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Title: Identification of ribosomal responses to translational pausing in Saccharomyces cerevisiae
Author: Corrigall, Holly
ISNI:       0000 0004 7967 6539
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2019
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Protein synthesis requires the coordinated interaction of translational components to supply charged transfer RNAs to the elongating ribosome. Failure to supply charged tRNAs efficiently can trigger mistranslation, frameshifting, and ribosome drop-off, with associated degradation of the mRNA and nascent peptide. To investigate the cellular response to ribosome pausing, a novel dicistronic reporter was constructed directing the expression of a translationally fused mRuby2-GFP protein. The reporter, with a stop codon inserted at the mRuby2-GFP junction, was successfully able to report stop codon readthrough in response to suppressor tRNAs and translational error-inducing drugs. Ribosomal responses to hybrid-state pausing were investigated using CGA arginine codons at the mRuby2-GFP junction. The GFP: mRuby2 ratio of the CGAcontaining fluorescent reporter was reduced by deletion of the LTN1 and RQC2 genes, indicating turnover of translation abandonment products by the ribosome quality control (RQC) pathway. Using depletion of glutamine tRNA synthetase, the reporter was then used to investigate induced pausing due to slow delivery of charged tRNAs. Ribosome drop-off was triggered at glutamine codons along the length of the reporter, reducing the expression of GFP relative to mRuby2 fluorescence. The GFP: mRuby2 ratio was predominantly resistant to loss of Rqc2 and Ltn1, except when additional copies of rare CAG glutamine codons were introduced, indicating a non-canonical translational abandonment pathway. Proteomic analysis of the fused fluorescent protein expressed under pause-induced conditions revealed evidence for mistranslation, and for the RQC-mediated addition of C-terminal alanine and threonine extensions, a known degradation signal. However only a subset of the RQC pathway proteins, including Slh1, Vms1, Rcq1, Cdc48 and Hbs1, were associated with ribosomes paused at slow codons. Overall the results indicate the possibility that an alternative quality control pathway is triggered by ribosomal stalling due to depletion of available tRNA, with characteristics distinct from responses to pausing in the ribosomal hybrid state.
Supervisor: Stansfield, Ian ; Romano, Mamen Sponsor: East of Scotland Bioscience Doctoral Training Partnership (EASTBIO)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Saccharomyces cerevisiae ; Proteins ; Ribosomes