Fig 1.
The nuclear genome of S. cerevisiae encodes four different tRNA Arg genes.
In yeast, nineteen genes encode four different tRNA Arg: tR1, 2, 3 and 4. The figure indicates for each isoacceptor gene, the primary sequence, the number of copies and the arginine codon(s) decoded by the corresponding transcript. Two areas of the tRNA Arg are particularly relevant for this study and are highlighted on the cartoon: nucleotide 20 in the D loop and nucleotides 35 and 36 in the anticodon loop. Both color codes and highlighted positions are consistent throughout the different figures. mcm5U34 in tR3 stands for 5-methoxycarbonylmethyluridine.
Fig 2.
Systematic assessment of nucleotide 20: description of the in vivo approach and the phenotypes of the mutants.
The left panel summarizes the principle of the genetic tools developed previously and the two observable phenotypes on solid media. The right panel displays, for chimeric tR1, 2 and 3, all the combinations of nucleotide at position 20 that were tested in YAL5. White and red nucleotides indicate functional and inactive mutants respectively. The background color highlights the wild type nucleotide in each tRNA construct.
Fig 3.
Impact of the UC20 substitution on the binding and the aminoacylation of tR2 by yArgRS.
(A) Yeast wild-type tR2 and UC20 mutant were individually transcribed in vitro, radiolabeled, incubated with 0 to 11 μM of yArgRS and separated on a 6% non-denaturing polyacrylamide gel. The signals corresponding to free and complexed tRNAs were quantified by phosphorimaging and processed to estimate the protein-tRNA dissociation constant. (B) The same protein-tRNA partners were tested through in vitro arginylation assays in order to establish both their Michaelis-Menten and rate equilibrium constants.
Fig 4.
Systematic assessment of nucleotide 35 and 36: quantification of in vivo aminoacylation levels by Northern blot.
The right panel displays the twelve tR4 anticodon mutants that were prepared, expressed in YAL5 and analyzed by Northern blot. Total RNAs were extracted and separated under acidic conditions before being transferred onto a nylon membrane and probed with a mixture of 32P-labeled oligonucleotides complementary to control (5S rRNA and tRNA Leu) and target (tR4 mutants) RNAs. Acylated (+) and deacylated (-) samples were loaded on separate lanes. Radioactive signals were quantified by phosphorimaging in order to estimate the cellular aminoacylation level of tR4 mutants relative to wild-type tR4. Relative in vivo arginylation levels are reported as shades of grey on the heat map. Arginine and leucine transferred on their cognate tRNAs, affect the migration to different extents. At pH 4.5, arginine has a net positive charge that significantly slows down the migration of tRNA Arg, yielding a wider shift. The left panel shows a modified YAL5 obtained after substituting pAL5 by a pRS314 plasmid expressing the chimeric tR3 competent for the translation of CGG codons but unable to hybridize a tR4-specific probe. This strain, free of endogenous tR4, enables the specific detection of tR4 mutants by Northern blot. Note: the actual substitution of pAL5 performed after the transformation with the mutants and before the analysis by Northern blot, is not depicted on this figure.
Fig 5.
Potential impact of the cellular expression of mistranslator tRNAs.
Three parameters influence arginine misincorporation in the proteome in response to the expression of mistranslating chimeras: codon representation in the genome, nature of the substitution, and the relative amount of mutant versus endogenous tRNA competing for the same codon. Gene copy number and BLOSUM 62 scores were used as a proxy for tRNA expression and substitution assessment respectively. Shades of red indicate potential toxicity on the heat map. A chimera competing with a low abundant endogenous tRNA for the mistranslation of a highly represented codon into an unfavorable amino acid, represents one of the most toxic scenarios.