Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA.
We have isolated TBP (TATA-binding protein)-interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP-agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein (Pk-TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk-TIP is composed of 224 amino acid residues (molecular weight of 25,558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk-TIP and recombinant Pk-TBP indicated that they interact with each other with an equilibrium dissociation constant, KD, of 1.24-1.46 microM. A gel mobility shift assay indicated that Pk-TIP inhibited the interaction between Pk-TBP and a TATA-DNA. Pk-TIP may be one of the archaeal factors which negatively regulate transcription.
Matsuda
T
T
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
Morikawa
M
M
Haruki
M
M
Higashibata
H
H
Imanaka
T
T
Kanaya
S
S
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019711:Pyrococcus / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016385:TATA Box / Q000235:genetics*; D013997:Time Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000235:genetics; D016335:Zinc Fingers
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C121585:TIP protein, Pyrococcus kodakaraensis; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII