title: The ribotoxin restrictocin recognizes its RNA substrate by selective engagement of active site residues.
abstract: Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein-RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Plantinga
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Korennykh
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Piccirilli
authors.forename: Carl C
authors.initials: CC
authors.lastname: Correll
mesh_terms: D000465:Algorithms; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; C036196:MITF protein, Aspergillus restrictus; D012313:RNA; D012260:Ribonucleases