FATE OF NORLEUCINE AS A REPLACEMENT FOR METHIONINE IN PROTEIN-SYNTHESIS

In vivo synthesised protein with norleucine occupying one half of the normal methionine loci was prepared using a methionine auxotroph of Escherichia coli K12. The extent of charging of the analogue onto both tRNAmet species and subsequent incorporation into soluble protein was monitored with a double-labelling system comprising [G-3H]norleucine and [35S]methionine. Further experiments established that norleucine can be formylated in vivo once charged onto the initiator tRNAfmet. An N-terminal analysis of the crude soluble protein revealed that formylnorleucyl-tRNAfmet can initiate protein synthesis and that the formyl group is then removed from the nascent polypeptide. We were also led to conclude that the N-terminal methionine-amino peptidase does not recognise the analogue in this position. Slow growth rates on the methionine analogue have been partly attributed to limiting levels of charged tRNAmmet, resulting in turn from the inefficiency of norleucine charging by methionyl-tRNA synthetase. Finally no evidence has been found for the production of aberrant protein as a result of norleucine incorporation, implying that limited growth on the analogue is due to its inability to replace methionine as the precursor of S-adenosyl methionine. © 1979.