Sturchler-Pierrat C, Hubert N, Totsuka T, Mizutani T, Carbon P, Krol A
Unité Propre de Recherche 9002 du CNRS, Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
J Biol Chem. 1995 Aug 4;270(31):18570-4. doi: 10.1074/jbc.270.31.18570.
Selenocysteine synthesis is achieved on a specific tRNA, tRNA(Sec), which is first charged with serine to yield seryl-tRNA(Sec). Eukaryotic tRNA(Sec) exhibits an aminoacyl acceptor stem with a unique length of 9 base pairs. Within this stem, two base pairs, G5a.U67b and U6.U67, drew our attention, whose non-Watson-Crick status is maintained in the course of evolution either through U6.U67 base conservation or base covariation at G5a.U67b. Single or double point mutations were performed, which modified the identity of either or both of the base pairs. Serylation by seryl-tRNA synthetase was unaffected by substitutions at either G5a.U67b or U6.U67. Instead, and quite surprisingly, changing G5a.U67b and U6.U67 to G5a-C67b/U6.G67 or G5a-C67b/C6-G67 gave rise to a tRNA(Sec) mutant exhibiting a gain of function in serylation. This finding sheds light on the negative influence born by a few base pairs in the acceptor stem of tRNA(Sec) on its serylation abilities. The tRNA(Sec) capacities to support selenocysteylation were next examined with regard to a possible role played by the two non-Watson-Crick base pairs and the unique length of the acceptor stem. It first emerges from our study that tRNA(Sec) transcribed in vitro is able to support selenocysteylation. Second, none of the point mutations engineered at G5a.U67b and/or U6.U67 significantly modified the selenocysteylation level. In contrast, reduction of the acceptor stem length to 8 base pairs led tRNA(Sec) to lose its ability to efficiently support selenocysteylation. Thus, our study provides strong evidence that the length of the acceptor stem is of prime importance for the serine to selenocysteine conversion step.
硒代半胱氨酸的合成是在一种特定的转运RNA（tRNA(Sec)）上完成的，该转运RNA首先被丝氨酸负载，生成丝氨酰-tRNA(Sec)。真核生物的tRNA(Sec)具有一个独特的9个碱基对长度的氨酰基接受茎。在这个茎中，两个碱基对，即G5a.U67b和U6.U67，引起了我们的注意，它们的非沃森-克里克状态在进化过程中通过U6.U67碱基的保守性或G5a.U67b处的碱基共变得以维持。进行了单点或双点突变，这些突变改变了一个或两个碱基对的特性。丝氨酰-tRNA合成酶的丝氨酰化作用不受G5a.U67b或U6.U67处替换的影响。相反，令人惊讶的是，将G5a.U67b和U6.U67变为G5a-C67b/U6.G67或G5a-C67b/C6-G67会产生一个在丝氨酰化方面表现出功能增强的tRNA(Sec)突变体。这一发现揭示了tRNA(Sec)接受茎中的一些碱基对对其丝氨酰化能力的负面影响。接下来，研究了tRNA(Sec)支持硒代半胱氨酸化的能力，涉及两个非沃森-克里克碱基对和接受茎独特长度可能发挥的作用。我们的研究首先表明，体外转录的tRNA(Sec)能够支持硒代半胱氨酸化。其次，在G5a.U67b和/或U6.U67处设计的任何点突变都没有显著改变硒代半胱氨酸化水平。相比之下，将接受茎长度减少到8个碱基对会导致tRNA(Sec)失去有效支持硒代半胱氨酸化的能力。因此，我们的研究提供了有力证据，表明接受茎的长度对于丝氨酸向硒代半胱氨酸的转化步骤至关重要。
