Fig. 3.
PSTK activity. (A) Aminoacylation and phosphorylation as independent reactions. Aminoacylation of synthetic tRNA[Ser]Sec with [3H]serine and phosphorylation of [3H]seryl-tRNA[Ser]Sec with [γ-32P]ATP were carried out. (B) Aminoacylation and phosphorylation as coupled reactions. Coupled reactions in which aminoacylation and phosphorylation occurred in the same reaction with [3H]serine and [γ-32P]ATP were carried out. Use of thioredoxin as a control protein in place of PSTK in the kinase reaction showed no phosphorylation activity of tRNA[Ser]Sec.(C) Divalent metal cation requirement. Reactions for determining the cofactor requirement were carried out with increasing concentrations of divalent cation. (D) Reversibility of the reaction. Aliquots were removed from reactions with or without ADP, and the amount of radioactivity remaining in O-[32P]phosphoseryl-tRNA[Ser]Sec was determined by the phosphocellulose filter assay at the time intervals shown. At the end of the reaction, the remaining sample was deacylated, and the radioactivity present in ATP and in O-phosphoserine was determined by measuring strips that were cut out from a developed TLC chromatogram. The reaction was reversed 26.6%, as determined by the radioactivity present in ATP (8.25 × 104 cpm) divided by the total counts present in ATP and O-phosphoserine (3.105 × 105 cpm). Details of the reactions involving aminoacylation, phosphorylation, cofactor requirement, and reversibility are given in Methods.