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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
112
28-POS
Board 14
Single-Molecule Imaging in Live Cells Reveals Kinetics of Transcription-Coupled Repair
Han N. Ho
, Harshad Ghodke, Antoine M. van Oijen.
University of Wollongong, Wollongong, NSW, Australia.
Transcription-coupled repair (TCR) is a highly conserved process responsible for the targeted
removal of lesions that stall RNA polymerases on the transcribed strand. In
Escherichia coli
,
TCR is mediated by the repair factor Mfd. On binding to stalled RNA polymerases, Mfd
displaces the polymerase and subsequently recruits the nucleotide excision repair machinery. The
molecular mechanisms of Mfd recruitment to transcription complexes remain to be elucidated in
live cells. Using single-molecule imaging, we imaged Mfd in live
E. coli
cells and found that a
significant fraction of Mfd associates with transcription elongation complexes even in the
absence of genotoxic stress. Further, we identify two populations of bound Mfd: a long-lived
fraction representing Mfd in TCR and a sub-second fraction, presumably representing Mfd in the
search mode. Using inhibitors of RNAP that promote stalling, we found that Mfd displays a new
kinetic mode, reflecting the kinetics of drug-stalled RNA polymerases. These results provide
insights into the mechanism of transcription-coupled repair in
E. coli
.