Escherichia coli NusG Links the Lead Ribosome with the Transcription Elongation Complex

Abstract

Summary It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.

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 ). In the NusG/NusA coupled complex, the RNAP β’ subunit contacts the 30S subunit protein S3, NusA simultaneously binds to α/β subunits and S2/S5, and finally NusG binds to β/β’ and S10 ( Figure 7 ).…”

“…The closely coupled ribosome prevents the formation of R-loops and RNAP backtracking, thereby promoting genome stability ( Gowrishankar and Harinarayanan, 2004 ; Proshkin et al, 2010 ; Stevenson-Jones et al, 2020 ) and inhibits factor-independent termination by blocking the formation of nascent RNA hairpins ( Roland et al, 1988 ). The coupled ribosome also prevents mRNA degradation, by blocking the access of RNaseE ( Iost and Dreyfus, 1995 ), or premature Rho termination, by sequestering NusG and shielding the nascent RNA ( Washburn et al, 2020 ). When the coupling is broken, e.g., by the ribosome pausing or stalling, Rho releases the nascent RNA, a phenomenon known as polarity ( Richardson, 1991 ).…”

Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises

“…A cryo-electron microscopy (cryo-EM) structure of a TEC·NusG complex did not show any defined density for the NusG CTD, suggesting that NusG mediates a flexible RNAP-ribosome association ( Kang et al, 2018b ). This was confirmed by another cryo-EM structure of a ribosome-bound NusG, that showed a defined density for the CTD but not for the NTD ( Washburn et al, 2020 ). This latter publication also reported NMR results supporting the association of an RNAP·NusG complex with S10.…”

“…Several types of data indicate that ribosome translocation along the nascent mRNA to the proximity of the TEC may be a prerequisite for the formation of a physically coupled expressome. Firstly, NusG association with TECs, which occurs only after substantial transcription ( Mooney et al, 2009a ), is facilitated by cotranscriptional translation ( Washburn et al, 2020 ). Additionally, the intranucleoidal ribosome concentration (2–8 μM; Bakshi et al, 2012 ; Sanamrad et al, 2014 ) is substantially lower than the NusG·ribosome dissociation constant (50 μM; Burmann et al, 2010 ), so the NusG·ribosome association is likely favored by the translocation of the leading ribosome towards the TEC along the nascent mRNA.…”

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control

“…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 ).…”