3h phenylalanine

In vitro aminoacylation assays were performed essentially as described [29] (link), in a 25 μl volume at 30 °C for 20 min using 50 μM ³H phenylalanine (1 mCi, 126.2 Ci/mmol; Perkin Elmer), 4 μg tRNA and 2–4 μg enzyme in buffer containing 50 mM Tris–HCl pH 7.75, 12 mM MgCl₂, 10 mM KCl, 0.2 μg/ml BSA and 2.5 mM ATP as described. Prior to addition, tRNAs were heated for 2 min at 60 °C and cooled down slowly to room temperature. At different time points, 5 μl aliquots were spotted onto Whatman 0.45 μM nitrocellulose membrane and precipitated in ice cold 5% trichloroacetic acid. Incorporation of radioactive phenylalanine was measured by liquid scintillation counting. Furthermore, to assess ATP binding only, reactions under similar conditions were performed using 2 mM γATP (250 μCi, 3000 Ci/mmol; Perkin Elmer) and 4 μg enzyme. Incorporation of radioactive ATP was measured by Cerenkov counter.

Poly(U), puromycin and tRNA^Phe from E. coli were from Sigma-Aldrich and L-[¹⁴C(U)]Phenylalanine (18 GBq/mmol) or [³H]Phenylalanine (4740 GBq/mmol) from Perkin Elmer. tRNA^Phe was aminoacylated using [¹⁴C(U)]Phenylalanine or [³H]Phenylalanine with an excess amount of partially purified phenylalanyl-tRNA synthetase from E. coli [21 (link)]. [³H]Phe-tRNA^Phe (128 GBq/mol) was acetylated following the method described by Haenni and Chapeville [22 (link)].