Codon-Dependent Translational Accuracy Controls Protein Quality in Escherichia coli but not in Saccharomyces cerevisiae

Abstract

In order to generate a functional proteome, gene expression pathways must assemble proteins accurately according to the rules of the genetic code. General gene expression accuracy is known to be high, but errors nevertheless occur with measurable frequencies. Here we develop a mass-spectrometry (MS) based assay for the detection of a particular type of gene expression error, amino acid misincorporation. This assay allows assessing a much broader range of misincorporation events compared to current, very sensitive but also very specific enzyme reporter assays. Our assay uncovers a remarkably rich pool of error products for a model protein expressed in E. coli, which depend quantitatively on codon usage in the expression construct. This codon usage dependence can be explained in part as a function of the composition of the tRNA pool in this organism. We further show that codon-dependent differences in error levels correlate with measurable changes in specific protein activity. In contrast to E. coli, error levels are lower, and appear not to be codon usage dependent, when the same model protein is expressed in S. cerevisiae.

Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates Lifespan

“…We argued that inadequate levels of aminoacyl-tRNA synthetases are expected to impair fidelity by limiting the availability of specific aminoacyl-tRNAs, leading to misincorporation of amino acids at the codons normally recognized by those charged tRNAs (due to use of a nearcognate but incorrect aminoacyl-tRNA). Similar findings were recently reported in yeast [20]. To test this, we knocked down selected aminoacyl-tRNA synthetases in C. elegans, a short-lived nematode worm that is widely used to study the control of lifespan.…”

“…In yeast, bacterial [19], and mammalian [9] cells, faster elongation rates compromise the accuracy of codon:anticodon recognition, although it has recently been reported that rates of translation elongation do not correlate with translation fidelity in yeast [20,21]. The above constructs allow us to assess the extent to which the rate of elongation affects the accuracy (fidelity) of mRNA translation, and explore what factors affect this.…”

“…Total RNA was extracted using TRIzol (Life Technologies). cDNA was synthesized using Super-Script III First-Strand (synthesis system for RT-PCR) from Life Technologies with oligo(dT) 20 . Samples were analyzed in triplicate (for each experiment) with SYBR green dye mix (PrimerDesign, Camberley, Hampshire, UK) on a CFX Connect Real-Time system (Bio-Rad Laboratories).…”