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

ABSTRACT 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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    ABSTRACT: Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.
    Molecular cell 05/2013; 51(1). DOI:10.1016/j.molcel.2013.04.021 · 14.02 Impact Factor
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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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    ABSTRACT: tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA(His) guanylyltransferase accumulate uncharged tRNA(His) lacking the G(-1) residue and subsequently accumulate additional 5-methylcytidine (m(5)C) at residues C(48) and C(50) of tRNA(His), due to the activity of the m(5)C-methyltransferase Trm4. We show here that the increase in tRNA(His) m(5)C levels does not require loss of Thg1, loss of G(-1) of tRNA(His), or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA(His) m(5)C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m(5)C in tRNA(His) after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m(5)C on tRNA(His) occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m(5)C appears specific to tRNA(His), as tRNA(Val(AAC)) and tRNA(Gly(GCC)) have much reduced additional m(5)C during these growth arrest conditions, although they also have C(48) and C(50) and are capable of having increased m(5)C levels. Thus, tRNA(His) m(5)C levels are unusually responsive to yeast growth conditions, although the significance of this additional m(5)C remains unclear.
    RNA 12/2012; 19(2). DOI:10.1261/rna.035808.112 · 4.94 Impact Factor
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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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    ABSTRACT: In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNA(Gln) (CUG) . A mutant allele, sup70-65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, ten novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA(Gln) (CUG) anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70-65 tRNA(Gln) (CUG) is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG-rich ORFs in the tRNA(Gln) (CUG) -depleted sup70-65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70-65 pseudohyphal phenotype was partly complemented by overexpressing CAA-decoding tRNA(Gln) (UUG) , an inefficient wobble-decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5' end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA(CUG) (Gln) constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.
    Molecular Microbiology 11/2012; 87(2). DOI:10.1111/mmi.12096 · 4.42 Impact Factor
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