Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.
2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.
Dong
B
B
Department of Cancer Biology, Research Institute, Cleveland Clinic Foundation, Ohio 44195.
Xu
L
L
Zhou
A
A
Hassel
B A
BA
Lee
X
X
Torrence
P F
PF
Silverman
R H
RH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004790:Enzyme Induction; D006801:Humans; D006868:Hydrolysis; D007372:Interferons / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009036:Moths; D011061:Poly A / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D011061:Poly A; D011072:Poly U; D008345:Manganese; D000255:Adenosine Triphosphate; D007372:Interferons; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D008274:Magnesium