Role of the leucine zipper in the kinetics of DNA binding by transcription factor USF.
USF is a transcription factor characterized by a helix-loop-helix (HLH) DNA-binding domain that has been highly conserved through evolution. Vertebrate USFs contain an additional C-terminal leucine zipper (LZ) immediately adjacent to the HLH domain. This LZ is essential for efficient DNA binding by human USF. However, sea urchin has a USF family member that lacks a LZ and yet binds DNA efficiently. To clarify the role of the LZ in DNA binding by USF, we compared the properties of human and sea urchin USFs and found that the two proteins interacted with their specific sites on the DNA with identical affinities but very different kinetics. Association and dissociation rate constants of sea urchin USF were about 10-fold those of human USF. Domain-swapping experiments revealed that the LZ was responsible for the slower kinetics of human USF. USF heterodimers containing a single LZ displayed rates intermediate between those of dimers containing either two or no LZ, indicating that zipper-zipper interactions within USF dimers were not important for DNA binding. Temperature effects on DNA-binding parameters revealed a very high energy barrier for binding of human USF to DNA. Presence of a LZ increased the activation energy of the reaction.
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Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007700:Kinetics; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012044:Regression Analysis; D012617:Sea Urchins; D013045:Species Specificity; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors