The yeast Saccharomyces cerevisiae decodes CAG codons using tRNAGlnCUG, encoded by the single-copy gene SUP70. On rich medium, the sup70-65 and sup70-33, alleles induce pseudohyphal growth, ordinarily a response to nitrogen limitation. sup70-65 tRNA is additionally a UAG stop codon suppressor. Characterisation of sup70 diploids revealed that they form pseudohyphal-like structures on both rich and N-deprived liquid, but not solid medium. Unlike canonical pseudohyphae, which bud in a unipolar fashion and are RAS2Val19 stimulated, sup70 pseudohyphal cells budded in a bipolar fashion that was RAS2Val19–resistant. Site-directed mutants of sup70-65 and sup70-33 that restored base complementarity in the mutant tRNA stems partially complimented the pseudohyphal phenotype and revealed that structural rigidity, rather than sequence identity, was the key phenotypic driver. A library of sup70 mutants, screened for novel nonsense UAG suppressor alleles, indentified several novel sites in the anticodon stem that induce tRNA distortion and UAG recognition. None of the new alleles induced formation of pseudohyphae. The sup70-65 pseudohyphal phenotype was weakly complemented through overexpression of the isoacceptor tRNAGlnUUG, which can inefficiently decode CAG via 3rd base wobble pairing, inferring pseudohyphal growth can be triggered by inefficient translation of CAG codons. Supporting this observation, tRNA Northern blotting revealed that all pseudohyphae-inducing sup70 mutants have reduced overall levels of tRNAGlnCUG, as well as reduced levels of tRNAGlnCUG charging. A systemic comparison of wild-type and sup70-65 proteomes using stable isotope labelling with amino acids revealed that 28 proteins showed significant changes in expression in the mutant, including 4 with known roles in control of bud size and/or budding pattern. This study revealed that pseudohyphae-inducing sup70 mutations compromise tRNAGlnCUG structure and amino acid charging and thus slow translation of cognate CAG codons, probably down-regulating the expression of a yet-to-be identified gene affecting the control of pseudohyphal differentiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:540316 |
| Date | January 2011 |
| Creators | Kemp, Alain |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=166224 |
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