Thursday, April 3, 2025
4/3/2025
Structural and functional studies of the mycobacterial transcription cycle - Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an ongoing global health crisis. The WHO reports 10 million active cases and over 1.5 million deaths annually. RNA polymerase (RNAP), the enzyme responsible for all transcription in bacteria, is the target for rifampicin, a first-line treatment for TB. RNAP is thus a proven and attractive target for developing new drugs. These facts highlight the importance of our recent structural and functional characterization of Mtb RNAP, and the essential transcription factors required for full transcriptional activity and intrinsic antibiotic resistance. In addition, we have found that studying diverse clades of bacteria deepens our understanding of general principles of transcription. Therefore, we study Actinobacteria, as the biophysical and molecular mechanisms of transcription in this clade are relatively understudied and because it includes important pathogens. We determined high-resolution cryo-EM structures of several Mtb initiation transcription complexes with essential transcription factors and important antibiotics in the previous funding period. Here my vision is to complete the characterization of initiation regulation and expand our focus to the post-initiation steps throughout the transcription cycle. These steps include elongation, pausing (a regulatory step where the RNAP temporarily halts at specific sequences, permitting input from diverse signals), and termination. We will also continue characterizing new RNAP inhibitors. To complete our understanding of initiation, we will continue our studies of the WhiB factors, focusing on the essential factor WhiB1, which is required for Mtb viability and response to stress. To characterize post-initiation steps, we plan to study the mechanisms of mycobacterial NusA and NusG. NusG in Mycobacteria increases transcriptional pausing and termination, yet it suppresses both in the well-characterized E. coli. This difference in activity emphasizes the need to study these factors in other clades of bacteria. Most of these studies will involve single-particle cryo-EM, but we plan to extend our studies in situ by using cryo-electron tomography to investigate cellular processes and organizations, such as ribosomal coupling to RNAP. To rigorously study how transcription factors act genome-wide, we will finish developing cell-free genomics: using genomic DNA as a substrate for purified RNAP and transcription factors, and quantifying resultant transcripts with RNA-seq. This method is necessary to identify native DNA sequences regulated by essential factors WhiB1, NusA, and NusG, whose regulons have remained elusive due to pleiotropy upon perturbation in cells. In sum, I envision using a multidisciplinary approach that includes structural, genetic, biochemical, genomic, and in situ experiments to understand the roles and mechanisms of each step in the transcription cycle and how they are regulated by cis and trans-acting elements. The results from this proposal have the potential to elucidate the molecular and biological mechanisms of the entire transcription cycle.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).