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In-cell architecture of an actively transcribing-translating expressome

By Francis J. O’Reilly, Liang Xue, Andrea Graziadei, Ludwig R Sinn, Swantje Lenz, Dimitry Tegunov, Cedric Blötz, Wim J. H. Hagen, Patrick Cramer, Jörg Stülke, Julia Mahamid, Juri Rappsilber

Posted 28 Feb 2020
bioRxiv DOI: 10.1101/2020.02.28.970111 (published DOI: 10.1126/science.abb3758)

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.

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