APO-ARAC ACTIVELY SEEKS TO LOOP

In the absence of arabinose and interactions with other proteins, AraC , the activator-repressor that regulates the araBAD operon in Escheric hia coli, was found to prefer participating in DNA looping interaction s between the two well-separated DNA half-sites, araI(1) and araO(2) a t their normal separation of 211 base-pairs rather than binding to the se same two half-sites when they are adjacent to one another. On the a ddition of arabinose, AraC prefer;ed to bind to the adjacently located half-sites. Inverting the distally located araO(2), half-site elimina ted the looping preference. These results demonstrate that apo-AraC po ssesses an intrinsic looping preference that is eliminated by the pres ence of arabinose. We developed a method for the accurate determinatio n of the relative affinities of AraC for the DNA half-sites araI(1), a raI(2), and araO(2) and nonspecific DNA. These affinities allowed accu rate calculation of basal level and induced levels of expression from p(BAD) under a wide variety of natural and mutant conditions. The calc ulations independently predicted the looping preference of apo-AraC. ( C) 1998 Academic Press Limited.