Mechanisms of biomolecular condensation in bacterial ribosomal RNA transcription

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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.