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. 2000 Apr 25;97(10):5173–5178. doi: 10.1073/pnas.090102597

Figure 2.

Figure 2

FlagGcd10p/Gcd14p complexes specifically catalyze synthesis of m1A in purified tRNAiMet. Initiator tRNAMet purified from yJA146 (gcd10Δ hcIMT4) was incubated with purified FlagGcd10p/Gcd14p complexes and [methyl-14C]AdoMet as described in Fig. 1, hydrolyzed to nucleosides, and resolved by HPLC chromatography as described (5). The fractions were monitored for UV absorbance at 254 nm (A254; solid line) and assayed for cpm [14C-methyl] by flow scintillation analysis as described (31) (dotted line). (A) Mock-treated tRNA. (B) tRNA treated with FlagGcd10p/Gcd14p complexes. (C) Double reciprocal plots of tRNA(m1A)MTase activity of purified FlagGcd10p/Gcd14p complexes as a function of tRNAiMet concentration (1.2–60 nM) (a) or Ado[14C]Met concentration (0.5–20 μM) (b). Assays contained 10 μM Ado[14C]Met (a) or 10 μM tRNAiMet (purified from the gcd10Δ hcIMT4 strain) (b) and approximately 0.3 μg of enzyme in each reaction (100 μl). Lines were fitted by using linear regression analysis.