2-5A-dependent RNase molecules dimerize during activation by 2-5A.
2-5A-dependent RNase is an interferon-inducible enzyme that requires 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) for its endoribonuclease activity against single-stranded RNAs. We demonstrate here that recombinant, human 2-5A-dependent RNase forms stable homodimers during its stimulation by 2-5A. The protein dimers were observed to form only upon binding to 2-5A, as shown using gel filtration chromatography and chemical cross-linking and after centrifugation in glycerol gradients. A monoclonal antibody to 2-5A-dependent RNase was prepared and used to probe the subunit structure of the enzyme in the presence or absence of 2-5A. Using oligoadenylates of different length, structure, and 5'-phosphorylation states we determined that conversion of 2-5A-dependent RNase from its monomeric, inactive form to its homodimeric, active form required the presence of functional 2-5A. These results demonstrate that the catalytically active form of 2-5A-dependent RNase is a homodimer.
Dong
B
B
Department of Cancer Biology, Cleveland Clinic Foundation, Ohio 44195.
Silverman
R H
RH
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001704:Biopolymers; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007313:Insecta; D009843:Oligoribonucleotides / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding
D000227:Adenine Nucleotides; D001704:Biopolymers; D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease