Figure 3.

Affinity-purified T.thermophilus TrmI protein methylates A58 of in vitro transcribed T.thermophilus tRNA^Asp. (A) Nucleotide sequence of T.thermophilus tRNA^Asp (33). m¹A58 is shown in a gray box. The adenosines 5′-adjacent to a guanosine are indicated by an asterisk. (B) Autoradiograms of 2-dimensional chromatograms of P1 or T2 hydrolysates of [α-³²P]ATP- or [α-³²P]GTP-labeled T.thermophilus tRNA^Asp transcripts incubated for 1 h at 60°C in the presence of the purified TrmI protein (see Materials and Methods for details). In the case of the chromatogram designated GTP/T2*, the T2 hydrolysis was carried out in the absence of carrier yeast tRNA. Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers. (C) Kinetics of in vitro formation of m¹A58 in T.thermophilus tRNA^Asp in the presence (closed circles) or absence (open circles) of the purified TrmI protein.