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Full‐length human TBP (hTBP) shows markedly different TATA‐box binding kinetics and thermodynamics from yeast TBP
Author(s) -
Whittington JoDell,
Wood Torri,
Daugherty Margaret,
Parkhurst Laura,
Parkhurst Lawrence
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a285-c
Subject(s) - kinetics , enthalpy , chemistry , tata binding protein , tata box , crystallography , binding energy , dna , thermodynamics , biochemistry , dna binding protein , transcription factor , gene , promoter , physics , quantum mechanics , nuclear physics , gene expression
Human TATA binding protein (TBP) and yeast TBP differ only by an Arg(hTBP)/ Lys(yTBP) substitution in the TATA‐binding domain; 29 other interacting amino acids (aa) are conserved. Conversely, hTBP N‐terminal domain (NTD, 159 aa with a Q 38 ‐tract) shows only 5% homology with the yTBP NTD (60 aa). X‐ray structures for the DNA‐bound C‐terminal domains are nearly identical; we attribute the markedly different kinetics to differing roles played by the NTDs. Time‐resolved FRET measurements show yTBP and hTBP bind and bend the DNA by 75° and 93°, respectively. The affinity of hTBP for the Adenovirus major late promoter is 4x that of yTBP, and the overall enthalpy change on binding is ~ 14 kcal/mole more endothermic. In both cases the association kinetics are bi‐phasic, reflecting formation of bent DNA intermediates. For hTBP, the activation energy for the first step is much less than in yTBP, the first intermediate is more highly reactive in both the forward and reverse directions and the overall association reaction is more rapid. While yTBP shows a complex pattern of entropy and enthalpy changes as the reaction proceeds through intermediates, hTBP shows a progressive increase in enthalpy and entropy. The markedly different kinetics for yTBP and hTBP show that the two NTDs interact quite differently with the TATA box at each step of the reaction. Grant Support: NIH GM 59346.