FIGURE 3.
A, 15% SDS-polyacrylamide gel analysis of wild-type TrmH and two C-terminal deletion mutant proteins. The gel was stained with Coomassie Brilliant Blue. B, methyl transfer activities of wild-type TrmH and two C-terminal deletion mutant proteins. Yeast tRNAPhe transcript was used as substrate tRNA. The L179Stop mutant protein, in which the C-terminal region was deleted, completely lost methyl transfer activity. C, kinetic parameters of C-terminal deletion mutant proteins for yeast tRNAPhe. The Q185Stop mutant protein has decreased activity with a relatively large Km value, suggesting that amino acid residues from Gln-185 to Lys-194 are involved in the initial binding process. D, gel mobility shift assay with wild-type and mutant TrmH proteins. The gel was double-stained with Coomassie Brilliant Blue and methylene blue for detections of protein and RNA, respectively. The two mutant proteins (L179Stop and Q185Stop) did not show a clear band corresponding to the protein-tRNA complex. However, these proteins have weakened affinity for tRNA, because the bands derived from tRNA were smeared.