In vitro expression of Escherichia coli ribosomal protein L10 gene: Tripeptide synthesis as a measure of functional mRNA

The in vitro synthesis of the initial tripeptide, fMet-Ala-Leu, of Escherichia coli ribosomal protein L10 has been studied in a plasmid DNA-directed system using purified factors. In contrast to the synthesis of the dipeptide, fMet-Ala, described recently (N. Robakis L. Meza-Basso N. Brot, and H. Weissbach, 1981, Proc. Nat. Acad. Sci. USA 78, 4261-4264), tripeptide formation leads to a stable peptidyl-tRNA:mRNA:70 S complex. Using mRNA for L10, a good correlation has been observed between the amount of tripeptide formed and the amount of fMet-tRNA bound to a mRNA:ribosome complex. These results indicate that tripeptide formation in this system can be used as a simple and specific assay for the amount of functional mRNA present.