Mechanisms of biomolecular condensation in bacterial ribosomal RNA transcription

随着核糖体RNA （rRNA）转录的基本过程发生在凝聚体中，类似于真核生物的核仁，通过生物分子凝聚对原核亚细胞组织的研究最近获得了动力。具体来说，据报道，NusA是一种重要的转录抗终止因子，在大肠杆菌中进行了液-液相分离（LLPS），并在快速生长的细胞中形成转录凝聚物的基础。然而，NusA如何驱动LLPS以及凝析油与转录效率的关系尚不清楚。我们自己的工作最近也表明，在基因组减少的病原体肺炎支原体中，NusA位于转录-翻译偶联的联系上。肺炎分枝杆菌NusA进化出高度无序的c端结构域，取代了大肠杆菌中结构化的c端结构域，而在枯草芽孢杆菌中完全不存在。我们发现肺炎支原体NusA也在体外相分离，并在大肠杆菌细胞中异种表达。在这篇论文中，我们的目标是利用三种不同细菌中NusA同源物序列的高度可变性来剖析由核糖体RNA转录NusA-含抗终止复合物介导的rRNA转录背景下的相分离原理、凝析成核机制、生长动力学、超分子结构和功能。我们将结合生化和功能分析，利用核磁共振波谱以及单分子荧光显微镜和低温电子断层扫描的新组合进行跨尺度的结构研究，以提供转录凝聚物的机制和定量理解。总的来说，我们将为社区提供新的方法来综合建模凝析油的分子结构和动力学。

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.