8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase.
The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.
Li
S W
SW
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Moskow
J J
JJ
Suhadolnik
R J
RJ
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism*; D000345:Affinity Labels; D000495:Allosteric Site; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D010777:Photochemistry; D011070:Poly I-C / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D011072:Poly U / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D012330:RNA, Double-Stranded; C063517:poly(I,8-azidoI).poly(C12,U); D011072:Poly U; C063516:poly(I,8-azidoI).poly(C); D011494:Protein Kinases; D015088:2',5'-Oligoadenylate Synthetase; D008836:Micrococcal Nuclease; D011070:Poly I-C