In vitro selection of integration host factor binding sites

Integration host factor (IHF) is a bacterial protein that binds and severel y bends a specific DNA target IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a resu lt of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics co nducive for both binding and bending with little regard for specific contac ts between the protein and the DNA. We have examined the sequence requireme nts of the 5' binding domain of the IHF binding target. Using a SELEX proce dure, we randomized and selected variants of a natural IHF site. We then an alyzed these variants to determine how the 5' binding domain affects the st ructure, affinity, and function of an IHF-DNA complex in a native system. D espite finding individual sequences that varied over 100-fold in affinity f or IHF, we found no apparent correlation between affinity and function.