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首页> 外文期刊>Molecular and Cellular Biology >TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.
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TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

机译：非洲爪蟾RNA聚合酶III转录因子IIIB的TATA-box DNA结合活性和亚基组成。

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The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.
机译：RNA聚合酶III转录起始因子TFIIIB包含TATA盒结合蛋白（TBP）和聚合酶III特异性TBP相关因子（TAFs）。先前的研究表明，含有共有TATA-box序列的DNA寡核苷酸会抑制聚合酶III的转录，这意味着TBP的DNA结合域暴露在TFIIIB中。我们已经研究了非洲爪蟾TFIIIB的TATA盒DNA结合活性，使用转录抑制测定和凝胶迁移率测定。用突变和非特异性DNA进行的凝胶位移竞争试验证明了TFIIIB-TATA盒DNA复合物的特异性。这种蛋白质与DNA相互作用的表观解离常数约为0.4 nM，类似于酵母TBP对相同序列的亲和力。在一系列四个离子交换色谱步骤中，TFIIIB转录活性和TATA-box结合活性共分馏，重组的转录反应表明TATA-box DNA-蛋白质复合物包含TFIIIB TAF活性。表观分子量为75和92 kDa的多肽与该复合物中的TBP相关。从十二烷基硫酸钠-凝胶洗脱后，将这些多肽复性，并单独或组合测试TFIIIB TAF活性。重组TBP以及包含75-kDa和92-kDa多肽的蛋白质级分足以重构TFIIIB转录活性和DNA结合活性，这表明非洲爪蟾TFIIIB由TBP和这些多肽组成。

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