Specific recognition of DNA by integration host factor. Glutamic acid 44 of the beta-subunit specifies the discrimination of a T:A from an A:T base pair without directly contacting the DNA.
Integration host factor (IHF) is a protein that binds to the H' site of bacteriophage lambda with sequence specificity. Genetic experiments implicated amino acid residue Glu(44) of the beta-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 45 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 H'45A mutant, was reduced >2000-fold relative to the wild-type site. Substitution of amino acid beta-Glu(44) with alanine prevented IHF from discriminating against the H'44A variant but not the other H' site substitution mutants. Further analysis with other substitutions at position beta44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44. Because the beta-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.
Departments of Microbiology and Biochemistry and College of Medicine, University of Illinois, Urbana, Illinois 61801, USA.
E K
EK
Read
R I
RI
Gumport
J F
JF
Gardner
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020029:Base Pairing; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018698:Glutamic Acid; D034982:Integration Host Factors; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D021122:Protein Subunits; D018698:Glutamic Acid; D004247:DNA