Facile characterization of translation initiation via nonsense codon suppression

被引:8
作者
Karginov, AV
Lodder, M
Hecht, SM
机构
[1] Univ Virginia, Dept Chem, Charlottesville, VA 22901 USA
[2] Univ Virginia, Dept Biol, Charlottesville, VA 22901 USA
关键词
D O I
10.1093/nar/27.16.3283
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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(CUA)(Phe) 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.
引用
收藏
页码:3283 / 3290
页数:8
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