Fos, Jun and CREB basic-domain peptides have intrinsic DNA-binding activity enhanced by a novel stabilizing factor.
Transcription factors with a 'leucine zipper' (LZ) domain bind to DNA cis-elements, often with palindromic structures. DNA-binding by such factors requires the presence of highly basic regions in the proteins found adjacent to the LZ domain. In order to determine if the observed DNA-binding specificity is programmed by the basic region itself, we developed a competitive binding assay to compare relative binding affinities of synthetic peptides to specific promoter elements. In this report we demonstrate that the basic domains of the oncoproteins Fos, Jun and the transcription factor CREB, possess the structural information necessary to compete for promoter-specific binding. To study the relative binding affinity of the basic motifs to specific promoter elements, we used synthetic peptides to compete for intrinsic Fos/Jun and CREB DNA-binding activity present in HeLa cell nuclear extracts. These studies demonstrate that the basic peptides of both Fos and Jun have higher affinity for the TPA responsive element (TRE) and the cAMP responsive element (CRE) relative to the corresponding peptide for CREB. The peptides showed virtually no affinity for either Sp1 or octamer consensus promoter sequences, demonstrating that these basic peptides also retained their promoter selectivity. We further demonstrate that the conserved dipolar arrangement of two basic amino acid clusters is required for selective, competitive binding. A second conserved feature critical for competitive binding relates to the distance separating the dipolar basic clusters. Our competition binding assay has also helped to identify a novel protein factor, termed ABP (auxilliary bridging protein), which interacts to stabilize Fos and Jun basic domain binding-interaction with specific promoter elements. The data suggest a mechanism in which ABP acts to promote more stable protein-DNA complexes.
Busch
S J
SJ
Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
Sassone-Corsi
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004355:Drug Stability; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors