GENETIC AND BIOCHEMICAL-ANALYSIS OF IHF HU HYBRID PROTEINS/

Integration host factor (IHF) is a small heterodimeric DNA-binding pro tein of E coli composed of two subunits, alpha and beta, encoded by th e himA and hip genes, respectively. IHF binds to DNA at a consensus se quence and bends DNA. HU protein, encoded by the hupA and hupB genes, is similar to IHF except that it does not bind to a specific DNA seque nce. To investigate the protein determinants for IHF specificity we ex changed progressively longer segments from the C-terminus of Hip with those of HupA, and followed the activity in vivo and in vitro of four such IHF/HU hybrids. Replacement of 11 residues from the C-terminal al pha helix of Hip by the complementary eight residues of HupA (hybrid 1 ), had only minor effects on the DNA binding activity of the protein. As progressively longer segments of Hip were replaced by HupA, a preci pitous decrease in IHF activity was observed. The hybrid with the long est substitution, hybrid 4, was totally inactive in vivo and could not be purified. None of the hybrid proteins could complement HU activity . Comparing the activities of hybrid 1, hybrid 2 and IHF point mutants , led us to conclude that the structural integrity of the C-terminal a lpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity. We favor the hypothesis tha t alpha helices 3 of both IHF subunits interact with the body of IHF s o as to anchor the arms. This interaction stabilizes the arms to permi t DNA binding specificity. Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.