Studies of Schizosaccharomyces pombe TFIIE indicate conformational and functional changes in RNA polymerase II at transcription initiation
Studies of Schizosaccharomyces pombe TFIIE indicate conformational and functional changes in RNA polymerase II at transcription initiation
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DOI：
10.1111/j.1365-2443.2005.00833.x
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发表时间：
2005-03-01
期刊：
影响因子：
2.1
通讯作者：
Ohkuma, Y
中科院分区：
文献类型：
--
作者：
Hayashi, K;Watanabe, T;Ohkuma, Y
通用转录因子 TFIIE 在 RNA 聚合酶 II (PolII) 的转录过程中发挥重要作用。尽管最近取得了进展，但其精确机制（包括生物学功能）的阐明还有待进一步表征。我们报告了粟酒裂殖酵母 TFIIE (spTFIIE) 的分离和表征。与人类和其他真核 TFIIE 蛋白一样，spTFIIE 由 α 和 β 亚基组成，编码这两个亚基的基因对于生存至关重要。染色质免疫沉淀测定表明 spTFIIE 在体内定位于启动子。 TFIIEbeta 的 C 端碱性螺旋环区域参与从转录起始到延伸的转变，对其进行的突变分析表明，该区域受影响的转录缺陷突变体也是冷敏感的。 spTFIIEbeta 亚基结合 spTFIIEbeta 和 spTFIIEalpha，但 spTFIIEalpha 仅结合 spTFIIEbeta。这些结果表明TFIIE形成α(2)β(2)异四聚体，其中两个alphata异二聚体通过β亚基连接。结合特异性的进一步分析表明，spTFIIEbeta 结合 PolII 的 Rpb2 和 Rpb12 亚基，而 spTFIIEalpha 主要结合 Rpb5，Rpb5 位于钳位区并在转录起始时改变构象。
The general transcription factor TFIIE plays essential roles in transcription by RNA polymerase II (PolII). Despite recent progress, the elucidation of its precise mechanisms including biological functions awaits further characterization. We report the isolation and characterization of Schizosaccharomyces pombe TFIIE (spTFIIE). Like human and other eukaryotic TFIIE proteins, spTFIIE consists of alpha and beta subunits and the genes encoding both subunits are essential for viability. Chromatin immunoprecipitation assays demonstrated that spTFIIE localizes to promoters in vivo. Mutational analysis of the C-terminal basic helix-loop region of TFIIEbeta, which is involved in the transition from transcription initiation to elongation, revealed that transcription-defective mutants affected in this region are also cold sensitive. The spTFIIEbeta subunit binds both spTFIIEbeta and spTFIIEalpha but spTFIIEalpha binds only spTFIIEbeta. These results indicate that TFIIE forms an alpha(2)beta(2) heterotetramer in which two alphabeta heterodimers are connected via beta subunits. Further analysis of binding specificities showed that spTFIIEbeta binds the Rpb2 and Rpb12 subunits of PolII, whereas spTFIIEalpha predominantly binds Rpb5, which is located at the clamp region and changes conformation upon transcription initiation.