INITIATION OF PROTEIN-SYNTHESIS IN MAMMALIAN-CELLS WITH CODONS OTHER THAN AUG AND AMINO-ACIDS OTHER THAN METHIONINE

Protein synthesis is initiated universally with the amino acid methion ine. In Escherichia coli, studies with anticodon sequence mutants of t he initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, howev er, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding rai sed the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we stu died the activities, in initiation, of four different anticodon sequen ce mutants of human initiator tRNA in mammalian COS1 cells, using repo rter genes carrying mutations in the initiation codon that are complem entary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, r espectively. CAG initiates protein synthesis with glutamine but extrem ely poorly, whereas UAG could not be used to initiate protein synthesi s with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons o ther than AUG for studying the many interesting aspects of protein syn thesis initiation in mammalian cells.