RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2
RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2
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DOI：
10.1074/jbc.m402394200
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发表时间：
2004-07-23
影响因子：
4.8
通讯作者：
Shuman, S
中科院分区：
文献类型：
--
作者：
Nandakumar, J;Ho, CK;Shuman, S
本文报道了噬菌体T4 RNA连接酶2（Rnl 2）是双链RNA或RNA中3 '-OH/5'-PO 4切口处RNA连接的有效催化剂。DNA混合体模板链在接近反应性3 '-OH和5'-PO 4末端中的关键作用通过当双链体底物含有缺口或翼而不是切口时Rnl 2的RNA密封活性的急剧降低来强调。RNA切口连接需要ATP和二价阳离子辅因子（Mg或Mn）。dATP、GTP、CTP和UTP都不能替代ATP。我们通过丙氨酸扫描在Rnl 2的N-末端核苷酸转移酶结构域内鉴定了7个功能上重要的氨基酸（Tyr-5、Arg-33、Lys-54、Gln-106、Asp-135、Arg-155和Ser-170），并基于AMP结合酶的晶体结构估算了这些残基的特定作用。Rnl 2的C-末端结构域中的14个保守残基的突变分析鉴定了3个氨基酸（Arg-266、Asp-292和Glu-296）为连接酶活性所必需的。我们的研究结果巩固了噬菌体Rnl 2和动质体原生动物的RNA编辑连接酶之间的进化联系。
Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/ 5'-PO4 nick in a double-stranded RNA or an RNA . DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO4 termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor ( either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyltransferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids ( Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.