title: Effect of flanking bases on the DNA specificity of EmBP-1.
abstract: EmBP-1 is a basic region leucine zipper (bZIP) protein found in many types of plants. In general, plant bZIP proteins bind selectively to DNA sequences containing ACGT core sequences with different immediate flanking nucleotides preferred by different proteins. I report that the distant flanking sequence also has a strong effect on the preference of EmBP-1 for internal bases and determine the residue governing this effect. EmBP-1 binds selectively to the 10 bp gcG-box palindrome GCCACGTGGC 18-fold more tightly than the gcC-box GTGACGTCAC, but when the outer flanking G/C residues were changed to A/T (i.e., ACCACGTGGT and ATGACGTCAT), an only 1.2-fold preference for G-box binding was observed. Analysis of a series of single-residue alanine mutants of EmBP-1 revealed that this effect is mediated by arginine 10. Mutation of this residue to alanine greatly reduces the affinity for the gcG-box while leaving the affinity for other sequences relatively unchanged. Partial retention of G-box specificity upon mutation of R10 to lysine indicates that the effect is reliant on the basic nature of the residue. Additional studies with other EmBP-1 protein mutants and with oligonucleotides containing the T/A and C/G flanking sequences demonstrate the complexity of the protein-DNA interaction and demonstrate that the mechanism of sequence selective DNA binding is highly dependent on the flanking sequence.
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, Ontario, Canada. antonia.dejong@mail.utoronto.ca
authors.forename: Antonia T
authors.initials: AT
authors.lastname: De Jong
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D018744:DNA, Plant / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010944:Plants / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D018744:DNA, Plant