title: Nonspecific deadenylation on sarcin/ricin domain RNA catalyzed by gelonin under acidic conditions.
abstract: Gelonin is a single-chain ribosome-inactivating protein that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the ribosomal SRD. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic conditions. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage style to wild-type SRD RNA, underscoring the important role of pH value in site specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the nonspecific deadenlyation by gelonin.
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, 320 Yue-yang Road, Shanghai 200031, China.
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: He
authors.forename: Shuang
authors.initials: S
authors.lastname: Tang
authors.forename: Wang-Yi
authors.initials: WY
authors.lastname: Liu
authors.forename: Fiorenzo
authors.initials: F
authors.lastname: Stirpe
mesh_terms: D000225:Adenine / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D005656:Fungal Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D051381:Rats; D012260:Ribonucleases / Q000235:genetics; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin / Q000235:genetics; D013379:Substrate Specificity
chemical_list: C105968:C-SAR protein, Aspergillus clavatus; D005656:Fungal Proteins; D009841:Oligonucleotides; D010940:Plant Proteins; D012339:RNA, Ribosomal, 28S; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum; D012276:Ricin; D012260:Ribonucleases; D000225:Adenine