title: A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome.
abstract: PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: B
authors.initials: B
authors.lastname: Schwer
authors.forename: C
authors.initials: C
authors.lastname: Guthrie
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007700:Kinetics; D009154:Mutation; D010641:Phenotype; D020365:RNA Helicases; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D005656:Fungal Proteins; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases