title: The catalytic efficiency of yeast ribonuclease III depends on substrate specific product release rate.
abstract: Members of the ribonuclease III (RNase III) family regulate gene expression by triggering the degradation of double stranded RNA (dsRNA). Hundreds of RNase III cleavage targets have been identified and their impact on RNA maturation and stability is now established. However, the mechanism defining substrates' reactivity remains unclear. In this study, we developed a real-time FRET assay for the detection of dsRNA degradation by yeast RNase III (Rnt1p) and characterized the kinetic bottlenecks controlling the reactivity of different substrates. Surprisingly, the results indicate that Rnt1p cleavage reaction is not only limited by the rate of catalysis but can also depend on base-pairing of product termini. Cleavage products terminating with paired nucleotides, like the degradation signals found in coding mRNA sequence, were less reactive and more prone to inhibition than products having unpaired nucleotides found in non-coding RNA substrates. Mutational analysis of U5 snRNA and Mig2 mRNA confirms the pairing of the cleavage site as a major determinant for the difference between cleavage rates of coding and non-coding RNA. Together the data indicate that the base-pairing of Rnt1p substrates encodes reactivity determinants that permit both constitutive processing of non-coding RNA while limiting the rate of mRNA degradation.
authors.affiliation: Département de biologie, Faculté de science, Université de Sherbrooke, Sherbrooke, Québec J1E 2R1, Canada.
authors.forename: Marc-Andre
authors.initials: MA
authors.lastname: Comeau
authors.affiliation: Département de biologie, Faculté de science, Université de Sherbrooke, Sherbrooke, Québec J1E 2R1, Canada Daniel.Lafontaine@USherbrooke.ca.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Lafontaine
authors.affiliation: Département de microbiologie et d'infectiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Pavillon de recherche appliquée sur le cancer, Sherbrooke, Québec J1E 4K8, Canada Sherif.Abou.Elela@USherbrooke.ca.
authors.forename: Sherif
authors.initials: S
authors.lastname: Abou Elela
mesh_terms: D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D055162:Biocatalysis; D005453:Fluorescence; D017930:Genes, Reporter; D007700:Kinetics; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III