Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654895
Title: Detailed analysis of sequence requirements within 2A translational recoding peptides
Author: Yan, Fu
ISNI:       0000 0004 5361 0056
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2014
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Abstract:
2A sequences are short 20 ~30 amino acid peptides initially characterised from foot and mouth disease virus (FMDV), but also encoded in a range of Picornaviruses and other viruses as well as non-LTR retrotransposons in a broad range of organisms. They direct a translational recoding event in which ribosomes that have reached the final codon of the 2A sequence pause and terminate translation in the absence of a stop codon and then restart translation. The effect is to separate a single ORF into 2 polypeptides, between the final 2 amino acids (glycine and proline) of 2A, ‘skipping’ a peptide bond. 2A sequences comprise 2 parts, a conserved GDV/IEXNPGP C-terminal motif, and a highly variable, but necessary, N-terminal portion. In the work described in this thesis, extensive mutagenesis studies were carried out across both the conserved and non-conserved portions of the FMDV 2A peptide, including alanine, glycine and proline scanning mutagenesis, site-directed mutagenesis screens. These data indicate that while the amino acid present at the first 2-3 positions of the peptide is not critical for activity, the residue at most other positions is important, including several positions that vary considerably between different 2A peptides. Intriguingly, results of alanine, glycine and proline scanning mutagenesis were very similar, suggesting that features of side chains, and not just the secondary structure propensity of the peptide, plays a critical role. Despite their great variability, each 2A peptide must provide a set of interactions between side chains within the peptide and with the ribosomal exit tunnel to allow the 2A reaction to proceed. Finally, the work also verified a previous finding that insertion of a stop codon in place of the final proline codon of 2A leads ribosomes to stall, unable to terminate translation. Efforts were made to establish a system for purification of such stalled ribosome-nascent chain complexes towards the eventual aim of structural characterisation. The data strongly support a model in which the interaction of the 2A peptide with the ribosome distorts ribosomal conformation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.654895  DOI: Not available
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