Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

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 .

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