Development of a tRNA-dependent in vitro translation system

Abstract

A method is described for depleting rabbit reticulocyte lysates and wheat germ extracts of endogenous tRNAs by affinity chromatography using a matrix generated by coupling ethanolamine to epoxy-activated Sepharose 6B. Greater than 90% depletion of tRNA is achieved with the result that translation becomes in effect absolutely dependent on added tRNA. This depletion procedure should prove very useful for studying the influence of tRNA concentration, and the spectrum of the tRNA population, on recoding events such as programmed frameshifting and readthrough of termination codons.

In vitro selection for sense codon suppression

“…4B), thereby demonstrating the efficiency with which the chromatographic step removes endogenous tRNAs. It should be noted that we did not observe a complete elimination of endogenous tRNAs, which suggests that the background counts measured by Jackson and coworkers for tRNA-dependent lysate without tRNA supplementation (Jackson et al 2001) can still support efficient translation of mRNA-peptide fusions.…”

“…If this is true, then reducing the overall tRNA concentration should increase suppression at all sense codons. Previously, Jackson and coworkers developed a protocol using ethanolamine-Sepharose to specifically deplete tRNAs from a translation extract (Jackson et al 2001). We subjected commercial rabbit reticulocyte lysate to ethanolamine-Sepharose to create a lysate whose endogenous tRNA population has been partially depleted, and then assayed GUA suppression by biocytintRNA UAC (Fig.…”

“…This result indicates that an optimal concentration or balance of endogenous tRNAs to the sense suppressor likely exists. Lysates totally lacking tRNA will not support translation (Jackson et al 2001), whereas lysates that contain large quantities of endogenous tRNA will require high concentrations of suppressor to incorporate an unnatural amino acid. In line with this hypothesis, adding back calf liver tRNA to 20 µg/mL (a lower concentration than the untreated, commercial lysate concentration of 60 µg/mL; Jackson and Hunt 1983) results in improved sense suppression approaching 50% (Fig.…”

“…In an effort to determine the impact of endogenous tRNA depletion in translation reactions on sense codon suppression, we passed our in-house rabbit reticulocyte lysate (prepared as described in Materials and Methods) over an ethanolamine-Sepharose column (Jackson et al 2001). The level of endogenous tRNA depletion is assessed by the fold change in total mRNA-peptide fusion (i.e., [ 35 S]-labeled material eluted from dT 25 -cellulose) on the addition of 1.5 µg calf liver tRNA (Fig.…”

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

“…Moreover, suppression of both amber and ochre stop codons has been demonstrated in mammalian cells [67]. Attempts have been made to use suppressors of rare codons in E. coli expression systems, and depletion of endogenous tRNAs from the cellfree extract has made this more tractable [68][69][70][71][72]. However, readthrough from tRNAs with near-anticodons and the need to heavily mutagenize genes to remove the selected sense codons makes this strategy problematic.…”