Lau Audrey O T, Smith Alias J, Brown Mark T, Johnson Patricia J
UCLA, School of Medicine, Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
Mol Biochem Parasitol. 2006 Nov;150(1):56-62. doi: 10.1016/j.molbiopara.2006.06.008. Epub 2006 Jul 13.
The core promoter that directs RNA polymerase to the start of transcription in the protist Trichomonas vaginalis is an initiator (Inr) element recognized by the Inr Binding Protein, IBP39. This nuclear protein is composed of two domains: a 14.5 kDa amino (N-terminal) and a 25 kDa carboxy terminal domain (C-domain). Here we describe the identification of an IBP39-interacting protein by screening a T. vaginalis expression library using a two-hybrid system with the IBP39 C-domain as bait. The CTD of the large subunit of RNAP II was found to specifically interact with the C-domain. The specificity and nature of the interaction between the CTD of RNAP II and the C-domain of IBP39 was validated by three independent biochemical methods: co-immunoprecipitation with epitope-tagged proteins, affinity chromatography and enzyme linked ligand sorbent (ELLSA) assays. Binding was shown to involve hydrophobic bonds and to have a disassociation constant (K(d)) of 690 nM (+/-55). These results confirm and extend our previous binding studies using a peptide composed of the last nine amino acids of RNAP II CTD [Schumacher MA, Lau AOT, Johnson PJ. Structural basis of core promoter recognition in a primitive eukaryote. Cell 2003;115:413-24] that predicted an interaction between the CTD and IBP39. These data further demonstrate that IBP39 minimally possesses two functional domains: a N-terminal DNA binding domain (that recognizes the Inr) [Liston DR, Johnson PJ. Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 1999;19:2380-8] and a C-terminal protein binding domain that recognizes the RNAP II CTD, an interaction that may be critical for recruiting RNAP II for initiation of transcription.
在原生生物阴道毛滴虫中，指导RNA聚合酶转录起始的核心启动子是一种起始子（Inr）元件，可被Inr结合蛋白IBP39识别。这种核蛋白由两个结构域组成：一个14.5 kDa的氨基（N端）结构域和一个25 kDa的羧基末端结构域（C结构域）。在此，我们描述了通过使用以IBP39 C结构域为诱饵的双杂交系统筛选阴道毛滴虫表达文库来鉴定一种与IBP39相互作用的蛋白。发现RNA聚合酶II大亚基的CTD与C结构域特异性相互作用。通过三种独立的生化方法验证了RNA聚合酶II的CTD与IBP39的C结构域之间相互作用的特异性和性质：用表位标签蛋白进行共免疫沉淀、亲和色谱和酶联配体吸附（ELLSA）测定。结果表明，结合涉及疏水键，解离常数（K(d)）为690 nM（±55）。这些结果证实并扩展了我们之前使用由RNA聚合酶II CTD的最后九个氨基酸组成的肽进行的结合研究[Schumacher MA, Lau AOT, Johnson PJ. 原始真核生物中核心启动子识别的结构基础。细胞2003；115：413 - 24]，该研究预测了CTD与IBP39之间的相互作用。这些数据进一步证明，IBP39至少具有两个功能结构域：一个N端DNA结合结构域（识别Inr）[Liston DR, Johnson PJ. 原始真核生物中普遍存在的启动子元件分析：起始子元件的早期进化。分子细胞生物学1999；19：2380 - 8]和一个C端蛋白结合结构域，该结构域识别RNA聚合酶II的CTD，这种相互作用可能对募集RNA聚合酶II启动转录至关重要。
