Control of translation efficiency in yeast by codon-anticodon interactions

Department of Biochemistry and Biophysics, University of Rochester Medical School, Rochester, New York 14642, USA.
RNA (Impact Factor: 4.94). 10/2010; 16(12):2516-28. DOI: 10.1261/rna.2411710
Source: PubMed


The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNA(Arg(UCG)), and is associated with generation of a smaller RNA fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.

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    • "Dual-Luciferase Assay Dual-luciferase reporter constructs were obtained from Elizabeth Grayhack (Letzring et al., 2010). Whole cell extracts from yeast transformants were assayed for firefly and Renilla luciferase activity (details are in the Supplemental Experimental Procedures). "
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    • "Bulk low molecular weight RNA was isolated from 150 to 300 OD of yeast cells that were grown in conditions described above, using a hot phenol extraction method, as described elsewhere (Kotelawala et al. 2008). Total RNA was extracted from stationary phase cells by lysis with glass beads, phenol-chloroform extraction, and ethanol precipitation, as described previously (Letzring et al. 2010). tRNAs were purified using 5 ′ biotinylated DNA oligomers complementary to the following: nt 48–72 for tRNA "
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    • "This is simply because the CAG-decoding tRNA is ninefold less abundant than CAA-decoding tRNA, inducing ribosomal queues at the 5′ end of the mRNA. Other researchers have also demonstrated that the introduction of multiple tandem CAG codons into the 5′ end of the luciferase ORF resulted in reduced translational expression in wild-type S. cerevisiae (Letzring et al., 2010). When the experiment was repeated in a sup70-65 mutant, expression of the CAG-engineered reporter was further reduced by 60% relative to the level achieved in a CAA-containing control construct (Fig. 7). "
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