Fig. 1: Biochemical characterisation of the F plasmid relaxosome.

a SDS-PAGE gel of the relaxosome. Purified TraI, TraY, IHF, and TraM were mixed with oriT₃₁₆ and unbound components were excluded by size-exclusion chromatography, leading to a purified relaxosome. Molecular Weight markers are labelled and n = 6 independent experiments. For the uncropped gel image see source data file. For structural studies, the complex was stabilised by cross-linking with glutaraldehyde. We do not believe that crosslinking introduces bias in our case, since our structure recapitulates prior biochemical and structural knowledge on individual proteins. b oriT sequences protected from DNase I nuclease cleavage. The sequenced fragments represented by blue lines are mapped against oriT₃₁₆. The length of each fragment is shown. The various binding sites for each relaxosome protein as defined by Ilangovan et al.¹⁴ for TraI¹⁴ and by Frost et al., and Lum et al.^11,69 for IHF, TraM, and TraY are shown under oriT₃₁₆ using boxes colour-coded red for IHF, yellow for TraY, dark blue and orange for TraI_helicase and TraI_TE, and magenta for TraM. When present in the DNA fragment, the position of the nic site is shown by a black filled triangle. c DNAs used in this study. The DNAs are labelled according to the following notation: DNAs are numbered relative to the nic site on the T-strand. Therefore, nic serves as the origin, with the bases or base pairs 3’ to it being negatively numbered, and the bases and base pairs 5’ to it being positively numbered. This notation provides instant recognition of the position of nic in the DNA used. Furthermore, “ss” and “ds” DNA is used to indicate which region of oriT is single-stranded or double-stranded in the DNA used. For example, ss_{-27_+8}ds_{+9_+143} is a DNA which is single-stranded from base +8 to −27 and double-stranded from +9 to +143. The various binding sites for each relaxosome proteins as discovered in this study are shown underneath. Box colour-coding is as in panel b except for the three TraY molecule train which are coloured pale yellow, bright yellow, and olive green for TraY1, TraY2, and TraY3, respectively. The position of the nic site (black arrow) is shown.