Dimer Dissociation and Thermosensitivity Kinetics of the Saccharomyces cerevisiae and Human TATA Binding Proteins
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- 作者：Amy J. Jackson-Fisher ; Sandeep Burma ; Matthew Portnoy ; Lumelle A. Schneeweis ; Robert A. Coleman ; Madhusmita Mitra ; Carmelata Chitikila ; and B. Franklin Pugh
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 31, 1999
- 年：1999
- 卷：38
- 期：35
- 页码：11340 - 11348
- 全文大小：128K
- 年卷期：v.38,no.35(August 31, 1999)
- ISSN：1520-4995
文摘
A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal inactivation of theyeast Saccharomyces cerevisiae and human TATA binding proteins (TBP) was conducted. We find thatyeast TBP dimers, like human TBP dimers, are slow to dissociate in vitro (t1/2 ~20 min). Mild mutationsin the crystallographic dimer interface accelerate the rate of dimer dissociation, whereas severe mutationsprevent dimerization. In the presence of excess TATA DNA, which measures the entire active TBPpopulation, dimer dissociation represents the rate-limiting step in DNA binding. These findings providea biochemical extension to genetic studies demonstrating that TBP dimerization prevents unregulatedgene expression in yeast [Jackson-Fisher, A. J., Chitikila, C., Mitra, M., and Pugh, B. F. (1999) Mol. Cell3, 717-727]. In the presence of vast excesses of TBP over TATA DNA, which measures only a verysmall fraction of the total TBP, the monomer population in a monomer/dimer equilibrium binds DNArapidly, which is consistent with a simultaneous binding and bending of the DNA. Under conditionswhere other studies failed to detect dimers, yeast TBP's DNA binding activity was extremely labile in theabsence of TATA DNA, even at temperatures as low as 0 
C. Kinetic analyses of TBP instability in theabsence of DNA at 30 C revealed that even under fairly stabilizing solution conditions, TBP's DNAbinding activity rapidly dissipated with t1/2 values ranging from 6 to 26 min. TBP's stability appeared tovary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBPinactivation.
C. Kinetic analyses of TBP instability in theabsence of DNA at 30 C revealed that even under fairly stabilizing solution conditions, TBP's DNAbinding activity rapidly dissipated with t1/2 values ranging from 6 to 26 min. TBP's stability appeared tovary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBPinactivation.