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. 2010 Jan 6;285(12):9018–9029. doi: 10.1074/jbc.M109.065698

FIGURE 5.

FIGURE 5.

tRNA-dG18dG19A20 variant was not methylated by TrmH. A, RNA sequencing of tRNA-dG18dG19A20 variant using the Donis-Keller method. Lane C, untreated control RNA; lane Al (left), alkaline hydrolysis (95 °C, 2 min); lanes RNase T1 included 0.03, 0.1, or 0.3 units of enzyme; lanes RNase U2 included 0.01, 0.03, or 0.1 units of enzyme; Lane Al (right), alkaline hydrolysis (95 °C, 4 min). Positions of G and A nucleotides are marked on both sides of the gel, and those of dG18, Gm19, and G20 are indicated with arrows. B, elimination of the methyl group acceptance activity by substitution of G18G19G20 by dG18dG19A20. Methylation of tRNA-dG18 monitored by 10% PAGE (7 m urea). TrmH was pre-incubated with [14C]AdoMet, and then with either wild-type tRNAPhe (left lanes) or tRNAPhe-dG18dG19A20 (right lanes) prior to analysis by 10% PAGE (7 m urea). The gel was stained with methylene blue (MB, left panel), and an autoradiographic image of the same gel was then captured using a Fuji BAS2000 imaging analyzer (right panel).