FIGURE 6.

Methyl transferase (MTase) activities of mutant _TkTrm10 compared to the WT. E. coli tRNA was incubated in the presence of 30 µg of purified enzyme and [methyl-¹⁴C]-SAM (see Materials and Methods) after which either total radioactivity incorporated in tRNA was counted after TCA precipitation or the 5′-phosphate mononucleosides, obtained after digestion of the tRNA by nuclease P1, were separated by 2D-TLC. The radioactive nucleotides were then identified by autoradiography. (A) Autoradiograms of the 5′-phosphate mononucleosides separated by 2D-TLC. Circles in dotted lines show the migration of the four canonical nucleotides used as UV markers. The _TkTrm10 variant is indicated at the top left corner of each autoradiogram. The radioactivity of the spots corresponding to pm¹A and pm¹G was measured by scintillation counting. (B) Total radioactivity incorporated in tRNA by the different _TkTrm10 variants related to WT (left panel) and the pm¹A/pm¹G ratio (right panel). Values given are the average ± SD of three independent experiments.