Tocchini-Valentini G, Saks M E, Abelson J
Division of Biology, California Institute of Technology, Pasadena, CA, USA.
J Mol Biol. 2000 May 19;298(5):779-93. doi: 10.1006/jmbi.2000.3694.
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. In 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(SerDelta); 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(Leu) 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.
转运RNA（tRNA）根据其可变环的结构分为两类。在大肠杆菌中，来自三个同功受体组的tRNA被归类为II型。亮氨酸tRNA就包括这样一组。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，我们比较了I型tRNA（Leu）变体与其II型对应物在体内和体外的特性。
至少需要六个保守的tRNA（Leu）核苷酸才能将II型tRNA（Ser）琥珀抑制子的氨基酸识别从丝氨酸改变为亮氨酸残基。其中五个核苷酸影响tRNA的三级结构；tRNA（Ser）中的G15 - C48三级“Levitt碱基对”被改变为A15 - U48；D环的α和β区域中的核苷酸数量被改变，以实现所有tRNA（Leu）中G18和G19的定位；在碱基配对的额外茎和T茎之间的47n位置插入了一个碱基；此外，tRNA（Ser）的G73“判别”碱基被改变为A73。这种最小程度改变的tRNA（Ser）只插入亮氨酸残基，并且是亮氨酰 - tRNA合成酶（LeuRS）的优良体外底物。在一个平行实验中，对插入谷氨酰胺的I型tRNA（RNA（SerDelta）；一种琥珀抑制子，其中II型tRNA（Ser）的额外茎环被一个共有I型环取代）进行了核苷酸替换。这种“I型”交换实验在体内和体外都成功了，但比II型交换需要更多的核苷酸替换。I型和II型交换揭示了tRNA（Leu）受体茎碱基对在tRNA（Leu）功能中的贡献差异：在I型而非II型折叠中，亮氨酸特异性取决于tRNA（Leu）受体茎序列的存在。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，从而可能给整个tRNA折叠引入细微变化，就有可能检测到受体茎序列对LeuRS识别的其他隐秘贡献。讨论了这种效应的可能原因。
