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bioRxiv · 10.1101/2025.09.05.674597

Structural basis for loading of Transcription Repair-Coupling factor Mfd onto stalled elongation complexes

Brewer, J.·Llewellyn, E.·Campbell, E. A.·Darst, S. A.
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Abstract

Transcription-coupled repair (TCR) is a nucleotide excision repair sub-pathway that preferentially removes lesions from the DNA template-strand stalling RNA polymerase (RNAP) elongation complexes (ECs). In bacteria, the superfamily 2 Mfd translocase mediates TCR by displacing stalled ECs and recruiting Uvr(A)B. Using cryo-electron microscopy, we previously visualized seven Mfd-EC complexes spanning the ATP-dependent Mfd loading and EC displacement pathway [L1 -> L2(ADP) -> C1(ATP) -> C2(ATP) -> C3(ADP) -> C4(ADP) -> C5(ATP)]. The first intermediate (L1) was poorly resolved (4.1 [A] nominal resolution) due to low particle occupancy. The pathway is characterized by very large Mfd structural transitions, notably the L1 -> L2 transition. Here, we pre-loaded Mfd with ATP in the presence of the{gamma} -phosphate mimic, BeF3-, limiting rounds of ATP hydrolysis. The resulting accumulation of early intermediates allowed us to resolve the L1 intermediate to 3.5 [A] nominal resolution, revealing bound ADP-BeF3-. We also identified a new intermediate between L1 and L2, L1.5, providing further insight into Mfd conformational changes during loading. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/674597v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1472098org.highwire.dtl.DTLVardef@acfca3org.highwire.dtl.DTLVardef@fe4424org.highwire.dtl.DTLVardef@12bc521_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Brewer, J., Llewellyn, E., Campbell, E. A., Darst, S. A.. 2025-09-06. Structural basis for loading of Transcription Repair-Coupling factor Mfd onto stalled elongation complexes. https://doi.org/10.1101/2025.09.05.674597

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