Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.
Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.
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DOI：
10.1093/nar/gkt720
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发表时间：
2013-11
影响因子：
14.9
通讯作者：
Basta T
中科院分区：
文献类型：
--
作者：
Perrochia L;Guetta D;Hecker A;Forterre P;Basta T
n6 -苏酰基氨基腺苷（t6A）是一种通用的tRNA修饰，对正常细胞生长和准确翻译至关重要。在古细菌和真核生物中，通用蛋白Sua5和保守的KEOPS/EKC复合物共同催化t6A的生物合成。KEOPS/EKC复合物由Kae1组成，Kae1是一种通用金属蛋白，属于ATPases的ASHKA超家族；Bud32，一个非典型蛋白激酶和两个小蛋白，Cgi121和Pcc1。在这项研究中，我们研究了KEOPS/EKC亚基在t6A生物合成中的需求和功能作用。我们证明Pcc1， Kae1和Bud32形成一个最小的功能单元，而Cgi121作为变构调节因子。我们证实了Pcc1促进KEOPS/EKC复合体的二聚化，并发现它与Kae1一起构成了该复合体的tRNA结合核心。Kae1以金属依赖的方式结合l-苏酰氨基甲酰amp中间体，并将l-苏酰氨基甲酰部分转移到底物tRNA上。令人惊讶的是，我们发现Bud32受Kae1调节，不作为蛋白激酶，而是作为可能参与tRNA解离的p环atp酶。总的来说，我们的数据支持一个机制模型，在这个模型中，t6A生物合成的最后一步依赖于一个严格的催化成分Kae1和三种二聚化、tRNA结合和调节所必需的伙伴蛋白。
N6-threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t6A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t6A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t6A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.
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影响因子：
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作者：
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作者：
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