Systematic detection of amino acid substitutions in proteome reveals a mechanistic basis of ribosome errors

Abstract

Translation errors limit the accuracy of information transmission from DNA to proteins. Selective pressures shape the way cells produce their proteins: the translation machinery and the mRNA sequences it decodes co-evolved to ensure that translation proceeds fast and accurately in a wide range of environmental conditions. Our understanding of the causes of amino acid misincorporations and of their effect on the evolution of protein sequences is largely hindered by the lack of experimental methods to observe errors at the full proteome level. Here, we systematically detect and quantify errors in entire proteomes from mass spectrometry data. Following HPLC MS-MS data acquisition, we identify E. coli and S. cerevisiae peptides whose mass and fragment ion spectrum are consistent with that of a peptide bearing a single amino acid substitution, and verify that such spectrum cannot result from a post-translational modification. Our analyses confirm that most substitutions occur due to codon-to-anticodon mispairing within the ribosome. Patterns of errors due to mispairing were similar in bacteria and yeast, suggesting that the error spectrum is chemically constrained. Treating E. coli cells with a drug known to affect ribosomal proofreading increased the error rates due to mispairing at the wobble codon position. Starving bacteria for serine resulted in specific patterns of substitutions reflecting the amino acid deficiency. Overall, translation errors tend to occur at positions that are less evolutionarily conserved, and that minimally affect protein energetic stability, indicating that they are selected against. Genome wide ribosome density data suggest that errors occur at sites where ribosome velocity is relatively high, supporting the notion of a tradeoff between speed and accuracy as predicted by proofreading theories. Together our results reveal a mechanistic basis for ribosome errors in translation.

Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates 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.…”

Global mistranslation increases cell survival under stress in Escherichia coli

“…More generally, high global (rather than proteinspecific) mistranslation rates could confer a selective advantage by generating a "statistical proteome"-a bet-hedging strategy whereby a few cells with some mistranslated proteins (rather than all cells with one specific mistranslated protein as in the previous example) can survive an environmental stress [15,16]. Such a mixed proteome can occur both as a consequence of baseline mistranslation (as shown by proteome analyses [1]) or through artificial or stress-induced mistranslation. One example of general, stress-induced proteome-wide beneficial mistranslation comes from work on mis-methionylation in E. coli.…”

“…The rate of protein mistranslation is amongst the highest known error rates in cellular biosynthetic processes, ranging from 1 in 10,000 to 1 in 100 mis-incorporated amino acids in E.coli [1,2]. As a result, 10 to 15% of all proteins in an actively growing E.coli cell are likely to carry at least one mis-incorporated amino acid [3,4], implying a high tolerance for mistakes.…”

Ribosome stoichiometry: from form to function.

“…A higher throughput means of detecting error rates is therefore highly desirable. A recent preprint has suggested a possible mass spectrometry-based approach [74], by exploiting the Maxquant software's [75] ability to perform a blind peptide modification search. While the sensitivity of the approach may limit it to study of the more abundant mis-translation error products, the authors data included altered error rates following perturbations such as amino acid starvation and the addition of an antibiotic known to effect ribosomal proofreading function, suggesting the method holds promise as a high-throughput means of investigating ribosomal error rates.…”