Dimer dissociation and thermosensitivity kinetics of the Saccharomyces cerevisiae and human TATA binding proteins

A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal ina ctivation of the yeast Saccharomyces cerevisiae and human TATA binding prot eins (TBP) was conducted. We find that yeast TBP dimers, like human TBP dim ers, are slow to dissociate in vitro (t(1/2) similar to 20 min). Mild mutat ions in the crystallographic dimer interface accelerate the rate of dimer d issociation, whereas severe mutations prevent dimerization. In the presence of excess TATA DNA, which measures the entire active TBP population, dimer dissociation represents the rate-limiting step in DNA binding. These findi ngs provide a biochemical extension to genetic studies demonstrating that T BP dimerization prevents unregulated gene expression in yeast [Jackson-Fish er, A. J., Chitikila, C., Mitra, M., and Pugh, B. F. (1999) Mol. Cell 3, 71 7-727]. In the presence of vast excesses of TBP over TATA DNA, which measur es only a very small fraction of the total TBP, the monomer population in a monomer/dimer equilibrium binds DNA rapidly, which is consistent with a si multaneous binding and bending of the DNA. Under conditions where other stu dies failed to detect dimers, yeast TBP's DNA binding activity was extremel y labile in the absence of TATA DNA, even at temperatures as low as 0 degre es C, Kinetic analyses of TBP instability in the absence of DNA at 30 degre es C revealed that even under fairly stabilizing solution conditions, TBP's DNA binding activity rapidly dissipated with t(1/2) values ranging from 6 to 26 min. TBP's stability appeared to vary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBP inactiva tion.