Anti-cooperative biphasic equilibrium binding of transcription factor upstream stimulatory factor to its cognate DNA monitored by protein fluorescence changes.
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.
Sha
M
M
Department of Chemistry, Hunter College, City University of New York, New York 10021-5024, USA.
Ferré-D'Amaré
A R
AR
Burley
S K
SK
Goss
D J
DJ
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D004247:DNA