THE CHARACTERIZATION OF A NEW SPECIES OF LEUCYL-sRNA FORMED DURING METHIONINE DEPRIVATION OF ESCHERICHIA COLI WITH RELAXED CONTROL

Ilost amino acid auxotrophs of Escherichia coli synthesize RNA at a significant rate only when the essential amino acid is supplied and, therefore, when the cells are capable of net protein synthesis.', However, Borek et al.2 have shown that a particular methionine auxotroph of E. coli (58-161) is capable of net RNA synthesis during periods of methionine deprivation and differs from other auxotrophs in that the control of RNA synthesis is "relaxed" rather than "stringent." Subsequent genetic analysis of this mutant3l 4 has shown that it contains at least two genetic lesions, one related to the methionine requirement, the other to the control of RNA synthesis. Investigations of the nature of the RNA formed during methionine deprivation of the relaxed control mutant have indicated that the sRNA does not contain the full complement of methylated purines and pyrimidines,5' 6 which are primarily localized in sRNA. The evidence accumulated to date indicates that despite this deficiency the sRNA formed during methionine starvation can be acylated with amino acids in reactions catalyzed by the homologous amino acid activating enzymes.7-9 Furthermore, methyl-deficient sRNA species active as a substrate for one amino acid activating enzyme are not substrates for other activating enzymes,9 indicating that the amino acid activating enzymes exhibit the same high degree of substrate specificity with methyl-deficient sRNA that they exhibit with normal sRNA. In a previous publication'0 it was shown that sRNA obtained from E. coli after methionine deprivation was a poor substrate for the leucine activating enzyme from yeast as compared to sRNA from normal E. coli cells. This change was attributed to the formation during methionine starvation of a species of sRNA which was a competent acceptor for leucine with the E. coli enzyme but not with the yeast enzyme. This proposal is supported by the fact that the elution profiles from methylated albumin columns (MAK) of leucyl-sRNA from normal and methyl-deficient sRNA were strikingly different and indicated that a new species of leucine acceptor sRNA appeared during methionine deprivation. In the present communication, evidence is presented indicating that the new chromatographic component is a methyl-deficient species of leucyl-sRNA. this new component accumulates during methionine deprivation but not during leucine deprivation of a relaxed control mutant auxotrophic for both of these amino acids Under conditions identical to those leading to the accumulation of the new component in the relaxed control mutant, a methionine auxotroph of E. coli with stringent control does not accumulate it. Furthermore, its accumulation during methionine deprivation parallels the increased ability of the sRNA to accept methyl groups from S-adenosylmethionine in the RNA methylase reaction. Materials and Methods.-Organisms: Cultures of the relaxed control methionine auxotroph