The Biology of Anticodon Stem-Loop Modifications of Yeast tRNAs

摘要

tRNAs play an essential role in translation by decoding mRNA codons to add the correct amino acids to the growing polypeptide chain. During biogenesis, tRNAs undergo extensive post-transcriptional modifications to ensure correct folding and high stability, and to ensure the accuracy and efficiency of translation. Modifications in the anticodon stem-loop (ASL) are highly conserved, and their lack is often associated with growth defects in the yeast Saccharomyces cerevisiae, and with human neurological or mitochondrial disorders. However, the precise function of many ASL modifications and the specificity of the corresponding modifying enzymes remain to be determined.;This thesis focused on the biology and specificity of three sets of ASL modifications in yeast tRNAs. One project focused on the importance of pseudouridine (Psi) at N38 and N39, introduced by Pus3, a modification that is found in all kingdoms of life. This work showed that the temperature sensitivity of pus3Delta mutants was primarily due to reduced function of tRNAGln(UUG), caused by the absence of Psi 38, coupled with the temperature-dependent loss of 2-thiolation at U34 that was found to occur in commonly used yeast strains. Additional experiments showed that tRNAGln(UUG) function was compromised in pus3Delta mutants at temperatures where U34 modification was intact, and showed that Pus3 also had a role in other tRNAs, including tRNATrp(CCA) and tRNALeu(CAA).;A second project focused on the substrate specificity of Trm140 for 3-methylcytidine modification. Biochemical and in vivo analysis demonstrated that Trm140 recognized its substrates by two distinct modes: tRNAThr substrates were recognized based on recognition of specific residues N35-N 36-N37 in the anticodon loop; whereas tRNASer substrates were recognized by interaction with seryl-tRNA synthetase, and through the variable loop of the tRNA.;A third project focused on why lack of 2'-O-methylation at C32 and N34, introduced by Trm7/FTSJ1, causes a severe growth defect in yeast and X-linked intellectual disability in humans. Several lines of genetic evidence were presented indicating that the trm7 Delta growth defect in S. cerevisiae was caused by reduced tRNAPhe charging and the consequent activation of the general amino acid control response, which appeared to be conserved in distantly related organisms, including Schizosaccharomyces pombe. .