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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH
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Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH

机译：在3'-末端磷酸和5'-OH之间连接RNA的连接酶RtcB的鸟苷酸化的结构和机理研究

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摘要

The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2,3-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn~(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn~(2+) alone (RtcB/ Mn~(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn~(2+)). The RtcB/ Mn~(2+) structure (at 1.6 A resolution) shows two Mn~(2+) ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn~(2+) complex (at 2.3 A resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn~(2+) ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (ⅰ) guanylylation of the enzyme, (ⅱ) guanylyl-transfer to the RNA substrate, and (ⅲ) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn~(2+) active center, which provides detailed insight into how the enzyme-bound GMP is tans-ferred to the 3-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5-hydroxyl of the'second RNA substrate, resulting in the ligated product and release of GMP.
机译：RtcB蛋白最近被鉴定为3'-磷酸RNA连接酶，可直接将以2,3-环磷酸为末端的RNA链连接到GTP / Mn〜（2+中的另一RNA链的5'-羟基上）依赖性反应。在这里，我们报告了火球菌RNA剪接连接酶RtcB的两个晶体结构，与单独的Mn〜（2+）（RtcB / Mn〜（2+））以及共价结合的GMP（RtcB-GMP / Mn〜（ 2+））。 RtcB / Mn〜（2+）结构（分辨率为1.6 A）在活性位点显示两个Mn〜（2+）离子，附近有一系列硫酸根离子，它们指示RNA磷酸骨架的结合位点。 RtcB-GMP / Mn〜（2+）配合物的结构（在2.3 A分辨率下）揭示了组氨酸404的鸟苷酸化的详细几何形状。关键残基在两个Mn〜（2+ ）离子，四种硫酸根和GMP在广泛的诱变和生化实验中得到了验证，这也为RtcB连接途径的三个步骤提供了全面的表征：（ⅰ）酶的鸟苷酸化，（ⅱ）鸟苷酸转移至RNA底物和（ⅲ）整体连接。这些结果表明，酶的底物诱导的GTP结合位点和假定的反应性RNA末端位于双核Mn〜（2+）活性中心附近，这为如何将酶结合的GMP鞣制为RNA底物的3-磷酸被第二RNA底物的5-羟基激活和随后的亲核攻击，导致连接的产物和GMP的释放。

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    Markus Englert; Shuangluo Xia; Chiaki Okada; Akiyoshi Nakamura; Ved Tanavde; Min Yao; Soo Hyun Eom; William H. Konigsberg; Dieter Soell; Jimin Wang;

  • 作者单位

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

    Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan;

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

    Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan;

    School of Life Sciences, Steitz Center for Structural Biology, Gwangju Institute of Science and Technology, Buk-gu, Gwangju 500-712, Republic of Korea;

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    RNA repair; tRNA splicing; two-metal-ion catalysis;

    机译：RNA修复;tRNA剪接;双金属离子催化;

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