Mutational analysis of human RNA polymerase II subunit 5 (RPB5): The residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein
Mutational analysis of human RNA polymerase II subunit 5 (RPB5): The residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein
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DOI：
10.1093/jb/mvi119
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发表时间：
2005-09-01
影响因子：
2.7
通讯作者：
Murakami, S
中科院分区：
文献类型：
--
作者：
Le, TTT;Zhang, SJ;Murakami, S
RNA 聚合酶 II (RNAPII) 亚基 5 (RPB5) 位于起始位点下游 DNA 附近，是与多个调节因子相互作用的位点。乙型肝炎病毒 X 蛋白 (HBx) 结合 RPB5 的中心部分来调节激活的转录，TFIIF 亚基 RAP30 与 RPB5 的相同部分相互作用，这对于 TFIIF 和 RNAPII 之间的关联至关重要。然而，这些相互作用所需的残基仍然未知。在这里，我们报告了 RPB5 中心部分的系统诱变，使用两步丙氨酸扫描文库来查明其与 TFIIF 复合物中的 RAP30 和/或 HBx 结合的关键残基，并在哺乳动物细胞和体外结合测定中鉴定了这些残基。四个残基 F76、1104、T111 和 5113 对于 TFIIF 和 HBx 结合都至关重要，表明相互作用位点的重叠性质。此外，HBx 结合需要 V74 和 N98，RAP30 结合需要 T56 和 L58。有趣的是，暴露于溶剂的残基 T111 和 5113 非常接近 DNA，这意味着有两个因素可能调节 DNA 和 RPB5 之间的相互作用。
RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76,1104, T111 and 5113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and 5113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5.