Figure 5

Crystal structure of the Mu transpososome and Mu DNA topology. The figures in (A) and (B) are reproduced with permission from Phoebe Rice [35]. (A) Structure of the Mu transpososome engaged with cleaved Mu ends joined to target DNA. (B) Schematic of the structure in A, illustrating positions of the various MuA domains and DNA segments. Full length MuA protein is 630 residues long. The polypeptide in the crystal structure includes residues 77 to 605; it is missing the regulatory N (Iα)- and C (IIIβ)-terminal domains that interact with the enhancer, and with proteins MuB and ClpX, respectively. Catalytic sites are marked as tan/yellow stars in B. The donor DNA in the crystallized complex consists of two equivalent right ends, each with two MuA binding sites. It shows one right-handed DNA crossing. (C) On native Mu DNA, the left (L) and right (R) ends have three MuA binding sites each, and are non-equivalent with regard to their spacing and orientation. A third enhancer (E) segment is essential for assembly of a functional transpososome. The enhancer is positioned closer to the L end on the Mu genome. In a transpososome assembled on this native configuration of the L, E, R segments, six MuA subunits bound via their Iβγ domains to the L (L1-L3) and R (R1-R3) ends, make bridging interactions with the enhancer (E: O1-O2) via their Iα domains to trap five supercoils - two L-R, two R-E and one L-E crossings - as indicated (see [37]). R-E interactions initiate assembly and are essential [38]. When the transpososome is treated with high salt or heparin, a stable tetrameric core remains, which still retains two L-R and one R-E DNA crossing (black arrow heads) [39]. Of the two L-R crossings, the one at the top of the diagram, is likely the one seen in the crystal structure (A, B). Placement of the second L-R crossing is arbitrary, but note that this crossing as well as the proximal R3-E crossing is maintained by the tetramer [39]. See [38] for details.