Indirect Bacterial Transcription-Translation Coupling Mechanism Revealed by In Situ Integrative Structural Biology

Abstract

In situ structural biology aims to resolve macromolecular structures and collective behaviors of macromolecular machineries within the cell. In bacteria, transcription mediated by RNA polymerase is known to be functionally coupled to the leading ribosome, a process crucial for efficient gene expression. Direct ribosome-RNA polymerase interaction has been demonstrated in vitro, but how these two gigantic molecular machines coordinate is yet unclear, especially within the context of crowded and complex cellular environments. Using Mycoplasma pneumonia as model system, we integrated cellular cryo-electron tomography followed by sub-tomogram analysis (STA) and in-cell crosslinking mass spectrometry (CLMS) to investigate the coupling in native cells, without resorting to labelling or cell disruption. By averaging sub-tomograms extracted in silico from cellular volumes, we solved the 70S ribosome structure at 6.4 Å resolution, so far the highest resolution reported for in-cell cryo-ET. After large-scale classification, we determined the structure of a super-complex consisting of ribosome, RNA polymerase and other auxiliary proteins. Protein-protein interaction networks revealed by in-cell CLMS indicated an essential transcription elongation factor bridging RNA polymerase and the translating ribosome. Integrative modelling suggests a novel, indirect transcription-translation coupling mechanism, which advances our understanding of key machineries of the central dogma of molecular biology in bacteria. Methodology wise, our work demonstrates the feasibility and superiority of integrative structural biology performed directly inside cells. Integration of two approaches, cryo-ET/STA and in-cell CLMS, holds great potential to delineate cellular processes in a quantitative and structural view.