FIGURE 2.
Incorporation of PyC75 to E. coli tRNACys. (A) A denaturing gel analysis (12% PAGE/7 M urea) of incorporation of PyC75 to the transcript of E. coli tRNACys. The final concentration of each component is indicated in parentheses. The full-length tRNA-A76 and the truncated tRNA-C74 were body labeled by transcription with α-32P-ATP, which provided a marker on the left. The body-labeled tRNA-C74 was used as the substrate for reactions 1–6, whereas the unlabeled tRNA-C74 was used with α-32P-ATP for reaction 7. The symbols “+” and “−” indicate the presence and absence of a reagent, respectively. The denaturing gel was run in 90 mM Tris-HCl (pH 8.3), 90 mM boric acid, and 2 mM EDTA at 1800 V for 2 h at 60°C. (B) RP-HPLC chromatogram of the CCA reaction on the C8 column developed by increasing concentration of methanol (shown in blue as percent in v/v). The elution profile of tRNA-C74 before the CCA reaction is shown in black, while that after the reaction is shown in red. (C) RP-HPLC chromatogram of the CysRS aminoacylation reaction of tRNA-CPyCA generated from extension of tRNA-C74 on the C8 column developed by increasing concentration of methanol (shown in blue as percent in v/v).