The GCN4 bZIP can bind to noncognate gene regulatory sequences.
We show that a minimalist basic region/leucine zipper (bZIP) hybrid, comprising the yeast GCN4 basic region and C/EBP leucine zipper, can target mammalian and other gene regulatory sequences naturally targeted by other bZIP and basic/helix-loop-helix (bHLH) proteins. We previously reported that this hybrid, wt bZIP, is capable of sequence-specific, high-affinity binding of DNA comparable to that of native GCN4 to the cognate AP-1 and CRE DNA sites. In this work, we used DNase I footprinting and electrophoretic mobility shift assay to show that wt bZIP can also specifically target noncognate gene regulatory sequences: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (Xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and the E-box (Enhancer box, 5'-CACGTG). Although wt bZIP still targets AP-1 with strongest affinity, both DNA-binding specificity and affinity are maintained with wt bZIP binding to noncognate gene regulatory sequences: the dissociation constant for wt bZIP in complex with AP-1 is 13 nM, while that for C/EBP is 120 nM, XRE1 240 nM, and E-box and HRE are in the microM range. These results demonstrate that the bZIP possesses the versatility to bind various sequences with varying affinities, illustrating the potential to fine-tune a designed protein's affinity for its DNA target. Thus, the bZIP scaffold may be a powerful tool in design of small, alpha-helical proteins with desired DNA recognition properties.
Fedorova
Anna V
AV
Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada L5G 4T8.
Chan
I-San
IS
Shin
Jumi A
JA
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024721:E-Box Elements / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D008958:Models, Molecular; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA