FIG. 2.

Analysis of the SAM-binding site. (A) Effects of mutations of the SAM-binding site on N7 methylation of WNV RNA cap. The substrate G*pppA-RNA, representing the first 190 nucleotides of the WNV genome (the asterisk indicates that the following phosphate was ³²P labeled), was incubated with WT or mutant MTase in the presence of SAM. The MTase variants are indicated at the top. Since the reactions were performed in N7 buffer (pH 7.0), which supports N7 methylation but not 2′-O methylation, G*pppA-RNA was converted to m⁷G*pppA-RNA without further 2′-O methylation (39). The reaction mixtures were digested with nuclease P1 to release m⁷G*pppA (product) or G*pppA (residual substrate) and then analyzed on a TLC plate. The methylation efficiencies of mutant MTases were compared with that of the WT MTase (set at 100%) and are indicated below the TLC results. (B) Effects of mutations of the SAM-binding site on 2′-O methylation. The experiments were performed as described for panel A, except that the substrate m⁷G*pppA-RNA was incubated in 2′-O buffer (pH 10, which supports 2′-O methylation), resulting in m⁷G*pppAm-RNA. ³²P-labeled markers, G*pppA, and m⁷G*pppA are indicated at the top of panel A. The position of the origin and the migration positions of the G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are shown to the left of the TLC images. (C) Summary of methylation results from panels A and B. Averages of three independent experiments are presented. (D) SAM-binding activities of mutant MTases. The UV-cross-linked ³H-SAM-MTase complex was analyzed by SDS-PAGE and quantified by fluorography (top). The relative cross-linking efficiencies of the WT and mutant MTases are indicated below the autoradiograph. Five micrograms of each indicated MTase was separated by SDS-PAGE and stained with Coomassie blue (bottom).