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Structural and functional studies of the core splicing factor Prp8 Open Access
- Other title
- Type of item
- Degree grantor
University of Alberta
- Author or creator
- Supervisor and department
MacMillan, Andrew (Biochemistry)
- Examining committee member and department
Owttrim, George (Biological Sciences)
MacMillan, Andrew (Biochemistry)
Glover, Mark J.N. (Biochemistry)
Kothe, Ute (Chemistry and Biochemistry, University of Lethbridge)
Schultz, Michael C. (Biochemistry)
Department of Biochemistry
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
More than 90% of human genes undergo a processing step called splicing, whereby noncoding introns are removed from initial transcripts and coding exons are ligated together to yield mature messenger RNA. Splicing involves two sequential transesterification reactions catalyzed by a large RNA/protein complex called the spliceosome. RNA components of the spliceosome are at least partly responsible for splicing catalysis. However, the precise architecture of the spliceosome active site and whether it includes protein components remain unknown. Numerous genetic and biochemical analyses have placed one of the largest and most highly conserved of nuclear proteins, Prp8, at the heart of the catalytic core of the splicing machinery during spliceosome assembly and through catalysis. Here we provide structural and functional evidence that the RNase H domain of Prp8 undergoes a conformational change during splicing which unmasks a metal-binding site required for the second step of splicing. We are able to demonstrate that a magnesium ion essential for the catalysis binds to the RNase H domain of Prp8 and a metal specificity switch which abrogates metal binding severely inhibits the second step of splicing. We also show that yeast prp8 first- and second-step alleles correspond to Prp8 mutants that favour one of the two distinct Prp8 conformations observed in the crystal structure of Prp8 RNase H domain. Together these data support the model of rearrangements within the spliceosome at the time of transition between the first and second step of splicing. Our findings also establish that Prp8 is a metalloprotein which promotes exon ligation and are consistent with the designation of the spliceosome as a ribonucleoprotein enzyme.
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- Citation for previous publication
Chilibeck et al., (2006) J. Biol. Chem.
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