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

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 Å 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 γ-phosphate mimic, BeF3- , limiting rounds of ATP hydrolysis. The resulting accumulation of early intermediates allowed us to resolve the L1 intermediate to 3.5 Å 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.

SUBMITTER: Brewer J 

PROVIDER: S-EPMC12424862 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature