tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily.
tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily.
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SPOUT 甲基转移酶超家族的细菌 tRNA Xm32 修饰酶对 tRNA 的识别
DOI：
10.1093/nar/gkv745
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发表时间：
2015-09-03
影响因子：
14.9
通讯作者：
Wang ED
中科院分区：
文献类型：
--
作者：
Liu RJ;Long T;Zhou M;Zhou XL;Wang ED
来自SPOUT甲基转移酶超家族的TrmJ蛋白是存在于细菌和古细菌中的tRNA Xm 32修饰酶。与古细菌TrmJ不同，细菌TrmJ需要全长tRNA分子作为底物。细菌TrmJ如何识别底物tRNA并特异性催化核糖32的2′-O修饰仍然未知。在本文中，我们证明了所有6个在32位具有2′-O-甲基化核苷的大肠杆菌（Ec）tRNA都是EcTrmJ的底物，并且我们表明tRNA手肘区域而不是氨基酸受体茎是甲基化反应所需的。我们的晶体学研究表明，全长EcTrmJ在不对称单元中形成不寻常的二聚体，催化SPOUT结构域和C-末端延伸形成单独的二聚体缔合。基于这些发现，我们使用电泳迁移率变动分析，等温滴定量热法和酶的方法，以确定氨基酸内的EcTrmJ参与tRNA结合。我们发现，tRNA识别EcTrmJ涉及的合作影响的保守残基从两个SPOUT和延伸结构域，这一过程是由柔性铰链区，连接这两个域。
TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2′-O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli (Ec) tRNAs with 2′-O-methylated nucleosides at position 32 are substrates of EcTrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding. We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.