Open AccessSimplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.
Author(s) -
Y. Cenatiempo,
Tomasz Twardowski,
Betty Redfield,
Brian R. Reid,
Harry Dauerman,
Herbert Weissbach,
Nathan Brot
Publication year - 1983
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.80.11.3223
Subject(s) - tripeptide , reticulocyte , messenger rna , in vitro , translation (biology) , protein biosynthesis , biology , transfer rna , microbiology and biotechnology , initiation factor , dipeptide , escherichia coli , globin , eif4a , biochemistry , chemistry , amino acid , rna , gene
An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val. Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.