FIGURE 5.
Point mutations in the T. thermophilus tRNAPhe transcript. A, 17 point mutations were introduced individually into the T. thermophilus tRNAPhe transcript. The arrows show the mutations and transcript numbers. The mutation sites and positions are indicated in the clover leaf structure. The transcripts of which the initial velocities of methyl group acceptance activity were more than 50% of that of the wild-type tRNAPhe transcript are enclosed with boxes. The kinetic parameters are summarized in Table 3. B, the methyl group acceptance activities of point mutants were analyzed with the gel assay. The upper panels show the gel stained with methylene blue. The lane numbers correspond to the transcript numbers. WT indicates the T. thermophilus wild-type tRNAPhe transcript. Incorporation of the [14C]methyl group was monitored by autoradiography of the same gels (lower panels). C, the preferred T-loop sequence for methylation by TrmI is depicted. The methylation site A58 is enclosed with a box.