title: Characterization of the cooperative function of inhibitory sequences in Ets-1.
abstract: DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.
authors.affiliation: Department of Oncological Sciences, Universtiy of Utah School of Medicine, Salt Lake City, 84132, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Jonsen
authors.forename: J M
authors.initials: JM
authors.lastname: Petersen
authors.forename: Q P
authors.initials: QP
authors.lastname: Xu
authors.forename: B J
authors.initials: BJ
authors.lastname: Graves
mesh_terms: D000494:Allosteric Regulation; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin
chemical_list: D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D014357:Trypsin