Prokaryotic Transcription Termination R37GM041376-R01GM041376 - Competitive Renewal Resubmission 1

PROJECT SUMMARY In the bacterium Escherichia coli, ~40% of transcription termination is factor-dependent termination, mediated by the ATP-dependent RNA translocase Rho, and ~60% of transcription termination is intrinsic termination, mediated by sequences at which transcription yields an RNA hairpin followed by an RNA U-tract. In the archaeon Thermococcus kodakarensis, most transcription termination is factor-dependent termination, mediated by the endoribonuclease and processive exoribonuclease aCPSF1 (archaeal cleavage and polyadenylation specificity factor 1). Bacterial and archaeal transcription initiation and transcription elongation and their regulation are well understood, but bacterial and archaeal transcription termination and its regulation are poorly understood. Structures of a minimal functional factor-dependent pre-termination complex, a minimal functional intrinsic pre-termination complex, and a post-termination complex have become available only very recently, and no structural information whatsoever is available for steps in termination between formation of a pre-termination complex and formation of a post-termination complex. We propose to determine structures of intact, complete functional bacterial and archaeal factor-dependent pre-termination complexes, to determine structures of intact, complete functional bacterial intrinsic pre-termination complexes, and, most important, to obtain structural and functional information for intermediates in termination between formation of a pre-termination complex and formation of a post-termination complex. Our results will test directly the "RNAP hypertranslocation," "RNA extraction," and "allosteric" hypotheses for termination, will distinguish directly between "external force" and "invasion" models for termination, and will define, comprehensively, the protein-DNA, protein-RNA, and protein-protein interactions in factor-dependent termination, intrinsic termination, and Q-dependent antitermination. The proposed work will use cryo-EM, single-molecule picometer-resolution nanopore tweezers (SPRNT), and deep sequencing to address three Specific Aims: Aim 1: Define the structural and mechanistic basis of factor-dependent transcription termination Aim 2: Define the structural and mechanistic basis of intrinsic transcription termination Aim 3: Define the structural and mechanistic basis of Q-dependent transcription antitermination The proposed work will contribute directly to understanding bacterial and archaeal transcription and transcriptional regulation. Because bacterial RNAP and, especially, archaeal RNAP show sequence, structural, and mechanistic similarities to eukaryotic RNAP, the results also will contribute to understanding eukaryotic transcription and transcriptional regulation.