SELENOCYSTEYLATION IN EUKARYOTES NECESSITATES THE UNIQUELY LONG AMINOACYL ACCEPTOR STEM OF SELENOCYSTEINE TRNA(SEC)

Selenocysteine synthesis is achieved on a specific tRNA, tRNA(Sec), wh ich 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, d rew our attention, whose non-Watson-Crick status is maintained in the course of evolution either through U6.U67 base conservation or base co variation at G5a.U67b. Single or double point mutations were performed , which modified the identity of either or both of the base pairs. Ser ylation by seryl-tRNA synthetase was unaffected by substitutions at ei ther 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 fin ding 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 pair s and the unique length of the acceptor stem. It first emerges from ou r study that tRNA(Sec) transcribed in vitro is able to support selenoc ysteylation. Second, none of the point mutations engineered at G5a.U67 b and/or U6.U67 significantly modified the selenocysteylation level. I n contrast, reduction of the acceptor stem length to 8 base pairs led tRNA(Sec) to lose its ability to efficiently support selenocysteylatio n. Thus, our study provides strong evidence that the length of the acc eptor stem is of prime importance for the serine to selenocysteine con version step.