子结构和动力学的综合建模的新方法。

The investigation of prokaryotic subcellular organization via biomolecular condensation has recently gained momentum with the demonstration that the fundamental process of ribosomal RNA (rRNA) transcription takes place within condensates, similar to the eukaryotic nucleolus. Specifically, NusA, an essential transcription anti-termination factor, was reported to undergo liquid-liquid phase separation (LLPS) in E. coli and to form the basis of transcription condensates in fast growing cells. However, how NusA drives LLPS and how condensates relate to transcriptional efficiency are yet to be described. Our own work has also recently shown that NusA sits at the nexus of transcription-translation coupling in the genome-reduced pathogen Mycoplasma pneumoniae. The M. pneumoniae NusA has evolved a highly disordered C-terminal domain, which substitutes the structured C-terminal domains in E. coli and is completely absent in B. subtilis. We show that M. pneumoniae NusA also phase separates in vitro and following heterologous expression in E. coli cells. In this proposal, we aim to take advantage of the sequence hypervariability among NusA homologues across the three different bacteria to dissect the principles of phase separation, mechanisms of condensate nucleation, growth dynamics, supramolecular structure and function in the context of rRNA transcription mediated by the ribosomal RNA transcription NusA-containing antitermination complex. We will combine biochemical and functional assays, structural studies across scales using nuclear magnetic resonance spectroscopy and a novel combination of single-molecule fluorescence microscopy and cryo-electron tomography to provide a mechanistic and quantitative understanding of transcription condensates. Overall, we will provide the community with novel approaches in integrative modeling of molecular structure and dynamics in condensates.