The bZIP targets overlapping DNA subsites within a half-site, resulting in increased binding affinities.
We previously reported that the wt bZIP, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes GCN4 cognate site AP-1 (TGACTCA) but also selectively targets noncognate DNA sites, in particular the C/EBP site (TTGCGCAA). In this work, we used electrophoretic mobility shift assay and DNase I footprinting to investigate the factors driving the high affinity between the wt bZIP and the C/EBP site. We found that on each strand of the C/EBP site, the wt bZIP recognizes two 4 bp subsites, TTGC and TGCG, which overlap to form the effective 5 bp half-site (TTGCG). The affinity of the wt bZIP for the overall 5 bp half-site is >or=10-fold stronger than that for either 4 bp subsite. Our results suggest that interactions of the wt bZIP with both subsites contribute to the strong affinity at the overall 5 bp half-site and, consequently, the C/EBP site. Accordingly, we propose that the wt bZIP undergoes conformational changes to slide between the two overlapping subsites on the same DNA strand and establish sequence-selective contacts with the different subsites. The proposed binding mechanism expands our understanding of what constitutes an actual DNA target site in protein-DNA interactions.
Chan
I-San
IS
Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.
Shahravan
S Hesam
SH
Fedorova
Anna V
AV
Shin
Jumi A
JA
D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods; D003850:Deoxyribonuclease I / Q000737:chemistry; D006801:Humans; D016350:Leucine Zippers / Q000502:physiology*; D008956:Models, Chemical; D011485:Protein Binding / Q000502:physiology; D020218:Response Elements / Q000502:physiology*
D050976:Basic-Leucine Zipper Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I