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

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 $\text{ATP}$ -dependent Mfd loading and EC displacement pathway $[L1\text{-}>L2(\text{ADP})\text{-}>C1(\text{ATP})\text{-}>C2(\text{ATP})\text{-}>C3(\text{ADP})\text{-}>C4(\text{ADP})\text{-}>C5(\text{ATP}\left)\right]$ . 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 $L1\text{-}>L2$ transition. Here, we pre-loaded Mfd with $\text{ATP}$ in the presence of the γ-phosphate mimic, ${{\text{BeF}}_3}^{\text{-}}$ , limiting rounds of $\text{ATP}$ hydrolysis. The resulting accumulation of early intermediates allowed us to resolve the $L1$ intermediate to 3.5 Å nominal resolution, revealing bound $\text{ADP-}{{\text{BeF}}_3}^{\text{-}}$ . We also identified a new intermediate between $L1$ and $L2$ , $L1.5$ , providing further insight into Mfd conformational changes during loading.