The alpha and beta subunits of the GA-binding protein form a stable heterodimer in solution - Revised model of heterotetrameric complex assembly

We have studied the assembly of GA-binding protein (GABP) in solution and e stablished the sole of DNA in the assembly of the transcriptionally active GABP alpha(2)beta(2) heterotetrameric complex. GABP binds DNA containing a single PEA3/Ets-binding site (PEA3/EBS) exclusively as the ap heterodimer c omplex, but readily hinds as the GABP alpha(2)beta(2) heterotetramer comple x on DNA containing two PEA3/EBSs. Positioning of the PEA3/EBSs on the same face of the DNA helix stabilizes heterotetramer complex binding. These obs ervations suggest that GABP alpha beta heterodimers are the predominant mol ecular species in solution and that DNA containing two PEA3/ EBSs promotes formation of the GABP alpha(2)beta(2) heterotetrameric complex. We analyzed the assembly of GABP alpha(2)beta(2) heteromeric complexes in solution by analytical ultracentrifugation. GABP alpha exists as a monomer in solution while GABP beta exists in a monomer-dimer equilibrium (K-d = 1.8 +/- 0.27 m u M). In equimolar mixtures of the two subunits, GABP alpha and GABP beta f ormed a stable heterodimer, with no heterotetramer complex detected. Thus, GABP exists in solution as the heterodimer previously shown to be a weak tr anscriptional activator. Assembly of the transcriptionally active GABP alph a(2)beta(2) heterotetramer complex requires the presence of specific: DNA c ontaining at least two PEA3/EBSs.