Abstract
Mutant human initiator tRNA genes carrying changes in each of the three features unique to eukaryotic initiator tRNAs have been constructed, and introduced into CV-1 monkey kidney cells using SV40 virus vectors. The mutant tRNA genes are expressed, and the mutant tRNAs can all be aminoacylated with both rabbit liver and Escherichia coli methionyl-tRNA synthetases. Based on aminoacylation levels, the tRNAs are expressed to 5-15-fold over the level of endogenous initiator tRNA. The activity of the mutant [35S]methionyl-tRNAs in initiation was studied in rabbit reticulocyte and wheat germ cell-free protein synthesis systems programmed with various mRNAs. Initiation is studied by using a mRNA that codes for a protein whose N-terminal methionine is stable and not removed by methionine aminopeptidase. Changing the A1:U72 base pair to a G1:C72 base pair greatly reduced activity of the tRNA in initiation. Changing the three consecutive G:C base pairs (G29G30G31:C39C40C41) in the anticodon stem to those found in elongator methionine tRNA also reduced initiation activity. Interestingly, changing the A54 and A60 residues in loop IV to T54 and U60 had less of an effect on activity. The tRNA with changes in all three conserved features had virtually no activity in initiation.
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