Multiple molecules of integration host factor (IHF) at a single DNA binding site, the bacteriophage lambda cos I1 site.
Integration host factor (IHF) is an E coli protein that binds DNA sequence-specifically and serves as a cofactor in many intracellular processes including lambda DNA packaging. In gel shift experiments, cos DNA, a DNA fragment containing the recognition signal for lambda DNA packaging, forms multiple protein-DNA complexes when combined with pure IHF. Copper(II)-1,10 orthophenanthroline footprinting of individual IHF-cos DNA complexes shows that multiple complex formation does not result from IHF binding to successive sites on the cos DNA fragment. Instead, the footprinting of DNA from two IHF-cos complexes shows protection at one site alone. DNA in the first complex is only partially protected from nucleolytic cleavage, while DNA in the second, slower-moving, complex is completely protected at the same binding site. Quantitative Western blotting experiments determined the relative stoichiometry of IHF to DNA in the two complexes. The results confirm that two molecules of IHF bind at a single site in the cos fragment. This site, cos I1, has two matches to the IHF consensus sequence, but the two matches overlap by eight of thirteen nucleotides. A search of the DNA sequence around cos, using an expanded IHF consensus sequence, has revealed additional, low-affinity consensus matches, contiguous to these. The extent of the copper(II)-1,10 orthophenanthroline footprint and the stoichiometry of the IHF-cos I1 complexes suggest that either two molecules of IHF bind to overlapping sites, or IHF binds to a site of low affinity contiguous to a strong site. Application of a thermodynamic model to the results of gel shift experiments with IHF and cos DNA suggests that multiple complex formation requires cooperative interaction between the two IHF binding sites.
Morse
B K
BK
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459-0175, USA.
Michalczyk
R
R
Kosturko
L D
LD
D001345:Autoradiography; D001426:Bacterial Proteins / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins