A quantitative UV laser footprinting analysis of the interaction of IHF with specific binding sites: re-evaluation of the effective concentration of IHF in the cell.
The integration host factor (IHF) of Escherichia coli is a major nucleoid-associated protein that binds to specific sites on DNA. Using gel retardation and competition experiments we have estimated that in vitro IHF binds specific sites 1000-10,000 times more tightly than non-specific, chromosomal DNA. We have analyzed the in vitro and in vivo interaction of IHF with three specific binding sites using UV laser footprinting. Because there is a strict correspondence between the intensity of the footprinting signal and the occupancy of a site, we can correlate in vitro association constants with in vivo site occupancy. From the fractional occupancy of various ihf sites in vivo, we then estimate the amount of free IHF in the cell. Exponentially growing cells contain only about 0.7 nM of free IHF, a value 20-fold smaller than the one previously deduced from DMS footprinting. As a consequence low affinity sites are only partially occupied and strong binding sites reach semi-saturation. In stationary phase the concentration of free IHF in the cell increases about sevenfold. These results show that only a very small fraction of total IHF is free in solution. Given the affinity of IHF for non-specific DNA our data imply that a large part of chromosomal DNA is accessible to IHF, and that IHF is a major contributor to chromosomal DNA condensation. The in vivo UV-laser footprinting method is of general interest, because it allows the measurement and the comparison of DNA-protein interactions in vitro and in vivo.
Murtin
C
C
Département de Biologie Moléculaire, Université de Genève, 30 Quai E. Ansermet, Genève 4, 1211, Switzerland.
Engelhorn
M
M
Geiselmann
J
J
Boccard
F
F
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D034982:Integration Host Factors; D007700:Kinetics; D007834:Lasers; D008969:Molecular Sequence Data; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors