Integration host factor (IHF) is a protein that binds to the H site of bacteriophage with sequence specificity. Genetic experiments implicated amino acid residue Glu 44 of the ␤-subunit of IHF in discrimination against substitution of A for T at position 44 of the TTR submotif of the binding site (Lee, E. C., Hales, L. M., Gumport, R. I., Gardner, J. F. (1992) EMBO J., 11, 305-313). We have extended this observation by generating all possible single-base substitutions at positions 43, 44, and

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... of the H site. IHF failed to bind these H site substitution mutants in vivo. The K d (app) value for each H site substitution, except for H45A mutant, was reduced >2000-fold relative to the wild-type site. Substitution of amino acid ␤-Glu 44 with alanine prevented IHF from discriminating against the H44A variant but not the other H site substitution mutants. Further analysis with other substitutions at position ␤44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44. Because the ␤-Glu 44 residue does not contact the DNA, this residue probably enforces binding specificity indirectly through interaction with amino acids that themselves contact the DNA.