In-cell architecture of an actively transcribing-translating expressome

Abstract

Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell cross-linking mass spectrometry, cellular cryo–electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.

Translational Control by Ribosome Pausing in Bacteria: How a Non-uniform Pace of Translation Affects Protein Production and Folding

“…In many bacteria, including E. coli , transcription and translation are coupled. The mRNA emerging from the RNA polymerase (RNAP) is recruited by the leading ribosome that resides in close proximity or is even physically linked to RNAP ( Landick et al, 1985 ; Burmann et al, 2010 ; Proshkin et al, 2010 ; Kohler et al, 2017 ; O’Reilly et al, 2020 ; Wang et al, 2020 ; Webster et al, 2020 ), forming the so-called expressome ( Kohler et al, 2017 ; Figure 1 ). The rates of transcription and translation in the expressome are coordinated and change together in response to growth conditions ( Vogel and Jensen, 1994 ).…”

NusG, an Ancient Yet Rapidly Evolving Transcription Factor

“…The TEC-ribosome complexes, stabilized by general transcription factors, have been observed in vitro using cryo-EM ( Wang C. et al, 2020 ; Webster et al, 2020 ) and analyzed inside cells using a combination of cross-linking mass spectrometry and cryo–electron tomography ( O’Reilly et al, 2020 ). Evidence suggests that coupling may occur initially via direct RNAP:ribosome contacts and then is aided by accessory factors ( Washburn et al, 2020 ).…”

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control

“…Leveraging this coupled system, bacteria have evolved a variety of motifs within the nascent mRNA, including riboswitches and specific sequence elements, that induce transcriptional pausing and backtracking (Zhang et al, 2010 ; Perdrizet et al, 2012 ; Steinert et al, 2017 ). Furthermore, Escherichia coli ( E. coli ) bacteria have even been shown to directly couple transcription and translation, with ribosomes binding to mRNA during active transcription elongation by RNAP (Kohler et al, 2017 ; O'Reilly et al, 2020 ). This feature allows for the precise orchestration of gene regulation through the formation of the tightly coupled and highly efficient machinery termed the “expressome” (Proshkin et al, 2010 ; Kohler et al, 2017 ; O'Reilly et al, 2020 ; Washburn et al, 2020 ).…”

“…Furthermore, Escherichia coli ( E. coli ) bacteria have even been shown to directly couple transcription and translation, with ribosomes binding to mRNA during active transcription elongation by RNAP (Kohler et al, 2017 ; O'Reilly et al, 2020 ). This feature allows for the precise orchestration of gene regulation through the formation of the tightly coupled and highly efficient machinery termed the “expressome” (Proshkin et al, 2010 ; Kohler et al, 2017 ; O'Reilly et al, 2020 ; Washburn et al, 2020 ). The signal of a small ligand affecting the local structure of a riboswitch can then be transduced into a profound change in expressome function through a wave of kinetic selection, creating a system analogous to the struggle of “David vs. Goliath,” where a tiny metabolic ligand has the ability to control the activity of the giant expressome (Ray et al, 2019 ).…”