Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins.
The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.
Edwalds-Gilbert
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Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Kim
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DH
Kim
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Tseng
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YH
Yu
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Lin
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RJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D053487:DEAD-box RNA Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D005690:Galactose / Q000378:metabolism; D005799:Genes, Dominant; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010449:Peptide Mapping; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012326:RNA Splicing / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism
D005656:Fungal Proteins; D012331:RNA, Fungal; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D005690:Galactose