A Structural Perspective on Bacterial Transcription

Author

Joshua John Brewer

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Transcription is a highly conserved process that lies at the very center of biology. As in the other two domains of the tree of life, bacterial transcription is a multistep process. In the first step, RNA polymerase (RNAP) contacts with the promoter DNA are established and then must be broken for the enzyme to transition into the elongation phase of RNA production; a process known as promoter escape. While single-molecule and biochemical observations report that promoter escape is a highly regulated and sometimes rate-limiting step in the transcription cycle, the structural details remain obscure. Promoter escape also serves as the target for the clinically important antibiotic rifampicin (Rif), used to treat tuberculosis. In this work, we first provide the structural details of M. tuberculosis RNAP escaping from a promoter using a de novo cryoelectron microscopy approach, revealing seven distinct intermediates. We reveal an unanticipated level of structural rearrangement that RNAP undergoes to clear the promoter, including those required to release the initiation factor, σ, providing mechanisms to decades of biochemical observations. These structures and supporting biochemistry are consistent with a model of promoter escape that includes unexpected conformations exploitable in the development of Rif-alternatives. After RNAP achieves promoter escape, it transitions into the elongation phase of transcription. This elongation phase is vulnerable to bulky helix-distorting DNA lesions capable of stalling elongating RNAP in its tracks. Transcription-coupled repair (TCR) is a sub-pathway of the nucleotide excision repair pathway that preferentially removes lesions from the DNA template-strand. These lesions stall RNAP elongation complexes (ECs). The SF2 MFD translocase mediates TCR in bacteria by removing stalled RNAP from DNA lesions and recruiting appropriate TCR factors. Previously, we used cryoelectron microscopy to visualize MFD engaging with and attempting to displace the EC, revealing seven MFD-EC complexes spanning the MFD loading and EC displacement pathway. However, the first MFD-EC loading intermediate (L1) was poorly resolved and the transition from L1 to the second loading intermediate (L2) was unclear. To investigate further, we pre-loaded MFD with ATP in the presence of a y-phosphate mimic, BeF_3- , limiting rounds of ATP hydrolysis by MFD before being trapped by BeF_3- binding. This biochemical strategy allowed us to improve L1 resolution, revealing the nucleotide occupancy (ATP). We also identified an additional loading intermediate between L1 and L2 (L1.5) that clarifies the transition from L1 to L2. After the elongation phase concludes, transcription terminates. Following transcript release during intrinsic termination, Escherichia coli (E. coli) RNAP often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from the DNA by the Swi2/Snf2 ATPase RapA. In this work, we determined PTC structures on negatively-supercoiled DNA as well as of RapA engaged to dislodge the PTC. We found that core RNAP in the PTC can unwind DNA and initiate RNA synthesis but is prone to producing R-loops. We show that RapA helps to control cytotoxic R-loop formation invivo, likely by disrupting PTCs. Nucleotide binding to RapA triggers a conformational change that opens the RNAP clamp, allowing DNA in the RNAP cleft to reanneal and dissociate. We suggest that analogous ATPases acting on PTCs to suppress transcriptional noise and R-loop formation may be widespread. These results hold significance for the bacterial transcription cycle and highlight a role for RapA in maintaining genome stability. Overall, these studies come together to provide a deeper and ultimately revised perspective on the mechanisms underlying bacterial transcription.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Brewer, Joshua John, "A Structural Perspective on Bacterial Transcription" (2024). Student Theses and Dissertations. 794.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/794