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Title: Analysis of propagation-defective mutations of the yeast (PSI+) prion
Author: Marchante, Ricardo Miguel Neto
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2011
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Analysis of how prions are propagated and transmitted in the yeast Saccharomyces cerevisiae has begun to reveal how an amyloid-forming protein can act as an epigenetic determinant of cell phenotype. Through the ability of prions to self propagate, genetic traits encoded by prions are inherently dominant, yet the underlying mechanism is only just beginning to emerge. One approach to elucidating the mechanism of prion propagation is to establish why certain mutations can impact negatively on inheritance of a prion-based trait. This thesis reports on a combined in vivo and structural analysis of one such class of mutant - PNM2-1 - a dominant negative mutation that inhibits propagation of [PS/+], the prion form of the translation termination factor Sup35p. The original PNM2-1 allele, a G58D mutation lies in one of a series of five oligopeptide repeats in the amino-terminal prion domain of the Sup35p (eRF3) and cells expressing this allele cannot efficiently propagate the [PS/+] prion. To establish the mechanism by which the PNM2-1 allele mediates this effect, a series of PNM2-1G58/G59/Y60 mutants of Sup35p was constructed. By combining genetic crossing, phenotypic analysis and solution NMR structural studies, a clear correlation between the conformational changes in the oligopeptide repeat caused by these mutations and the relative impact of these mutations on the in vivo propagation of [PS/+] was demonstrated. The conformational constraints associated with these mutations were also shown to the affect the ability of the protein to form and/or incorporate different of prion variants. These findings provide a molecular explanation of the dominant negative effects of PNM2-1 mutation on the maintenance of the [PS/+] prion and provide important new insights into the importance of conformation in prion propagation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available