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How mutations in tRNA distant from the anticodon affect the fidelity of decoding.

ABSTRACT: The ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNA(Trp) harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNA(Trp), G24A tRNA(Trp) or the miscoding A9C tRNA(Trp)) bound to cognate UGG or near-cognate UGA codons, determined at 3.2-Å resolution. The A9C and G24A mutations lead to miscoding by facilitating the distortion of tRNA required for decoding. A9C accomplishes this by increasing tRNA flexibility, whereas G24A allows the formation of an additional hydrogen bond that stabilizes the distortion. Our results also suggest that each native tRNA will adopt a unique conformation when delivered to the ribosome that allows accurate decoding.

SUBMITTER: Schmeing TM

PROVIDER: S-EPMC3072312 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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How mutations in tRNA distant from the anticodon affect the fidelity of decoding.

Schmeing T Martin TM Voorhees Rebecca M RM Kelley Ann C AC Ramakrishnan V V

Nature structural & molecular biology 20110306 4

The ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNA(Trp) harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNA(Trp), G24A tRNA(Trp) or the miscoding A9C tRNA(Trp)) bound to cognate U ...[more]

PMID: 21378964

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Project description:tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2-encoded tRNAThrCGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five nonsynonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNALysCUU and tRNAeMetCAU, were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data could not detect any significant mistranslation, and reverse transcription-quantitative PCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis.IMPORTANCE In this work, we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e., high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few single nucleotide polymorphisms (SNPs), which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNAThrCGU at high temperature or switching the anticodon of tRNALysCUU or tRNAeMetCAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains.

Dataset Information

How mutations in tRNA distant from the anticodon affect the fidelity of decoding.

Publications

How mutations in tRNA distant from the anticodon affect the fidelity of decoding.

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