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PRINCIPAL PUBLICATION AND AUTHORS

Structural insights into RNA-mediated transcription regulation in bacteria, S. Dey (a,b,c,d), C. Batisse (a,b,c,d), J. Shukla (a,b,c,d), M.W. Webster (a,b,c,d), M. Takacs (a,b,c,d), C. Saint-André (a,b,c,d), A. Weixlbaumer (a,b,c,d), Mol. Cell 82(20), 3885-3900 (2022); https:/doi.org/10.1016/j.molcel.2022.09.020 (a) Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch (France) (b) Université de Strasbourg, IGBMC UMR 7104 UMR-S 1258, Illkirch (France) (c) CNRS, UMR 7104, Illkirch (France) (d) Inserm, UMR-S 1258, Illkirch (France)

RNA sequence, which signals RNAP to pause transcription and release the transcript. In bacteria, the RNA transcript additionally contains an RNA hairpin structure that accelerates RNA release. However, hairpins are not a universal requirement, and transcription termination by RNAP and U-rich signals occurs without the need for hairpin structures in eukaryotes, and in the absence of additional protein factors. RNA is even more versatile than that, because rather than terminating its own synthesis, it can also promote transcription and prevent termination. For example, a regulatory RNA called putL suppresses transcriptional pausing by RNAP, prevents transcription termination and therefore stimulates its own synthesis.

Experiments conducted at cryo-electron microscopy (cryo-EM) beamline CM01 crucially contributed to the understanding of how regulatory RNA elements can modulate RNAP without the need for additional protein factors and thus, the very enzyme responsible for its own synthesis. RNAP was trapped during the synthesis of putL RNA at a U-rich pause signal, and single-particle cryo-EM was used to visualise it at high resolution

(Figure 42a). The three-dimensional reconstruction made it possible to build and interpret an atomic model. The regulatory putL RNA binds RNAP in a way that stabilises the active form of the enzyme and disfavours adoption of the paused state. This was confirmed by obtaining structures with an altered putL RNA that lost its functionality. It was observed that RNAP adopts a pause- prone conformation at the U-rich signal. Unexpectedly, it was also found that a subset of RNAP molecules adopted a conformation that appeared to be trapped just before RNA transcript release. To confirm this, another set of cryo-EM reconstructions were determined in the complete absence of the regulatory RNA, and it was possible to visualise RNAP for the first time before and after RNA transcript release (Figure 42b).

The overall analysis of these cryo-EM structures provides a framework to understand how RNA is capable of stimulating its own synthesis. They further highlight intermediate states that occur during intrinsic transcription termination, a universally employed mechanism to release finished RNA products from RNAP.