Functional importance of Ψ38 and Ψ39 in distinct tRNAs, amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

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 (Psi) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3 Delta mutants of the yeast Saccharomyces cerevisiae and find that, although Psi(38) or Psi(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 Psi(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 degrees C. Additional examination of the role of Psi(38,39) provides evidence that Psi(38) is important for function of tRNA(Gln(UUG)) at permissive temperature, and indicates that Psi(39) is important for the function of tRNA(Trp(CCA)) in trm10 Delta pus3 Delta mutants and of tRNA(Leu(CAA)) as a UAG nonsense suppressor. These results provide evidence for important roles of both Psi(38) and Psi(39) in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.