Extended Data Fig. 3: Structural interrogation of UvrD CTD interactions in TCRC.

Supplementary to Fig. 3a. a–g, Structural analysis of UvrD CTD–RNAP interactions. a, UvrD and RNAP β-subunit domains observed in crystals structure were coloured and labelled. b, The overall structure of the UvrD CTD–RNAP β2i4 (PDB: 7EGS, Extended Data Table 1) complex. The major interface is highlighted by a rectangle. The ‘N’ and ‘C’ termini of UvrD CTD are numbered. c, Detailed UvrD CTD–RNAP β2i4 interactions. Oxygen, nitrogen, and water atoms are coloured in red, blue, and orange, respectively. Blue dash, H-bond. d, Yeast two-hybrid assay results show that alanine substitution of interface residues on UvrD CTD or βi4 impairs the interaction of UvrD and RNAP β pincer. The potential interactions were selected on SD (-HALW) plates, and the growth on SD (-LW) plates was used as input control. e, Strep-tag pull down results show that alanine substitution of interface residues of UvrD CTD or βi4 impairs RNAP–UvrD interaction. f, Sequence alignments of UvrD CTD and RNAP β2i4 from 316 non-redundant proteobacteria that contain βi4 insertion on RNAP. Key interface residues were labelled with asterisks and numbered as in E. coli. g, Cys pair cross-linking results demonstrate direct proximity of UvrD CTD and RNAP βi4. Wild-type or mutated UvrD-RNAP complexes were incubated in oxidative (CuCl₂) or reducing (DTT) condition and separated by SDS-PAGE.The asterisk marks two major impurity bands. h–m, Structural analysis of UvrD CTD–UvrB interactions. h, UvrD and UvrB domains (numbered as in E. coli). The domains observed in crystal structure are highlighted in colours. i, UvrD CTD interacts with UvrB-1a/1b/2domain (or UvrD NTD), consistent with a previous report³⁵. E. coli UvrD and UvrB were fused to GAL4-AD and GAL4-DBD, respectively. The potential interactions were selected on SD (-HALW) plates, and the growth on SD (-LW) plates was used as input control. j, A 2.6-Å crystal structure of T. thermophilus UvrD CTD–UvrB NTD complex (PDB: 7EGT, Extended Data Table 1). UvrB-1a docks on a shallow groove of UvrD CTD. UvrD CTD, UvrB-1a, UvrB-1b, and UvrB-2 are coloured in purple, cyan, orange, and light green, respectively. k, Detailed UvrD CTD–UvrB-1a interactions. Residues H654, R656, K680, R681, S683 of UvrD CTD make a H-bond network with D27, E29, R30, Q383 of UvrB-1a (residues are numbered as in T. thermophilus; the corresponding residues in E. coli are indicated in parentheses). Y686 of UvrD CTD makes stacking interaction with R55 and Q383 of UvrB-1a. l, Structural superimposition of UvrB NTD/UvrD CTD complex (coloured as above) and UvrB/dsDNA complex (grey and red; PDB: 6O8F)¹⁰² shows that UvrD CTD binds the opposite surface of UvrB dsDNA-loading cleft, implicating UvrD doesn’t affect dsDNA loading Of UvrB. m, Structural comparison between UvrD CTD/UvrB NTD (left) and UvrD CTD/RNAP β2i4 (right, PDB: 7EGS) shows that UvrB and RNAP β2i4 binds at the same cleft of UvrD CTD, and thereby suggests that the interactions of UvrB and RNAP to UvrD are mutually exclusive.