从一种嗜热古菌中分离出抑制TBP与TATA-DNA结合的TBP相互作用蛋白（TIP）。

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

作者信息

Matsuda T, Morikawa M, Haruki M, Higashibata H, Imanaka T, Kanaya S

机构信息

Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.

出版信息

FEBS Lett. 1999 Aug 20;457(1):38-42. doi: 10.1016/s0014-5793(99)01005-4.

DOI:10.1016/s0014-5793(99)01005-4

PMID:10486559

Abstract

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.

摘要

我们通过用TBP-琼脂糖亲和层析法从嗜热古菌柯达嗜热栖热菌（Pyrococcus kodakaraensis KOD1）的细胞裂解物中分离出了TBP（TATA结合蛋白）相互作用蛋白（TIP）。基于内部氨基酸序列信息，合成了PCR引物并用于扩增编码该蛋白的基因（Pk-TIP）。核苷酸序列的测定和重组蛋白的表征表明，Pk-TIP由224个氨基酸残基组成（分子量为25,558），并以二聚体形式存在。对重组Pk-TIP和重组Pk-TBP之间相互作用的BIAcore分析表明，它们彼此相互作用的平衡解离常数KD为1.24 - 1.46 microM。凝胶迁移率变动分析表明，Pk-TIP抑制了Pk-TBP与TATA-DNA之间的相互作用。Pk-TIP可能是负调控转录的古菌因子之一。