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首页> 外文期刊>The FEBS journal >Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro.
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Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro.

机译：变形虫变形杆菌TATA盒结合蛋白在体外与不同的TATA变体结合。

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摘要

The ability of Entamoeba histolytica TATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in supershift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found in E. histolytica gene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (+or-0.39) x 10-11 and 1.60 (+or-0.37) x 10-10 M. TATTTAAA and TAT_ _AAA motifs presented the lowest KD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding.
机译：变形虫变形杆菌的TATA结合蛋白（EhTBP）与基因启动子中不同TATA盒相互作用的能力可能是在此人类寄生虫中进行有效转录的关键因素之一。在本文中，我们使用了几种TATA变体来研究体外EhTBP DNA结合活性并确定TATA-EhTBP的解离常数。 EhTBP在由核提取物（NE）和TATTTAAA寡核苷酸形成的复合物中的存在，该复合物对应于溶组织性大肠杆菌的典型TATA盒，通过凝胶位移测定法得以证明。在这些实验中，检测到单个NE-TATTTAAA寡核苷酸复合物。在超移位实验中，复合物被抗EhTBP Igs阻滞，并且抗体在Western blot分析中也识别出交联的复合物。重组EhTBP与溶组织性大肠杆菌基因启动子中发现的TATA变体以及通过TATTTAAA序列突变产生的其他TATA变体形成了特异性复合物。 TATA变体的重组EhTBP的解离常数介于1.04（+或-0.39）x 10-11和1.60（+或-0.37）x 10-10 M之间。TATTTAAA和TAT__AAA基序呈现最低的KD值。有趣的是，重组EhTBP对TATA变体的亲和力强于其他报道的TBP。另外，EhTBP比人和酵母TBP更为混杂，这可能是由于涉及TBP-DNA结合的氨基酸的修饰。

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