Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro.
Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro.
复制标题
牛痘病毒核苷三磷酸磷酸水解酶 I C 端区域的选定突变对与病毒 RNA 聚合酶 H4L 亚基结合和体外早期基因转录终止的影响。
DOI：
10.1016/s0042-6822(03)00092-8
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发表时间：
2003
期刊：
影响因子：
3.7
通讯作者：
Niles,EdwardG
中科院分区：
文献类型：
--
作者：
Piacente,SarahC;Christen,LindaA;Mohamed,MohamedRagaa;Niles,EdwardG
牛痘病毒核苷三磷酸磷酸水解酶I （NPH I）是一种重要的早期基因转录终止因子。NPH I的c端与病毒粒子RNA聚合酶的H4L亚基（RAP94）的n端结合。这种相互作用是转录终止和转录释放所必需的。为了完善我们对nphi c端与H4L结合的特定氨基酸的理解，并开发一系列具有不同程度活性的突变用于体内研究，我们准备了一组短缺失，并将带电氨基酸簇替换为丙氨酸，或将大容量疏水氨基酸簇替换为丙氨酸突变。这些NPH I突变蛋白被表达、纯化，并测试了atp酶活性、与H4L的结合和转录终止活性。609 ~ 631氨基酸的大部分突变表现出活性降低。删除末端5个氨基酸（627-631），或用丙氨酸或谷氨酸取代y629，可显著减少NPH I介导的转录终止。删除末端F631，或用丙氨酸取代F631，减少与H4L的结合并消除末端活性。这些观察结果表明，末端5个氨基酸直接参与RNA聚合酶的结合和基因转录的早期终止。
Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) is an essential early gene transcription termination factor. The C-terminal end of NPH I binds to the N-terminal end of the H4L subunit (RAP94) of the virion RNA polymerase. This interaction is required for transcription termination and transcript release. To refine our understanding of the specific amino acids in the C-terminal end of NPH I involved in binding to H4L, and to develop a collection of mutations exhibiting various degrees of activity to be employed in in vivo studies, we prepared a set of short deletions, and clustered substitutions of charged amino acids to alanine, or bulky hydrophobic amino acids to alanine mutations. These NPH I mutant proteins were expressed, purified, and tested for ATPase activity, binding to H4L, and transcription termination activity. Most mutations in amino acids 609 to 631 exhibited reduced activity. Deletion of the terminal five amino acids (627–631), or substitution of Y629with alanine or glutamic acid, dramatically reduced NPH I mediated transcription termination. Deletion of the terminal F631, or substitution of F631with alanine, reduced binding to H4L and eliminated termination activity. These observations demonstrate that the terminal five amino acids directly participate in binding to RNA polymerase and in early gene transcription termination.