Effects of varying lengths of double-stranded RNA on binding and activation of 2'-5'-oligoadenylate synthetase.
We investigated the effects of varying the length of the double-stranded (ds) RNA cofactor on activation of 2'-5'-oligoadenylate [2-5 (A)] synthetase. dsRNA of 40, 55, 67, 85, and 110 bp lengths were generated by in vitro transcription of complementary strands and annealing and trimming of the single-stranded overhangs. The dsRNA molecules were radiolabeled by polynucleotide kinase and purified by gel electrophoresis. Their abilities to bind to recombinant mouse 9-2 2-5 (A) synthetase, as monitored by electrophoretic mobility shift assays, were comparable. When used at a saturating concentration of 25 microg/ml, all dsRNA molecules were equally effective in activating the enzyme. At a subsaturating concentration, however, longer RNAs were better activators. At 100 nM concentration, there was a linear relationship between the length of the dsRNA and its ability to activate 2-5 (A) synthetase.
Desai
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SY
Department of Molecular Biology, The Cleveland Clinic Foundation, Research Institute, OH 44195, USA.
Sen
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GC
D015088:2',5'-Oligoadenylate Synthetase / Q000187:drug effects*; D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D016014:Linear Models; D051379:Mice; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000187:drug effects
D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D015088:2',5'-Oligoadenylate Synthetase