In Vitro Selection of Integration Host Factor Binding Sites

In solution, B-form DNA is isotropically flexible only when it vastly exceeds its persistence length (150 bp; reviewed in reference 8). Nevertheless, DNA binding proteins often impose an anomalous structure onto their DNA targets despite typically contacting fewer than 30 bp. These deformations can be dramatic, however, such that the generated bend itself has a functional consequence. Since these DNA bending proteins generally require no coupled energy source for binding, the deformations are probably derived from the favorable thermodynamics of protein-DNA interactions. Proteins accomplish this feat by using various mechanisms, for example, charge neutralization of phosphates and/or destabilization of DNA base-stacking interactions (25, 30). What role does the DNA sequence play in its own distortion? While specific protein-DNA interactions are most often mediated via specific hydrogen bonding between DNA bases and amino acid residues, there is an ever-growing class of proteins that select their target site based on indirect readout, i.e., a structure or subset of structures that are specifically recognized by a protein (34). The bacterial protein integration host factor (IHF) epitomizes such proteins. It is a small heterodimeric protein consisting of homologous subunits, α and β, that binds and bends DNA specifically. Although known DNA targets are selected over bulk DNA by at least 1,000-fold (40), there is an inherent degeneracy of sequence in the target selection. IHF was originally discovered as a protein required for efficient integration of the bacteriophage lambda into the Escherichia coli chromosome (36). Subsequently, IHF has been shown to participate in virtually every type of nucleoprotein system (e.g., transcription, replication, and recombination). While its role is always that of an accessory factor, its involvement can be anything from a nominal 2-fold influence, as in promoter activation (23), to as great as a 10,000-fold effect, as in lambda integrative recombination. It is the complex of IHF with DNA that makes it unique among bending proteins; the recently solved cocrystal structure of IHF bound to one of its natural binding sites shows that the DNA is bent by 180° into a virtual U-turn (24, 25). IHF typically binds to a 30- to 35-bp sequence which can be divided into at least two domains. The 3′ region is significantly conserved, and sites share the consensus WATCAANNNNTTR (where W is A or T, R is purine, and N is any base). The lone cytosine base is conserved in every known natural site. Unlike the 3′ region, the 5′ region seems almost random; natural sites are typically AT rich, but no obvious patterns of sequence emerge as conserved. How does the IHF binding site accommodate specific binding and bending? Of particular interest is the 5′ binding domain, since it appears variant in each sequence. Recent evidence suggests that this domain is most successful at binding when an appropriately positioned run of adenines, or an A-tract, is present (11). A-tracts typically consist of three to six consecutive adenines that, in the proper sequence context, create an intrinsically rigid structure with a narrow minor groove; adjacent sequences typically possess an anisotropic bend. What facets of A-tracts, if any, attract IHF for preferred binding? We have attempted to answer these questions by performing a systematic evolution of ligands by exponential enrichment (SELEX) analysis. Selective pressure in vitro for high-affinity binding was applied to a population of IHF binding sites where the wild-type 3′ domain was held constant and the 5′ domain was randomized. Individual sequences were then compared for binding affinity, gross structure of nucleoprotein complexes, and the ability to function in bacteriophage lambda site-specific recombination. We found that the 5′ region can vary the affinity for IHF at least 100-fold. Although comprehensive rules for these sequence determinants could not be deduced, this region contributes significantly to the structure but little to the function of these nucleoprotein complexes. Finally, we found that while the base composition of this region is skewed in native sites, there appears to be no gross base composition advantage for either affinity or function.