Key result
Fluorescent amino acid-based in vitro system successfully monitors translation initiation efficiency at AUG codons.
A novel in vitro fluorescence-based assay enables facile characterization of eukaryotic translation initiation mechanisms.
Offers a tool to study translation mechanisms; extends basic research but leaves clinical cardiovascular applications open.
A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.
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Karginov et al. (1999) studied this question. In vitro translation system with non-natural fluorescent amino acid incorporation was evaluated on Translation initiation efficiency. An in vitro translation system incorporating a non-natural fluorescent amino acid successfully monitored translation initiation efficiency at an AUG codon in different contexts.
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