FIG. 4.

Effects of h-mtTFB1 RNA methyltransferase motif point mutations on SAM binding. A schematic diagram of the 346-amino-acid h-mtTFB1 protein is shown at the top of the figure with its RNA methyltransferase motifs I to VIII (17, 19) indicated by the black boxes. The positions of the three point mutations characterized in this study (G65A, motif I; N141A, motif IV; and K220A, motif VIII) are indicated by the arrows. The results of a solid-phase SAM-binding assay that involves the binding of radiolabeled ligand to immobilized GST::h-mtTFB1 proteins are shown in the bar graph. The ordinate represents the amount of labeled SAM (in counts per minute) bound to beads containing GST peptide alone (GST), GST::h-mtTFB1 (h-mtTFB1), and each mutated GST::h-mtTFB1 fusion protein (G65A, N141A, and K220A). That similar amounts of control and h-mtTFB1 proteins were loaded onto the beads was confirmed by Western analysis prior to each assay (data not shown).