Genetic and biochemical analysis of IHF/HU hybrid proteins.
Integration host factor (IHF) is a small heterodimeric DNA-binding protein of E coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to DNA at a consensus sequence 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 sequence. To investigate the protein determinants for IHF specificity we exchanged 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 alpha 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 precipitous decrease in IHF activity was observed. The hybrid with the longest 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 alpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity. We favor the hypothesis that alpha helices 3 of both IHF subunits interact with the body of IHF so as to anchor the arms. This interaction stabilizes the arms to permit DNA binding specificity. Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.
Goldenberg
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Department of Molecular Genetics, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Giladi
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Oppenheim
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C040330:histone-like protein HU, bacteria; D003850:Deoxyribonuclease I