Facile characterization of translation initiation via nonsense codon suppression

Andrei V. Karginov, Michiel Lodder, Sidney M. Hecht

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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 tRNA(Phe)(CUA) 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.

Original languageEnglish (US)
Pages (from-to)3283-3290
Number of pages8
JournalNucleic acids research
Volume27
Issue number16
DOIs
StatePublished - Aug 15 1999
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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