TABLE 1.

In vitro methyl esterification catalyzed by the Trm9 methyltransferase^a

Reaction component(s)	Radioactivity as 3H ester (cpm ± SD)
Expt 1
trm9 mutant extract and saponified tRNA	270 ± 71
Wild-type extract and saponified tRNA	3,546 ± 530
Trm9-overexpressing extract and saponified tRNA	5,138 ± 1,100
trm9 mutant extract and tRNA	428 ± 187
Wild-type extract and tRNA	668 ± 261
Trm9-overexpressing extract and tRNA	846 ± 257
Saponified tRNA alone	340 ± 141
tRNA alone	236 ± 13
trm9 mutant extract alone	314 ± 159
Wild-type extract alone	340 ± 173
Trm9-overexpressing extract alone	334 ± 122
Expt 2
Trm9 immunoprecipitate and tRNA	623
Trm9 immunoprecipitate alone	11
tRNA alone	80
Expt 3
Control immunoprecipitate (YPD) and tRNA	51
Control immunoprecipitate (YPD) alone	39

^aIn experiment 1, reaction mixtures containing 3 μl of [³H]AdoMet (final concentration, 0.8 μM), 50 μg of the designated protein extract, and 50 μg of either a commercial preparation of tRNA from baker's yeast or its saponified derivative (prepared as described in Materials and Methods) were incubated at 30°C for 30 min in a final volume of 50 μl containing 100 mM Tris-HCl, pH 8.0. Reactions were stopped by adding 1 volume of phenol and 1 volume of chloroform. tRNA was precipitated from the aqueous phase by adding 2 volumes of ethanol, and its [³H]methyl ester content was assayed as described in Materials and Methods. In experiment 2, in vitro reaction mixtures were prepared as described in Materials and Methods. The enzyme source was the monoclonal anti-HA immunoprecipitated protein (26) from strain HKY111 cells grown in YPG medium to express the HA-Trm9 fusion protein. In experiment 3, in vitro reaction mixtures were prepared as in experiment 2 except that the HKY111 cells were grown in YPD medium so that the HA-Trm9 fusion protein was not expressed.