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Title: A mutational analysis of the yeast protein splicing factor, Prp2
Author: Anderson, Susan Isobel
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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The Prp2 protein of Saccharomyces cerevisiae is a 100kDa non-snRNP splicing factor with amino acids common to the DEAD/H-box family of ATP-dependant RNA helicases. Prp2, Prp16 and Prp22 proteins have extensive sequence similarity, and function at sequential steps in the splicing pathway. Prp2 and Prp16 form transient reactions with the spliceosome, Prp2 at step 1 and Prp16 at step 2. In addition to the conserved motifs of the DEAD/H-box family, Prp2 also contains a sequence resembling a zinc finger motif. This sequence has been extensively mutagenised, and two mutations were found by in vivo complementation assays to affect the function of Prp2. In order to study the molecular interactions of Prp2 at step 1, several dominant negative PRP2 alleles had been generated previously in this laboratory by site-directed mutagenesis. When overexpressed from an inducible promoter, these cause accumulation of pre-mRNA in the presence of wild-type PRP2. These dominant negative alleles and wild-type PRP2 were subjected to deletion analyses. In the case of one dominant negative mutant which has a GKT-AKT change in the ATP-binding motif, some dominant activity was retained despite the removal of 19kDa from the protein. It has been suggested that the Prp16 protein plays a proof-reading role in splicing, as a mutant of PRP16, prp16-1, suppresses an A-C change of the branch nucleotide. A mutation analogous to the prp16-1 allele was created in PRP2 to determine if Prp2 plays a role in influencing the fidelity of splicing. Although no effect was found for this and other mutants of PRP2, it was discovered that overexpression of wild-type PRP16 had an effect similar to that of the prp16-1 mutant on the fidelity of splicing. A new hypothesis is proposed for the mechanism of suppression of branchpoint mutants by Prp16 whereby the Prp16 protein might mediate the binding of an unknown proof-reading factor to the branchpoint.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available