Structural basis of transcription-translation coupling and collision in bacteria

Abstract

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The pioneering ribosome potentially contacts RNA polymerase (RNAP), forming a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here we present a series of structures representing uncoupled, coupled and collided expressome states determined by electron cryomicroscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, stabilizing an otherwise variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance-channel to the RNAP exit-channel. In this collided complex, NusG-linkage is no longer possible. These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.

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). If the ribosome approaches the RNAP further, the collided state, in which the ribosome translocation and the factor-mediated coupling are no longer possible, forms (Wang C. et al, 2020;Webster et al, 2020). Preventing such unproductive collisions may be another function of NusA and NusG.…”

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World

“…Attempts to gain structural insights into bacterial expressomes were based on two approaches: (i) cryo-EM of samples formed by direct reconstitution of purified Escherichia coli components on mRNA substrates, which direct a precise spacing between RNAP and the 70S ribosome ( Wang et al, 2020 ; Webster et al, 2020 ), or (ii) direct visualization using cryo-ET in combination with in-cell cross-linking mass spectrometry in Mycoplasma pneumoniae ( O’Reilly et al, 2020 ). With sufficient mRNA separating the two machineries, RNAP adopts a wide range of orientations, the assembly is highly flexible, and the mRNA is the only consistent connection ( Figure 1A ).…”

“…With sufficient mRNA separating the two machineries, RNAP adopts a wide range of orientations, the assembly is highly flexible, and the mRNA is the only consistent connection ( Figure 1A ). In E. coli , adding NusG restrains RNAP and aligns the mRNA with the ribosomal helicase ( Figure 1B ), proposed to prevent secondary structure formation in the transcript ( Webster et al, 2020 ). Addition of the TF NusA stabilizes the NusG-coupled expressome ( Wang et al, 2020 ; Figures 1D , E ).…”

“…However, recent work in Bacillus subtilis showed that coupling of transcription and translation is not conserved across all bacteria ( Johnson et al, 2020 ). Recently, single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) was used to elucidate structural details of the coupled bacterial RNAP and ribosome, a macromolecular assembly termed “expressome.” It highlighted roles of transcription elongation factors NusG and/or NusA that physically connect RNAP with the ribosome ( Demo et al, 2017 ; Kohler et al, 2017 ; O’Reilly et al, 2020 ; Wang et al, 2020 ; Webster et al, 2020 ).…”