In vivo Footprinting Demonstration of Looping
The lac operon was the first well-understood gene system. The genes of the lac operon are turned on when lac repressor dissociates from the lac operator and the genes are turned off when the repressor binds. Every-one came to believe that the turning on or off of gene activity was accomplished by the acts of binding or dissociation of proteins from DNA. In the ara operon the repression that occurs in the absence of arabinose involves the araI site, for mutations in araI can interfere with repression. In the presence of arabinose the araI site is also required for induction. These facts require that at least part of the araI site be occupied by AraC protein both in the presence and absence of arabinose. Induction results from changing the state or conformation of AraC protein, and not the de novo binding or dissociation of the protein. A crucial test of the looping model that made these predictions was that araI be occupied by AraC protein in the absence of arabinose.
How can one test for the binding of a specific protein to a specific site in growing cells? Dimethylsulfate can be used for footprinting in addition to DNAse. In contrast to DNAse, dimethylsulfate easily enters cells. Its rate of methylation of guanines at a protein’s binding site can be either increased or decreased by the presence of the protein. Follow-ing a brief treatment with dimethylsulfate, analogous to light nicking by DNAse in DNA footprinting, the frequency of methylation at different guanines can be measured by isolating, labeling, cleaving at the posi-tions of methylations and separation of fragments on a sequencing gel.
In vivo footprinting showed that AraC protein occupies araI both inthe absence and presence of arabinose, thus fulfilling an essential requirement of the looping model. The footprinting experiments showed a second fact. The araO2 site, to which AraC protein binds only weakly in vitro, is also occupied in vivo. This is as required by the looping model. It is not, however, occupied when the araI and araO1 sites are deleted. That is, its occupancy depends on the presence of sites located more than a hundred nucleotides away. This is, of course, what happens in looping. It is the cooperativity generated by looping that leads to the occupancy of araO2. The binding of AraC protein to araI increases its concentration in the vicinity of araO2 to such an extent that this second site is occupied.
The degree of cooperativity generated by the looping can be roughly estimated. AraC protein is present at about 20 molecules per cell. This is a concentration of about 2 × 10-8 M. The concentration of araO2 in the presence of araI can be estimated to be at least 10-6 M. Thus, by virtue of looping, the concentration of AraC protein near araO2 can be in-creased more than 100-fold.