Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30°C, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 μM. Association is highly cooperative, with octamer formation favored by ~7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer⇆tetramer and tetramer⇆octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], ~3 mole equivalents of ions are released at each association step, while at high [KCl], ~3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.
|Number of pages||11|
|Journal||Journal of Molecular Biology|
|State||Published - Jan 29 1999|
Bibliographical noteFunding Information:
Supported by NSF Grant DMB 91-48816 to M.G.F., a Four Diamonds Fund Postdoctoral Grant to M.A.D. and NIH GM51506 and GM39929 to M.B.
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology