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bioRxiv · 10.1101/2024.01.11.575193

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase

Qayyum, M. Z.·Imashimizu, M.·Leanca, M.·Vishwakarma, R. K.·Riaz-Bradley, A.·Yuzenkova, Y.·Murakami, K.
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Abstract

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, {beta}1 and {beta}2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the {beta}2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms. Significance statementCellular RNA polymerase (RNAP) carries out RNA synthesis and proofreading reactions utilizing a mobile catalytic domain known as the trigger loop/helix. In cyanobacteria, this essential domain acquired a large Si3 insertion during the course of evolution. Despite its elongated shape and large swinging motion associated with the transition between the trigger loop and helix, Si3 is effectively accommodated within cyRNAP, with no impact on the fundamental functions of the trigger loop. Understanding the significance of Si3 in cyanobacteria and chloroplasts is expected to reveal unique transcription mechanism in photosynthetic organisms.

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Qayyum, M. Z., Imashimizu, M., Leanca, M., Vishwakarma, R. K., Riaz-Bradley, A., Yuzenkova, Y., Murakami, K.. 2024-01-11. Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase. https://doi.org/10.1101/2024.01.11.575193

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