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-a ssociated protein that binds to specific sites on DNA. Using gel retardatio n and competition experiments we have estimated that in vitro IHF binds spe cific sites 1000-10,000 times more tightly than non-specific, chromosomal D NA. 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 str ict 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 small er than the one previously deduced from DMS footprinting. As a consequence low affinity sites are only partially occupied and strong binding sites rea ch semi-saturation. In stationary phase the concentration of free IHF in th e 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 n on-specific DNA our data imply that a large part of chromosomal DNA is acce ssible to IHF, and that IHF is a major contributor to chromosomal DNA conde nsation. The in vivo UV-laser footprinting method is of general interest, b ecause it allows the measurement and the comparison of DNA-protein interact ions in vitro and in vivo. (C) 1998 Academic Press.