Facile characterization of translation initiation via nonsense codon suppression

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

doi:10.1093/nar/27.16.3283

Facile characterization of translation initiation via nonsense codon suppression

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

Research output: Contribution to journal › Article › peer-review

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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 language	English (US)
Pages (from-to)	3283-3290
Number of pages	8
Journal	Nucleic acids research
Volume	27
Issue number	16
DOIs	https://doi.org/10.1093/nar/27.16.3283
State	Published - Aug 15 1999
Externally published	Yes

ASJC Scopus subject areas

Genetics

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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.",

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AU - Lodder, Michiel

AU - Hecht, Sidney M.

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AB - 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.

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Facile characterization of translation initiation via nonsense codon suppression

Abstract

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