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 motifs on certain promoters and enhancers, as well as the beta-globin locus control region. We have used gel filtration chromatography, velocity 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 A) and a high frictional ratio (1.64), consistent with an asymmetric elongated oligomer. Under a variety of conditions, the only detectable USF43 species in solution and bound to DNA is a dimer. The carboxyl-terminal leucine zipper is absolutely essential for a stable dimer but not for the elongated conformation. We used a protease footprinting assay to demonstrate that, when USF43 binds to DNA, a approximately 15-kDa USF43 domain becomes resistant to cleavage with trypsin. This domain includes sequences that are not expected to interact with the DNA helix, suggesting that trypsin cleavage sites are masked by a conformational change. Our results show that the oligomerization state of USF43 does not change upon binding to DNA, and the helix-loop-helix oligomerization motif of USF43 is not itself sufficient to form a high affinity dimerization interface.
Bresnick
E H
EH
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Felsenfeld
G
G
D000595:Amino Acid Sequence; D001704:Biopolymers; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012996:Solutions; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D001704:Biopolymers; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D012996:Solutions; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D004247:DNA