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 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.
Jackson-Fisher
A J
AJ
Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16803, USA.
Burma
S
S
Portnoy
M
M
Schneeweis
L A
LA
Coleman
R A
RA
Mitra
M
M
Chitikila
C
C
Pugh
B F
BF
D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008301:Maleimides; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein; D013816:Thermodynamics; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008301:Maleimides; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C075594:1,6-bismaleimidohexane; D004247:DNA