Mizutani T, Tanabe K, Yamada K
Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
FEBS Lett. 1998 Jun 12;429(2):189-93. doi: 10.1016/s0014-5793(98)00589-4.
In Escherichia coli, selenocysteine biosynthesis and incorporation into selenoproteins requires the action of four gene products, including the specialized selenocysteine tRNA(Sec) and elongation factor SELB, different from the universal EF-Tu. In this regard, the situation is less clear in eukaryotes, but we previously reported the existence of SePF, a putative SELB homologue. The secondary structure of the tRNA(Sec) differs slightly in eukaryotes, due to a change in the lengths of several stems. Two non-Watson-Crick base pairs, G5a x U67b and U6 x U67, reside in the acceptor stem and are conserved in the course of evolution. Since it has already been reported that changing them to Watson-Crick base pairs did not affect the serylation or selenylation levels of tRNA(Sec), we asked whether these non-Watson-Crick base pairs are required for the interaction with SePF. To this end, tRNA(Sec) variants carrying Watson-Crick changes at these positions were tested for their ability to maintain the interaction with SePF. In these assays, the tRNA(Sec)-SePF interaction was determined by the protective action it confers against hydrolysis of the amino acid ester bond, under basic conditions. All the changes introduced at U6 x U67 did not significantly affect the interaction. Interestingly, however, the G5a x U67b to G5a-C67b substitution was sufficient, by itself, to lead to unprotection of the ester bond. Therefore, our finding strongly suggests that SePF is unable to interact with a tRNA(Sec) mutant version carrying a Watson-Crick G5a-C67b instead of the wild-type G5a x U67b base pair, establishing that G5a x U67b constitutes a structural determinant for SePF interaction.
在大肠杆菌中，硒代半胱氨酸的生物合成以及掺入硒蛋白需要四种基因产物的作用，包括特殊的硒代半胱氨酸tRNA（Sec）和延伸因子SELB，后者不同于通用的EF - Tu。在这方面，真核生物中的情况尚不清楚，但我们之前报道过存在一种假定的SELB同源物SePF。由于几个茎的长度发生了变化，真核生物中tRNA（Sec）的二级结构略有不同。两个非沃森 - 克里克碱基对，G5a x U67b和U6 x U67，位于受体茎中且在进化过程中保守。由于已经报道将它们改变为沃森 - 克里克碱基对不会影响tRNA（Sec）的丝氨酰化或硒代丝氨酰化水平，我们询问这些非沃森 - 克里克碱基对对于与SePF的相互作用是否必要。为此，测试了在这些位置携带沃森 - 克里克变化的tRNA（Sec）变体维持与SePF相互作用的能力。在这些实验中，tRNA（Sec） - SePF相互作用是通过其在碱性条件下对氨基酸酯键水解的保护作用来确定的。在U6 x U67处引入的所有变化均未显著影响相互作用。然而，有趣的是，仅G5a x U67b到G5a - C67b的取代就足以导致酯键去保护。因此，我们的发现强烈表明SePF无法与携带沃森 - 克里克G5a - C67b而非野生型G5a x U67b碱基对的tRNA（Sec）突变体版本相互作用，这表明G5a x U67b构成了SePF相互作用的结构决定因素。
