tRNA leucine identity and recognition sets
tRNA leucine identity and recognition sets
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DOI：
10.1006/jmbi.2000.3694
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发表时间：
2000-05-19
影响因子：
5.6
通讯作者：
Abelson, J
中科院分区：
文献类型：
--
作者：
Tocchini-Valentini, G;Saks, ME;Abelson, J
转移 RNA (tRNA) 根据其可变环的结构分为两类。在大肠杆菌中，来自三个同功接受基团的 tRNA 被归类为 II 型。亮氨酸 tRNA 就包含这样的一组。我们使用体内和体外方法来确定 tRNA(Leu) 功能所需的核苷酸。此外，为了研究 tRNA 折叠的作用，我们将 I 型 tRNA(Leu) 变体与 II 型对应物的体内和体外特征进行了比较。至少需要 6 个保守的 tRNA(Leu) 核苷酸才能将 II 型 tRNA(Ser) 琥珀抑制子的氨基酸身份和识别从丝氨酸改变为亮氨酸残基。其中 5 个核苷酸影响 tRNA 三级结构； tRNA(Ser)中的G15-C48三级“Levitt碱基对”更改为A15-U48；改变 D 环的 α 和 β 区域中的核苷酸数量，以实现所有 tRNA（Leu）中发现的 G18 和 G19 的定位，在碱基配对的额外茎和 T 茎之间的位置 47n 处插入碱基；此外，tRNA（Ser）的G73“鉴别器”碱基更改为A73。这种经过最小程度改变的 tRNA(Ser) 仅插入亮氨酸残基，是 LeuRS 的极好体外底物。在平行实验中，在插入谷氨酰胺的 I 型 tRNA（RNA(Ser Delta)；一种琥珀抑制子，其中 tRNA(Ser) II 型额外茎环被共有 I 型环取代）中进行了核苷酸取代。这种“I 型”交换实验在体内和体外均取得成功，但需要比 II 型交换更多的核苷酸取代。 I 型和 II 交换揭示了 tRNA(Leu) 受体茎碱基对对 tRNA(Leu) 功能的贡献差异：在 I 型折叠中，亮氨酸特异性取决于 tRNA 受体茎序列的存在，但在 II 型折叠中则不然。本研究中使用的 I 型和 II 型 tRNA 仅在可变环的序列和结构上有所不同。通过改变这个环，从而可能在整个 tRNA 折叠中引入微妙的变化，就有可能检测受体茎序列对 LeuRS 识别的神秘贡献。讨论了造成这种影响的可能原因。 (C) 2000 年学术出版社。
Transfer RNAs(tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function. Tn addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA(Leu) variants with their type II counterparts.A minimum of six conserved tRNA(Leu) nucleotides were required to change the amino acid identity and recognition of a type II tRNA(Ser) amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA(Leu) a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 "discriminator" base of tRNA(Ser) was changed to A73. This minimally altered tRNA(Ser) exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA(Ser Delta); an amber suppressor in which the tRNA(Ser) type II extra-stem-loop is replaced by a consensus type I loop). This "type I" swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap.The type I and II swaps revealed differences in the contributions of the tRNA(Leu) acceptor stem base-pairs to tRNA(Leu) function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed. (C) 2000 Academic Press.