Figure 3.
D210 is not essential for m1G9 activity of Trm10. (A) Activity assay of ScTrm10 with G9-labeled tRNAGly. Reactions contain 10-fold serial dilutions of purified ScTrm10; WT (wild-type) (3 μM-0.3 nM); D210N (3–0.003 μM); D210K (3–0.03 μM); D210A (3–0.003 μM); or no enzyme (–). Positions of the labeled m1G product (p*m1G) and unreacted substrate (p*G) are indicated. Specific activity of each enzyme was calculated based on the amount of each enzyme required to produce 20% product in one hour at 30°C and is labeled under the relevant panel for each enzyme. (B) Complementation of the yeast trm10Δ phenotype. An S. cerevisiae trm10Δ strain was transformed with plasmids expressing either wild-type ScTrm10 or human TRMT10A, or the human TRMT10A D210N variant, as indicated. Control strains (TRM10 and trm10Δ) transformed with empty vector are shown in the top two rows of each panel. Two independently isolated colonies for each strain were analyzed by replica plating onto S-Gal-leu media containing the indicated concentrations of 5-fluorouracil (0–25 ug/ml) and grown at either 30 or 37°C for 2–3 days before photographing.