| Multi-subunit RNA polymerases (RNAPs) catalyze a series of key reactions to initiate transcription from promoters, extend the RNA transcripts, select correct substrates, respond to regulatory signals such as pause signals, proof-read nascent RNA transcripts by hydrolytic cleavage, and terminate transcription at ends of transcription units. However, the underlying catalytic-center mechanisms and dynamics responsible for these essential functions are poorly understood. Recent crystal structures of transcription elongation complexes unveiled potential key functions of an active-site proximal, conformationally flexible protein module termed the trigger loop/helices. Research work described in this thesis investigates and helps define the biochemical functions of the trigger loop/helices module in transcript elongation, pausing, and proof-reading. Specifically, transient formation of the trigger helices (an alpha-helical hairpin) stabilized by an adjacent bridge helix via a metastable three-helix-bundle provides up to 10,000x rate assistance in achieving the transition state for nucleotidyl transfer, and does so by sterically positioning/aligning reactants in the catalytic center. Interference of such trigger loop/helices dynamics contributes to the regulation of elongation by transcriptional pauses, and is exploited by small molecules (antibiotics, toxins) to directly shut off transcript elongation. In stark contrast, the trigger helices module does not directly contribute to hydrolytic cleavage of RNA but likely unfolds to facilitate backtracking and engagement of cleavage factors. Finally, evolutionarily acquired, lineage-specific sequence insertions in RNAP modify and extend the function and regulation of the trigger loop/helices module. |
