Fig. 2.

A trimer of Xis cooperatively binds DNA. (A) Cooperative Xis binding to long attR DNA fragments. Increasing concentrations of Xis were incubated with a ³²P-labeled attR fragment (−220 to +43) and electrophoresed in a native polyacrylamide gel. (B) Xis binding to short attR DNA fragments (−104 to −51) exhibits reduced cooperativity. 1X, 2X, and 3X represent DNA complexes postulated to contain one, two, and three Xis monomers, respectively. (C) Cross-linking of Xis–attR complexes. ³²P-labeled Xis^HMK was incubated with fluorescein-labeled attR DNA (−104 to −54), subjected to cross-linking with BS³, and electrophoresed in a native gel as in B. Xis complexes representing 2X and 3X were extracted from the native gel and subjected to SDS/PAGE. The number of cross-linked Xis monomers in each product band is based on their apparent molecular mass. (D) Cross-linking of ^Δ55Xis–attR complexes. Protocol was the same as in C except that ³²P-labeled ^Δ55Xis^HMK was subjected to cross-linking with BS³ (11-Å spacer) or DSG (8-Å spacer), and the cross-linked products were extracted from the dominant slowest migrating complex. (E) Binding of Int^1–64 and two Xis protomers to attR fragments missing the X2 site. Xis (0.53 μM) and ^HisInt^1–64 (3 μM) were incubated with a ³²P-labeled 45-bp probe (−121 to −77) containing the P2 and X1 binding sites as designated and electrophoresed in a native polyacrylamide gel. The identities of each of the bands based on their electrophoretic mobilities are denoted. The sequences of the oligonucleotide substrates are given in SI Fig. 6.