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. 2000 Jul 15;14(14):1750–1764.

Figure 4.

Figure 4

(A,B) Analysis of protein-directed DNA bending by permutation gel electrophoresis. (A) Probes (each 140 bp) contain fbe site at positions shown; probes 3 and 4 contain SRY target site 5′-TTTGTT-3′. GMSA in B demonstrates very weak dependence of DM–fbe complexes c1 and c2 on fbe position in probe (lanes 1–4). Lanes 6 and 7 illustrate SRY control using probes 3 and 4 (Geise et al. 1992). The weak band under major SRY–DNA complex arises from proteolytic fragment; more slowly migrating species represent higher order complexes. Free probe 3 is shown in lane 5. (C) Tail mutant R91Q impairs specific DNA binding but not cooperativity. GMSA used 33P-labeled 33-bp fbe probe (1.5 nm). (Lanes 2–7) Native DSX domain (respective protein concentrations 4, 8, 12, 18, 24, and 48 nm); (lanes 8–16) R91Q variant domain (respective protein concentrations 12, 24, 48, 96, 120, 156, 240, 480, and 560 nm); the free probe is shown in lane 1. At 48 nm concentrations the native domain is 52% shifted, whereas shift of variant domain is negligible (<1%). Complexes c1 and c2 represent 1:1 and 2:1 binding of domain to dsxA; c3 represents a higher order complex observed at high protein concentrations (>100 nm).