DNA-binding properties of cloned TATA-binding protein from potato tubers.
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.
Holdsworth
M J
MJ
Molecular Genetics Department, John Innes Centre for Plant Science Research, Norwich, Norfolk, UK.
Grierson
C
C
Schuch
W
W
Bevan
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism*; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D012313:RNA; D004247:DNA