Monomeric and dimeric bZIP transcription factor GCN4 bind at the same rate to their target DNA site.
Basic leucine zipper (bZIP) transcription factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites. Dimerization of bZIP transcription factor GCN4 is linked to the folding of its C-terminal leucine zipper domain. However, monomeric GCN4, lacking a folded leucine zipper, also recognizes the DNA site with dimerization taking place on the DNA. Here we report the kinetics of DNA recognition by unfolded monomeric and folded dimeric derivatives of GCN4 using a 19 bp double-stranded DNA containing a palindromic CRE site. The rate of DNA binding of both monomeric and dimeric GCN4 has a bimolecular rate constant of 3-5 x 10(8) M(-1) s(-1), which is near the diffusion limit. Because the rate of dimerization of GCN4 is slower (1.7 x 10(7) M(-1) s(-1)) than the rate of DNA association, the formation of the dimeric GCN4-DNA complex through consecutive binding of two monomers (monomer pathway) is faster when starting from free monomers. Thus, the results presented here support facilitated and rapid target recognition by the monomeric transcription factor. However, DNA binding of preformed folded dimeric GCN4 is as rapid as complex formation through the monomer pathway. Therefore, the monomer and dimer pathways are kinetically equivalent if monomeric and dimeric GCN4 are at equilibrium. Hence, the dimer pathway may also have a role under in vivo conditions. The lower affinity of GCN4 in which two DNA contacting residues have been mutated is due exclusively to the faster dissociation of the mutant protein-DNA complex and not to slower complex formation.
Cranz
Susanne
S
Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Berger
Christine
C
Baici
Antonio
A
Jelesarov
Ilian
I
Bosshard
Hans Rudolf
HR
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D002151:Calorimetry; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D051790:G-Box Binding Factors; D007700:Kinetics; D016350:Leucine Zippers; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D017362:Cyclic AMP Response Element-Binding Protein; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases