In vitro methylation of tobacco mosaic virus RNA with ribothymidine-forming tRNA methyltransferase. Characterization and specificity of the reaction.
A novel method has been developed for the detection and study of tRNA-like moieties in viral RNAs. Tobacco mosaic virus RNA is an acceptable substrate for crude Escherichia coli ribothymidine-forming tRNA methyltransferase. Under optimum reaction conditions at least 85% of the methylation product is ribothymidine (rT). The reaction is essentially quantitative, 1 mol of rT being formed per mol of tobacco mosaic virus RNA. The optimum reaction conditions include the presence of 6.6 micrometers S-adenosyl-L-[Me-3H]methionine, 25 micrometers spermine, 25 mM ammonium acetate, and 50 mM HEPES, pH 8.0. Sequence analysis of (Me-3H)-labeled tobacco mosaic virus RNA shows that all of the methylation occurs at a single site and strongly suggests that this site is the 32nd residue from the 3'-end of tobacco mosaic virus RNA. This site closely resembles the normal position of rT in transfer RNA.
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D012367:RNA, Viral; D012260:Ribonucleases; D013096:Spermine / Q000494:pharmacology; D014027:Tobacco Mosaic Virus; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000378:metabolism*
D012367:RNA, Viral; C009182:ribothymidine; D013096:Spermine; D012359:tRNA Methyltransferases; D012260:Ribonucleases; D014529:Uridine