Fig. 5: Hub architecture of the ssDNA-27_+8ds+9_+143-R fully assembled relaxosome structure: overview of hubs 2 and 3, and footprint of relaxosome proteins on DNA.
a Hub2. Top: protein-protein interactions between TraY molecules. Residues at protein-protein interfaces are shown in spheres. Bottom: TraYs-DNA interactions. Boundary base pairs for each binding site are reported. TraY’s binding to DNA is typical of RHH dimers with the β1 and β2 strands interacting with the major groove of the DNA binding site79. But binding of TraY1 and TraY2 (but not TraY3 which is inverted) also extends to the minor groove. TraY1 interaction with TraY2 involves two clusters, but only one is involved in interactions between TraY2 and TraY3 (detailed in Supplementary Fig. 8). b Hub3. TraI TE interactions with DNA. Left: view highlighting ssDNA-binding with primarily the β6-β7, αH-αI and β10-β11 loops but also dsDNA-binding, primarily with the αC-β3 loop. Right: view 180° away, highlighting dsDNA interaction with primarily with the αC-β3, β4-β5, and β9-αF loops. The nic site is shown as are the binding site boundaries on the DNA (ssDNA between -2 and +9 and dsDNA between +10 and +15). The unpaired +9 base is labelled. c Hub3 (continued): protein-protein interactions between TraI TE and VH2A+2B/2B-like domains. TraI TE is shown in orange ribbon except the residues interacting with VH2A+2B/2B-like shown in green. VH2A+2B/2B-like is in ribbon and residues interacting with the TE and R-strand are shown in orange and light blue sticks. Residues and secondary structures involved in binding are labelled. One particular loop (αC-β3) stabilises 122 Å2 of surface area via interactions with TraI VH2B/2B-like residues in β10 and the αN-β14 loop. d Footprint of relaxosome proteins on DNA. ssDNA and dsDNA bases involved in binding relaxosome components (including TraM, details in Supplementary Fig. 9c) are colour coded according to the protein they contact (red and pink letters for IHFαβ, green letters for TraIVH2A+2B/2B-like, orange letters for TraI TE, and pale yellow, bright yellow, and olive green highlights for TraY1, TraY2, and TraY3, respectively, and magenta highlights for TraM). When two proteins have overlapping binding sites, the sequence is repeated above and coloured accordingly. The TraM footprint is shown above and under the R- and T-strand, respectively, in the sbmC region. Solid lines above the R-strand TraM site reports on previous findings based on hydroxyl radical protection assays.
