The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites.
We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.
Chan
I-San
IS
Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.
Fedorova
Anna V
AV
Shin
Jumi A
JA
D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000502:physiology*; D022762:CCAAT-Enhancer-Binding Proteins / Q000502:physiology; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology*
D050976:Basic-Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors