STRUCTURAL BIOLOGY

46 ESRF

Structural basis of transcription-translation coupling and collision in bacteria, M.W. Webster (a), M. Takacs (a), C. Zhu (a), V. Vidmar (a), A. Eduljee (a),

M. Abdelkareem (a) and A. Weixlbaumer (a), Science 369, 1355-1359 (2020); https:// doi.org/10.1126/science.abb5036. (a) Institut de Génétique et de Biologie

Moléculaire et Cellulaire (IGBMC), Illkirch (France)

[1] K. McGary & E. Nudler, Curr. Opin. Microbiol. 16, 112-117 (2013). [2] B.M. Burmann et al., Science 328, 501-504 (2010). [3] R. Kohler et al., Science 356, 194-197 (2017).

PRINCIPAL PUBLICATION AND AUTHORS

REFERENCES

While its importance has long been appreciated, the molecular mechanisms that support transcription-translation coupling have been unclear. The transcription factor NusG has been proposed to mediate the physical coupling of ribosomes with RNAP as it can bind each of the two machineries [2]. However, more recent studies have indicated that the ribosome may also interact directly with RNAP. A transcribing- translating complex in which the ribosome is closely associated with RNAP has been reconstituted and termed the expressome , as it contains all stages of bacterial gene expression within a single supramolecular assembly [3]. However, a reconstruction of this complex revealed an architecture that did not permit NusG to form a bridge. The role of the expressome and of NusG in mediating transcription-translation coupling therefore remained uncertain.

This study aimed to improve understanding of the molecular mechanism of coupling. Single- particle cryo-EM at beamline CM01 was used to visualise the events that occur when a trailing ribosome contacts RNAP. A series of ribosome- RNAP complexes were assembled from purified E. coli components with different mRNA lengths connecting the two complexes, simulating a translating ribosome approaching RNAP. Complexes were also prepared with and without the transcription factor NusG to test if, and how, it forms a bridge.

Three-dimensional reconstructions of expressomes were determined at near-atomic resolution (Figure 31). All key stages of bacterial gene expression, from mRNA synthesis in the RNAP active site to mRNA decoding and peptide transfer in the ribosome, are visible in the structures. Importantly, a comparison of the different samples revealed a series of structurally distinct states and the basis of their interchange (Figure 32). This distinguishes between the collided expressome , which is the state that had been previously observed, and the NusG- coupled expressome , which had not.

In the first sample, the mRNA connecting the ribosome and RNAP was long, and NusG was not present. Here, the machineries are structurally uncoupled, with the positions of each complex varying relative to the other among the set of imaged particles. By contrast, complexes bound by the transcription factor NusG are more stable due to the formation of a physical bridge between the NusG N-terminal domain bound to RNAP and the NusG C-terminal domain bound to ribosomal protein uS10. In this configuration, RNAP is no longer positioned directly in line with the ribosome mRNA entrance channel, and the intervening mRNA is instead bound to a basic surface of ribosomal protein uS3. By binding the mRNA as it exits RNAP, it is likely that the ribosome inhibits the formation of mRNA secondary structures that are known to inhibit both transcription and translation.

When the mRNA connecting the complexes is shortened, a substantial rearrangement of the complex occurs, forming the collided state . RNAP is positioned close to the entrance channel of the ribosome, allowing the mRNA to transit directly from the site of synthesis to the site of decoding. In this state, NusG does not form a bridge between the complexes as the mRNA is not long enough to span the distance observed for the NusG-coupled complex. The surfaces of the ribosome and RNAP in this arrangement are strikingly complementary in shape. These insights into the structure and dynamics of expressome complexes provide a valuable framework for future investigations into the role of coupling in gene expression.

Fig. 32: Interchange between molecular states during transcription-translation coordination.