Dimer Dissociation and Thermosensitivity Kinetics of the Saccharomyces cerevisiae and Human TATA Binding Proteins

Abstract

A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal inactivation of the yeast Saccharomyces cerevisiae and human TATA binding proteins (TBP) was conducted. We find that yeast TBP dimers, like human TBP dimers, are slow to dissociate in vitro (t(1/2) approximately 20 min). Mild mutations in the crystallographic dimer interface accelerate the rate of dimer dissociation, 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 findings provide a biochemical extension to genetic studies demonstrating that TBP dimerization prevents unregulated gene expression in yeast [Jackson-Fisher, A. J., Chitikila, C., Mitra, M., and Pugh, B. F. (1999) Mol. Cell 3, 717-727]. In the presence of vast excesses of TBP over TATA DNA, which measures 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 simultaneous binding and bending of the DNA. Under conditions where other studies failed to detect dimers, yeast TBP's DNA binding activity was extremely labile in the absence of TATA DNA, even at temperatures as low as 0 degrees C. Kinetic analyses of TBP instability in the absence of DNA at 30 degrees 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 inactivation.

DNA Bends in TATA-binding Protein·TATA Complexes in Solution Are DNA Sequence-dependent

“…The protein remains fully active in DNA binding even after 24 h at 0°C. These and similar results obtained by other assays 3,4 contrast sharply with a recent report of the loss of the "vast majority" of the DNA binding activity of S. cerevisiae TBP after 0.3 min of incubation at 30°C and all binding activity after 45 min even at 0°C (21).…”

Engineering Dimer-stabilizing Mutations in the TATA-binding Protein

“…Experiments were performed at least three times, and representative data are shown. Kinetic time courses were limited to about 3 h since TBP is intrinsically unstable when not bound to DNA (6). Irreversible inactivation and multiple dimer equilibria in these immobilized binding reactions preclude a meaningful interpretation of apparent rate constants.…”

“…1A) fused to glutathione S-transferase. Previously, we had demonstrated that, with both human and yeast TBP, slow binding of TBP to the GST-181C resin reflects slow dimer dissociation followed by rapid capture by resin-bound GST-181C (6,9).…”

Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus

“…For instance, dimerization of cI repressor monomers is required for high affinity binding to bacteriophage operator DNA (22). In contrast, dimerization inhibits DNA binding by the TATA-binding proteins (23). DNA MTases have also been shown to exist in alternate oligomeric states and to possess a variable active form.…”