Frank D J, Tyree C M, George C P, Kadonaga J T
Department of Biology, University of California at San Diego, La Jolla 92093-0347.
J Biol Chem. 1995 Mar 17;270(11):6292-7. doi: 10.1074/jbc.270.11.6292.
To study the mechanism of basal transcription by RNA polymerase II, a cDNA encoding the Drosophila homologue of the small subunit of TFIIF (also referred to as TFIIF30, RAP30, factor 5b, and gamma) was isolated. The Drosophila TFIIF30 gene is located at region 86C on the right arm of the third chromosome. The protein encoded by the cDNA, termed dTFIIF30, was synthesized in Escherichia coli and purified to greater than 95% homogeneity. In reconstituted transcription reactions with purified basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, designated dF30(119-276), which contains the carboxyl-terminal 158 amino acid residues of dTFIIF30, was found to possess approximately 50% of the transcriptional activity as full-length dTFIIF30. The interaction of dTFIIF30 with the large subunit of TFIIF (also referred to as TFIIF74, RAP74, factor 5a, and beta) was investigated by glycerol gradient sedimentation analyses. In these experiments, dTFIIF30, but not dF30(119-276), assembled into a stable heteromeric complex with TFIIF74. These results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxylterminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.
为了研究RNA聚合酶II的基础转录机制，我们分离了一个编码果蝇TFIIF小亚基（也称为TFIIF30、RAP30、因子5b和γ）同源物的cDNA。果蝇TFIIF30基因位于第三条染色体右臂的86C区域。该cDNA编码的蛋白质称为dTFIIF30，在大肠杆菌中合成并纯化至纯度大于95%。在与纯化的基础因子进行的重组转录反应中，dTFIIF30的比活性与其人类同源物相同。此外，发现一个羧基末端片段，命名为dF30(119 - 276)，它包含dTFIIF30的羧基末端158个氨基酸残基，其转录活性约为全长dTFIIF30的50%。通过甘油梯度沉降分析研究了dTFIIF30与TFIIF大亚基（也称为TFIIF74、RAP74、因子5a和β）的相互作用。在这些实验中，dTFIIF30而非dF30(119 - 276)与TFIIF74组装成稳定的异源复合物。这些结果与之前关于TFIIF的研究结果相结合，支持了一个关于TFIIF30功能的模型，其中羧基末端区域构成一个功能结构域，可与RNA聚合酶II相互作用以介导基础转录，而氨基末端包含一个与TFIIF74相互作用的结构域。
