Participation of the flank regions of the integration host factor protein in the specificity and stability of DNA binding.
The heterodimeric integration host factor (IHF) protein is a site-specific DNA-binding protein from Escherichia coli that strongly bends the DNA. It has been proposed (Yang, C., and Nash, H.A. (1989) Cell 57, 869-880; Granston, A. E., and Nash, H. A. (1993) J. Mol. Biol 234, 45-59; Lee, E. C., Hales, L. M., Gumport, R. I., and Gardner, J. F. (1992) EMBO J. 11, 305-313) that the wrapping of the DNA around the protein is stabilized through interactions between the flanks of the protein and the DNA. In order to elucidate which domains of the IHF protein are involved in these interactions, we have constructed mutant proteins in which the C-terminal part of one of the subunits has been deleted. We observed that the C-terminal alpha 3 helix of HimD is involved in the stability of DNA binding, but not in the specificity. In contrast the corresponding alpha 3 helix of HimA is essential for the sequence specificity, since an IHF mutant lacking this domain only binds to the DNA in a non-specific way. The possible role of the two C-terminal alpha-helical structures in complex formation will be discussed. We also examined the properties of an IHF mutant that has an amino acid substitution between beta sheets beta 1 and beta 2 of the HimD subunit (R46H). The occupancy of the ihf site by the mutant and wild type proteins differ in the 3' part of the ihf site and as a result the bend introduced in the DNA by the mutant protein is less pronounced. We propose that the arginine 46 in the HimD subunit is in vicinity of the TTR region of the consensus and that through contacts within the minor groove the DNA bend introduced by IHF is stabilized.
Zulianello
L
L
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
van Ulsen
P
P
van de Putte
P
P
Goosen
N
N
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D016415:Sequence Alignment; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors