Abstract
Upstream stimulatory factor USF is a human transcriptional activation factor, which uses a basic/helix-loop-helix/ leucin zipper (b/HLH/Z) motif to homodimerize and recognize specific sequences in the promoter region of both nuclear and viral genes transcribed by RNA polymerase II. Steady state fluorescence spectroscopy demonstrated that the basic/helix-loop-helix/leucin zipper domain of USF binds its DNA targets with high affinity and specificity, whereas removal of the leucine zipper yielding the basic/helix-loop-helix minimal DNA binding region reduces both affinity and specificity. Stopped flow method provided kinetic evidence for a two-step binding process involving rapid formation of a protein-DNA intermediate followed by a slow isomerization step, which is consistent with the basic region undergoing a random coil to alpha-helix folding transition on specific DNA recognition. The leucine zipper is also necessary for USF to function as a bivalent homotetramer, capable of binding two distinct recognition sites simultaneously and mediating DNA looping under physiologic conditions. Titration studies revealed that the first binding event has a equilibrium constant Keq = (2.2 +/- 2.0) x 10(9) M-1 for major late promoter DNA, whereas the second binding event occurs with a remarkable reduced affinity, Keq = (1.2 +/- 0.8) x 10(8) M-1. This anticooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in class II nuclear and viral gene promoters.
Highlights
M-1 This anticooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in class II nuclear and viral gene promoters
The leucine zipper motif is not involved in direct contact with DNA during blHLH/Z binding, it has some crucial effects on DNA binding to the protein
The fact that blHLH/Z had 1000-100-fold higher affinity to MLP than blHLH suggests that the leucine zipper is necessary for high affinity DNA binding
Summary
M-1 This anticooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in class II nuclear and viral gene promoters. USF belongs to the basic (hj/helix-loop-helix (HLH)/leucine zipper (Z) family of transcription factors and stimulates transcription by binding to a specific E-box motif (CACGTG) upstream of many cellular and viral promoters. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114. ** To whom correspondence should be addressed: Dept. ** To whom correspondence should be addressed: Dept. of Chemistry, Hunter College of the City University of New York, 695 Park Ave., New York, NY 10021-5024
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