Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes
Figure 5
All reactions were carried out under anaerobic conditions and are detailed in Materials and Methods. Synthetic tRNA[Ser]Sec was used in those reactions employing tRNA and its synthesis, aminoacylation with 3H-serine and phosphorylation were carried out as given [20]. Cloning of mouse sps2, and preparation of the Sec-to-Cys sps2 mutant, and cloning of E. coli SelA and mouse SecS are given in Materials and Methods.
(A) Sec biosynthesis using SeP as the active selenium donor with O-phosphoseryl-tRNA[Ser]Sec or seryl-tRNA[Ser]Sec and either mSecS or SelA as SecS is shown.
(B) Sec biosynthesis using mSPS2-Cys and selenide (HSe−) to provide SeP as the active selenium donor with O-phosphoseryl-tRNA[Ser]Sec or seryl-tRNA[Ser]Sec and either mSecS or SelA as SecS is shown. HSe− was maintained in the reduced state in reactions in B with DTT as described in Materials and Methods. Migration of control amino acids and pyruvate are indicated below the graphs in (A) and (B).
(C) The rate of Sec synthesis is shown. Reactions were terminated at 0, 1.25, 2.5, 5, 10, 20, 40, and 80 min. After chromatography and counting of samples in a liquid scintillation counter as given in Materials and Methods, the counts from the peaks of Sec, O-phosphoserine, or the degraded intermediate (the peak migrated after alanine and chromatographed with pyruvate) were pooled together for quantification at each time point. The analyses in this figure were carried out on deacylated products.