PROJECT SUMMARY/ABSTRACT This application describes our research into essential molecular pathways of the human pathogen, Mycobacterium tuberculosis (Mtb), including studies of transcription regulation and DNA repair. Infection with Mtb results in over 10 million new cases of tuberculosis and 1.5 million deaths annually, making it the deadliest infection in the world. In addition, this health crisis continues to be exacerbated by the emergence of drug- resistant strains, which demands the discovery of new antibiotic agents. In addition, we are deepening and broadening our biophysical work elucidating mechanisms of eukaryotic transcription initiation via both ensemble and single-molecule experiments coupled with kinetic modeling of the process in both yeast and humans. Transcription is responsible for changes in gene expression patterns during development or in adaptation to environmental conditions. The recruitment of RNA polymerase (RNAP) to particular genes at particular times is performed by sets of general and gene-specific transcription factors during transcription initiation. We are studying the essential, operator-independent, global transcription factors of Mycobacterium tuberculosis, CarD and RbpA. These factors act by modulating the rates of isomerization into and out of the open complex intermediate in initiation and, contrary to intuition, appear able to act as either activators or repressors without recognizing DNA sequence directly. We will answer critical questions in the field regarding the sequence- and sigma-factor (i.e., stress-response) dependence of these factors as well as their roles in post-initiation phases of transcription. We are also studying links between the transcription and DNA repair in Mtb. Mycobacteria lack classically conserved mismatch repair pathways (MMR) and possess repair factors not seen in E. coli. In addition, we have recently uncovered a novel oxidative switch that activates the Mtb nucleotide excision repair enzyme (NER), UvrD1. We are currently investigating the biophysical nature of this switch, alternative activation pathways, and the ability of UvrD1 to interact with RNAP during transcription-coupled NER. Of particular interest, and providing a link between our studies, is the shared RNAP-binding site used by both CarD and UvrD1. Lastly, we are continuing our investigations of the kinetic intermediates underlying pre-initiation-complex (PIC) dependent transcription initiation. Specifically, we are determining the mechanism of DNA bubble expansion during initial transcription in both yeast and humans. Our single-molecule magnetic-tweezers experiments will provide high-resolution views of the mechanism of PIC function. We are also following up on our recent discoveries of differences between the activities of yeast and human TFIIH (the general transcription factor required for promoter unwinding) that may underly the distinct usage of transcription-start sites in these organisms. As PIC function...