ABSTRACT
Basic region leucine zipper (bZIP) proteins represent a class of transcription factors that bind DNA using a simple, dimeric, alpha-helical recognition motif. The cAMP response element-binding protein (CREB) is a member of the CREB/ATF subfamily of bZIP proteins. CREB discriminates effectively in vivo and in vitro between the 10 bp cAMP response element (ATGACGTCAT, CRE) and the 9 bp activating protein 1 site (ATGACTCAT, AP-1). Here we describe an alanine scanning mutagenesis study designed to identify those residues within the CREB bZIP element that control CRE/AP-1 specificity. We find that the preference of CREB for the CRE site is controlled in a positive and negative way by acidic and basic residues in the basic, spacer and zipper segments. The CRE/AP-1 specificity of CREB is increased significantly by four glutamic acid residues located at positions 24, 28, 35 and 41; glutamic acid residues at positions 10 and 48 contribute in a more modest way. Specificity is decreased significantly by two basic residues located at positions 21 and 23; basic residues at positions 14, 18, 33 and 34 and V17 contribute in a more modest way. All of the residues that influence specificity significantly are located on the solvent-exposed face of the protein-DNA complex and likely participate in interactions between and among proteins, not between protein and DNA. The finding that the CRE/AP-1 specificity of CREB is dictated by the presence or absence of charged residues has interesting implications for how transcription factors seek and selectively bind sequences within genomic DNA.
Subject(s)
Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Response Elements/genetics , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , Binding Sites , Crystallography, X-Ray , Cyclic AMP Response Element-Binding Protein/genetics , DNA/genetics , DNA/metabolism , Glutamic Acid/genetics , Glutamic Acid/metabolism , Leucine Zippers , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Conformation , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Conformation , Solvents , Static Electricity , Substrate Specificity , ThermodynamicsABSTRACT
The cyclic AMP response element (CRE site, ATGACGTCAT) is the DNA target for transcription factors whose activities are regulated by cyclic AMP (1). Recently, we discovered that the CRE site is bent by 10-13 degrees toward the major groove (2). Little or no bend is detected in the related AP-1 site (ATGACTCAT), which differs from the CRE site by loss of a single, central, C.G base pair (2, 3). Here we describe experiments designed to identify which base pairs within the CRE site induce the bent structure in an attempt to understand the origins of the dramatically different conformations of the CRE and AP-1 sites. Our data indicate that the intrinsic CRE bend results from distortion within the TGA sequence found in each CRE half site (ATGAC). These two TGA sequences are located in phase with one another in the CRE sequence but are not (completely) in phase in the AP-1 sequence. This difference in phasing leads to the overall difference in bend as detected by gel (2) and cyclization methods (S. C. Hockings, J. D. Kahn, and D. M. Crothers, unpublished results; M. A. Fabian and A. Schepartz, unpublished results). Our results confirm earlier predictions of altered structure within TG steps, provide insight into the structural reorganizations induced in DNA by bZIP proteins, and lead to a revision of the relationship between the structures of the free and bZIP-bound forms of the CRE and AP-1 sites.
Subject(s)
Cyclic AMP Response Element-Binding Protein/genetics , Nucleic Acid Conformation , Regulatory Sequences, Nucleic Acid , Base Composition , Ligands , Nucleic Acid Heteroduplexes/chemistry , Oligonucleotides/chemistry , Transcription Factor AP-1/geneticsABSTRACT
BACKGROUND: HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. RESULTS: Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. CONCLUSIONS: The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.
