Purification, cloning, and characterization of the 16S RNA m5C967 methyltransferase from Escherichia coli.

The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed to be distinct due to a DNA sequencing error. The gene, here named rsmB, encodes a 429-amino acid protein that has a number of homologues in prokaryotes, Archaea, and eukaryotes. C-Terminal sequencing of the overexpressed and affinity-purified protein by mass spectrometry methods verified the sequence expected for the gene product. The recombinant protein exhibited the same specificity as the previously described native enzyme; that is, it formed only m5C and only at position 967. C1407, which is also m5C in natural 16S RNA, was not methylated. In vitro, the enzyme only recognized free 16S RNA. 30S ribosomal subunits were not a substrate. There was no requirement for added magnesium, suggesting that extensive secondary or tertiary structure in the RNA substrate may not be a requirement for recognition.

Purification, cloning, and characterization of the 16 S RNA m2G1207 methyltransferase from Escherichia coli.

The methyltransferase that forms m2G1207 in Escherichia coli small subunit rRNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme as the open reading frame yjjT (SWISS-PROT accession number ). The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid protein that has homologs in prokaryotes, Archaea, and possibly also in lower eukaryotes. The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corresponding free RNA. By hybridization and protection of appropriate segments of 16 S RNA that had been extracted from 30 S subunits methylated by the enzyme, it was shown that of the three naturally occurring m2G residues, only m2G1207 was formed. Whereas close to unit stoichiometry of methylation could be achieved at 0.9 mM Mg2+, both 2 mM EDTA and 6 mM Mg2+ markedly reduced the level of methylation, suggesting that the optimal substrate may be a ribonucleoprotein particle less structured than a 30 S ribosome but more so than free RNA.

Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.

Time dependence of ethionine-induced changes in rat liver transfer RNA methylation.

Methyl-deficient transfer RNA (tRNA) and subnormal levels of tRNA-methylating enzymes were found in the livers of female rats that had received injections of 250 mg DL-ethionine per kg body weight per day and 120 mg adenine per kg body weight per day for 2 days. Adenine alone had no effect. When the ethionine plus adenine injections were continued for longer periods of time, liver tRNA-methylating enzyme activity measured in vitro gradually increased and exceeded that of the controls. Concurrently, the relative methyl deficiency of liver tRNA decreased. The latter was evident because of the decreased ability of the tRNA to accept methyl groups during in vitro methylation catalyzed by homologous enzymes. Liver tRNA from animals that were treated with ethionine for 7 days could accept only about 40% as many methyl groups as could tRNA from animals that had received ethionine for only 2 days. No further significant change in methyl deficiency of the tRNA was seen when ethionine administration was extended to a total of 14 days. Enzyme preparations from ethionine-treated, but not control, rat livers contained dialyzable substances that inhibited the tRNA methylases and altered the base specificity of these enzymes. Although S-adenosylhomocysteine and S-adenosylethionine were found to be present in the liver preparations, neither of these substances could account for the observed changes in specificity.

Inhibition of protein synthesis in intact HeLa cells.

Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 x 10(-7) M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [(14)C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.