Holdsworth M J, Grierson C, Schuch W, Bevan M
Molecular Genetics Department, John Innes Centre for Plant Science Research, Norwich, Norfolk, UK.
Plant Mol Biol. 1992 Jun;19(3):455-64. doi: 10.1007/BF00023393.
A full-length cDNA clone encoding the TATA-binding protein (TBP), the DNA-binding component of the general transcription factor TFIID was cloned from potato tubers. The DNA sequence of this cDNA indicated that the predicted potato protein was very similar to cloned TBP from other species. Genomic southern analysis showed that TBP is encoded in the potato genome as a low-copy-number sequence. The potato TBP cDNA clone was shown to encode a functional protein that interacts in a sequence-specific way with the promoter region of a class-1 potato patatin gene. Functional analysis of carboxy-terminal truncated derivatives of potato TBP showed that important components of DNA binding were located within the carboxy-terminal 54 amino acids. Kinetic and thermodynamic properties of in vitro synthesised potato TBP were investigated, and demonstrated strict salt and temperature preferences for maximum DNA binding activity. In addition on and off-rate measurements showed that both association and dissociation of TBP from DNA is slow. The specific and the non-specific equilibrium constants Ks and Kn were calculated as 5 x 10(9) M-1 and 3.65 x 10(4) M-1 respectively. These results indicate that the interaction of potato TBP with the patatin promoter is highly specific.
从马铃薯块茎中克隆出一个全长cDNA克隆，其编码通用转录因子TFIID的DNA结合成分——TATA结合蛋白（TBP）。该cDNA的DNA序列表明，预测的马铃薯蛋白与从其他物种克隆的TBP非常相似。基因组Southern分析表明，TBP在马铃薯基因组中作为低拷贝数序列编码。马铃薯TBP cDNA克隆被证明编码一种功能性蛋白，该蛋白以序列特异性方式与1类马铃薯patatin基因的启动子区域相互作用。对马铃薯TBP羧基末端截短衍生物的功能分析表明，DNA结合的重要成分位于羧基末端的54个氨基酸内。研究了体外合成的马铃薯TBP的动力学和热力学性质，结果表明最大DNA结合活性对盐和温度有严格的偏好。此外，结合和解离速率测量表明，TBP与DNA的结合和解离都很缓慢。特异性和非特异性平衡常数Ks和Kn分别计算为5×10⁹ M⁻¹和3.65×10⁴ M⁻¹。这些结果表明，马铃薯TBP与patatin启动子的相互作用具有高度特异性。
