Studies on microbial RNA: II. Transfer of methyl groups from methionine to the RNA of a ribonucleoprotein particle

Abstract

When Escherichia coli W6 (a strain with a relaxed control of RNA synthesis) is starved for methionine, a ribonucleoprotein particle accumulates. These particles were partially purified, and the preparation was investigated by zone centrifuga-tion. It was found that the particles contained about 70% ribosomal RNA (R-RNA) and about 30% protein. The preparation was not homogeneous and contained additional protein and traces of S-RNA. These particles produced by methionine starvation of E. coli W6 were used as a substrate for the methylation of their own RNA. In the presence of ATP and a supernatant fraction, methyl groups from methionine were incorporated into a product obtained after phenol extraction and alcohol precipitation. Cold S-adenosylmethionine quenches this incorporation. [14C]methyl groups from S-adenosylmethionine were incorporated in the absence of supernatant liquid and ATP. The reaction product was hydrolysed by either 0·3 M -potassium hydroxide, pancreatic RNase or venom diesterase. Labelled RNA was prepared from a reaction mixture and fractionated by chromatography on Sephadex G200. Both R-RNA and S-RNA were methylated. The incorporation was proportional to the amount of methionine-starvation particles added. About one methylated base per 5000 bases was found as saturation value for R-RNA. The pattern of methylated nucleosides was found to be the same for R-RNA labelled in vivo and in vitro. This pattern differed from that for S-RNA. The dependence of the rate of methylation on the supernatant liquid was investigated with either methionine or S-adenosylmethionine as methyl donor. With the former, the supernatant liquid gave up to a tenfold stimulation; with the latter the stimulation was just detectable. For reactions with purified RNA from particles produced by methionine starvation of E. coli, the rate of methylation of R-RNA was strongly dependent on the amount of ribosomes present. These results suggest that R-RNA methylating enzymes are bound to particles and that the methylation of R-RNA is a step in the biosynthesis of ribosomes.