SUMMARY: Two methionine auxotrophs () of with the structural genes of the regulon intact have been studied. They possessed abnormal growth kinetics and the effect of the mutation on protein, ribonucleic acid and DNA synthesis suggested that these strains were impaired in their ability to synthesize protein; since they were able to synthesize methionine, but still required it for growth, they might have been defective in methionine activation for protein synthesis. To test this, the! activity of methionyl-sRNA synthetase (L-methionine: sRNA ligase (AMP) EC 6.I.I.10) was determined in enzymic extracts of wild-type and strains. By using an acylation reaction the activation of methionine for protein synthesis was shown to be very decreased in extracts and this was reflect- ed by a decreased level of charged methionyl-sRNA in mutant bacteria; in the pyrophosphate exchange assay mutants showed greatly increased (methionine) values. The release of [H]methionine from [H]methionyl-sRNA was catalysed by wild-type extract, provided that pyrophosphate was present in the assay mixture, but not by the mutant extract. These results are discussed in relation to the two-part reaction catalysed by methionyl-sRNA synthetase. Mutant and wild-type enzyme behaviour differed at different pH values but not when subjected to chromatography on DEAE-cellulose or gel-filtration on Sephadex-G. mutants grown with limiting methionine had decreased values of all the biosynthetic enzymes except cystathionase, which was apparently de-repressed, suggesting that methionyl-sRNA was not the co-repressor for the methionine biosynthetic pathway.


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