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Principal Investigator: Eric A Galburt
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2024
Award: $466,500
Funding agency: National Institute of General Medical Sciences
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 underlies gene expression, our unique approaches will provide important advances
in the study of human biology.
Terms: <Antibiotic Agents><Antibiotic Drugs><Antibiotics><BTF2 transcription factor><Basal Transcription Factor><Basal transcription factor genes><Binding Sites><Biochemical Pathway><Biochemistry><Biological Chemistry><Biophysics><Cessation of life><Combining Site><Complex><Coupled><DNA><DNA Damage Repair><DNA Repair><DNA Sequence><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Death><Deoxyribonucleic Acid><Dependence><Development><Drug resistance><E coli><E. coli><Escherichia coli><Eukaryota><Eukaryote><Expression Signature><Gene Action Regulation><Gene Expression><Gene Expression Profile><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Transcription><Genus Mycobacterium><Health><Human><Human Biology><Infection><Intuition><Investigation><Isomerism><Kinetics><Knowledge><Link><M tb><M tuberculosis><M tuberculosis infection><M. tb><M. tb infection><M. tuberculosis><M. tuberculosis infection><M.tb infection><M.tuberculosis infection><MTB infection><Magnetism><Metabolic Networks><Miscellaneous Antibiotic><Mismatch Repair><Modern Man><Molecular><Mycobacterium><Mycobacterium tuberculosis><Mycobacterium tuberculosis (MTB) infection><Mycobacterium tuberculosis infection><Nature><Nucleotide Excision Repair><Organism><Pathway interactions><Phase><Post-Replication Mismatch Repair><Process><RNA Expression><RNA Polymerases><Reactive Site><Research><Resolution><Role><Sigma Element><Sigma Factor><Sigma Initiation Factor><Sigma Subunit><TB infection><TFIIH><Transcription><Transcription Factor Proto-Oncogene><Transcription Initiation><Transcription Initiation Site><Transcription Regulation><Transcription Start Site><Transcription factor genes><Transcription-Coupled Nucleotide Excision Repair><Transcription-Coupled Repair><Transcriptional Control><Transcriptional Regulation><Tuberculosis><Unscheduled DNA Synthesis><Work><Yeasts><bacteria pathogen><bacterial pathogen><basic transcription factor 2><biological adaptation to stress><biophysical foundation><biophysical model><biophysical principles><biophysical sciences><developmental><disseminated TB><disseminated tuberculosis><drug resistant><experiment><experimental research><experimental study><experiments><gene expression pattern><gene expression signature><human pathogen><infection due to Mycobacterium tuberculosis><interest><intuitive><isomer><kinetic model><living system><magnetic><mtb><novel><pathogenic bacteria><pathway><promoter><promotor><reaction; crisis><recruit><repair><repair endonuclease><repair enzyme><repaired><resistance strain><resistance to Drug><resistant strain><resistant to Drug><resolutions><single molecule><social role><stress response><stress; reaction><transcription factor><transcription factor IIH><transcription factor TFIIH><transcriptional profile><transcriptional signature><tuberculosis infection><tuberculous spondyloarthropathy>