[Keywords: Biochemical genomics; protein microarray; proteome; high-throughput expression; galactose lethality; glycosylation] Supplemental material is available at http://www.genesdev.org.

Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4 oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4 oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4 oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 59 ends, mutations that sensitize SUP4 oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U 38 and U 39 in the anticodon stem-loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ 38 or Ψ 39 is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA Gln(UUG) , which normally has Ψ 38 . Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s 2 U moiety of mcm 5 s 2 U 34 of tRNA Gln(UUG) and the other two cytoplasmic species with mcm 5 s 2 U 34 , that the reduced s 2 U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s 2 U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ 38,39 provides evidence that Ψ 38 is important for function of tRNA Gln(UUG) at permissive temperature, and indicates that Ψ 39 is important for the function of tRNA Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ 38 and Ψ 39 in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1 ؊ sam2 ؊ mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 Å by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1 ؊ sam2 ؊ strain grown in selenomethionine. Six of eight selenium residues were located in the structure.x-ray crystallography ͉ methionine ͉ Saccharomyces cerevisiae

Modification defects in the tRNA anticodon loop often impair yeast growth and cause human disease. In the budding yeast Saccharomyces cerevisiae and the phylogenetically distant fission yeast Schizosaccharomyces pombe, trm7Δ mutants grow poorly due to lack of 2'-O-methylation of C32 and G34 in the tRNAPhe anticodon loop, and lesions in the human TRM7 homolog FTSJ1 cause non-syndromic X-linked intellectual disability (NSXLID). However, it is unclear why trm7Δ mutants grow poorly. We show here that despite the fact that S. cerevisiae trm7Δ mutants had no detectable tRNAPhe charging defect in rich media, the cells constitutively activated a robust general amino acid control (GAAC) response, acting through Gcn2, which senses uncharged tRNA. Consistent with reduced available charged tRNAPhe, the trm7Δ growth defect was suppressed by spontaneous mutations in phenylalanyl-tRNA synthetase (PheRS) or in the pol III negative regulator MAF1, and by overexpression of tRNAPhe, PheRS, or EF-1A; all of these also reduced GAAC activation. Genetic analysis also demonstrated that the trm7Δ growth defect was due to the constitutive robust GAAC activation as well as to the reduced available charged tRNAPhe. Robust GAAC activation was not observed with several other anticodon loop modification mutants. Analysis of S. pombe trm7 mutants led to similar observations. S. pombe Trm7 depletion also resulted in no observable tRNAPhe charging defect and a robust GAAC response, and suppressors mapped to PheRS and reduced GAAC activation. We speculate that GAAC activation is widely conserved in trm7 mutants in eukaryotes, including metazoans, and might play a role in FTSJ1-mediated NSXLID.