FIG. 1.

The product of the E. coli yggH ORF catalyzes the formation of m⁷G in tRNA. (A) SDS-PAGE of the purified YggHH6 protein. Lane 1, molecular mass markers in kilodaltons (Pharmacia Biotech); lane 2, purified protein. The thick and thin arrows indicate the recombinant YggHH6 protein and its minor contaminant, respectively (see the text for details). (B) Autoradiography of a two-dimensional chromatogram of 5′ phosphate nucleotides on a thin-layer cellulose plate. Total tRNA (100 μg) from the methionine-starved P4X-SB25 strain was incubated in a 200-μl reaction mixture containing 50 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]-Na (pH 7.0), 4 mM MgCl₂, 10 μM [methyl-¹⁴C]AdoMet (53 mCi/mmol), and 0.4 μg of the purified YggHH6 protein. After a 30-min incubation at 37°C, the tRNA was recovered and digested by nuclease P1, and the resulting nucleotides were analyzed as described previously (12).