The in vitro O2'-methylation of RNA in the ribonucleoprotein precursor-particles from a relaxed mutant of Escherichia coli.

Abstract

Ribonucleoprotein "precursor-particles" were isolated from a methionine auxotroph of E. coli (RCrelaxed), which had been cultured under conditions of methionine deprivation. Following incubation of the "particles" in vitro with [methyl-14C]-S-adenosyl methionine, the ribonucleates were extracted from the particles, and precipitated from aqueous 2 M sodium chloride solution at 0°, before analysis for the incorporation of O2′-[methyl-14C]-substituents into the RNA. It was found that two of the four principal alkali-stable dinucleotides, known to be present in alkali hydrolysates of E. coli rRNA, contained O2′-[methyl-14C]-substituents: O2′Me-GpGp and O2′Me-CpCp, whereas the two remaining dinucleotides, N4,O2′diMe-CpCp and O2′Me-UpGp, did not contain significant radioactivity. It has been concluded that highly specific enzymic O2′-methylation of E. coli RNA can occur subsequent to polynucleotide synthesis. Allied studies are reported concerning the characterization of N4,O2′-[dimethyl-14C]-cytidine 5′-phosphate that was recovered from whole snake venom hydrolysates of E. coli rRNA, which had been labeled in vivo with [methyl-14C].