Isolation of TBP‐interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA‐DNA

FEBS Letters

Haruki, Mitsuru²  Matsuda, Tomoki²  Kanaya, Shigenori²  Imanaka, Tadayuki³  Morikawa, Masaaki²  Higashibata, Hiroki¹ 

[1] Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan;Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan;Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan

关键词: TATA-binding protein;    TATA-binding protein-interacting protein;    Hyperthermophilic archaeon;    In-gel digestion;    Gel mobility shift assay;    TBP;    TATA-binding protein;    TIP;    TBP-interacting protein;   

DOI  :  10.1016/S0014-5793(99)01005-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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, K _D, of 1.24–1.46 μM. 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.

【 授权许可】

Unknown