A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys
A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys
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DOI：
10.1021/bi991061g
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发表时间：
1999-10-05
期刊：
影响因子：
2.9
通讯作者：
Florentz, C
中科院分区：
文献类型：
--
作者：
Helm, M;Giegé, R;Florentz, C
我们之前通过化学和酶的结构探测表明，与天然的人类线粒体tRNA（Lys）相反，相应的体外转录物不会折叠成预期的tRNA特异性三叶草结构。这种RNA折叠成一个凸起的发夹，包括一个扩展的氨基酸受体茎，一个超大的环代替t茎和环，以及一个反密码子样结构域。因此，天然tRNA中存在的六个修饰核苷酸中的一个或几个是必需的，并负责其三叶草结构。系统发育比较和变异转录本的结构分析表明m(1)A9是折叠过程中最可能的重要修饰核苷酸。本文描述了以m(1)A9为唯一修饰碱基的嵌合tRNA（Lys）的合成及其化学和酶探的结构分析。将这种结构与未修饰的RNA、完全修饰的天然tRNA和设计成模仿m(1)A9效应的变体的结构进行比较，表明嵌合RNA确实折叠成类似于天然tRNA的三叶草结构。因此，由于沃森-克里克碱基对的破坏，一个甲基就足以诱导这种不寻常的tRNA的三叶草折叠。这是第一个直接证明修饰的核苷酸在RNA折叠中的作用的证据。
We have previously shown by chemical and enzymatic structure probing that,opposite to the native human mitochondrial tRNA(Lys), the corresponding in vitro transcript does not fold into the expected tRNA-specific cloverleaf structure. This RNA folds into a bulged hairpin, including an extended amino acid acceptor stem, an extra large loop instead of the T-stem and loop, and an anticodon-like domain. Hence, one or several of the six modified nucleotides present in the native tRNA are required and responsible for its cloverleaf structure. Phylogenetic comparisons as well as structural analysis of variant transcripts had pointed to m(1)A9 as the most likely important modified nucleotide in the folding process. Here we describe the synthesis of a chimeric tRNA(Lys) With m(1)A9 as the sole modified base and its structural analysis by chemical and enzymatic probing. Comparison of this structure to that of the unmodified RNA, the fully modified native tRNA, and a variant designed to mimic the effect of m(1)A9 demonstrates that the chimeric RNA folds indeed into a cloverleaf structure that resembles that of the native tRNA. Thus, due to Watson-Crick base-pair disruption, a single methyl group is sufficient to induce the cloverleaf folding of this unusual tRNA. This is the first direct evidence of the role of a modified nucleotide in RNA folding.