title: The alpha and beta subunits of the GA-binding protein form a stable heterodimer in solution. Revised model of heterotetrameric complex assembly.
abstract: We have studied the assembly of GA-binding protein (GABP) in solution and established the role of DNA in the assembly of the transcriptionally active GABPalpha(2)beta(2) heterotetrameric complex. GABP binds DNA containing a single PEA3/Ets-binding site (PEA3/EBS) exclusively as the alphabeta heterodimer complex, but readily binds as the GABPalpha(2)beta(2) heterotetramer complex on DNA containing two PEA3/EBSs. Positioning of the PEA3/EBSs on the same face of the DNA helix stabilizes heterotetramer complex binding. These observations suggest that GABPalphabeta heterodimers are the predominant molecular species in solution and that DNA containing two PEA3/EBSs promotes formation of the GABPalpha(2)beta(2) heterotetrameric complex. We analyzed the assembly of GABPalpha(2)beta(2) heteromeric complexes in solution by analytical ultracentrifugation. GABPalpha exists as a monomer in solution while GABPbeta exists in a monomer-dimer equilibrium (K(d) = 1.8 +/- 0.27 microM). In equimolar mixtures of the two subunits, GABPalpha and GABPbeta formed a stable heterodimer, with no heterotetramer complex detected. Thus, GABP exists in solution as the heterodimer previously shown to be a weak transcriptional activator. Assembly of the transcriptionally active GABPalpha(2)beta(2) heterotetramer complex requires the presence of specific DNA containing at least two PEA3/EBSs.
authors.affiliation: Department of Biochemistry, University of Missouri at Columbia, Columbia, Missouri 65212, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chinenov
authors.forename: M
authors.initials: M
authors.lastname: Henzl
authors.forename: M E
authors.initials: ME
authors.lastname: Martin
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004742:Enhancer Elements, Genetic; D051268:GA-Binding Protein Transcription Factor; D016350:Leucine Zippers; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011120:Polyomavirus; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012996:Solutions; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012996:Solutions; D014157:Transcription Factors; D004247:DNA