THE LEUCINE-ZIPPER IS NECESSARY FOR STABILIZING A DIMER OF THE HELIX-LOOP-HELIX TRANSCRIPTION FACTOR USF BUT NOT FOR MAINTENANCE OF AN ELONGATED CONFORMATION

The basic helix-loop-helix transcription factor USF43 binds to E box m otifs on certain promoters and enhancers, as well as the beta-globin l ocus control region. We have used gel filtration chromatography, veloc ity centrifugation, and chemical cross-linking methods to investigate the stoichiometry and shape of USF43 in solution and when bound to DNA . USF43 has a very large Stokes' radius (44 Angstrom) and a high frict ional ratio (1.64), consistent with an asymmetric elongated oligomer. Under a variety of conditions, the only detectible USF43 species in so lution and bound to DNA is a dimer. The carboxyl-terminal leucine zipp er is absolutely essential for a stable dimer but not for the elongate d conformation. We used a protease footprinting assay to demonstrate t hat, when USF43 binds to DNA, a approximate to 15-kDa USF43 domain bec omes resistant to cleavage with trypsin. This domain includes sequence s that are not expected to interact with the DNA helix, suggesting tha t trypsin cleavage sites are masked by a conformational change. Our re sults show that the oligomerization state of USF43 does not change upo n binding to DNA, and the helix-loop-helix oligomerization motif of US F43 is not itself sufficient to form a high affinity dimerization inte rface.