Cleavage of oligoribonucleotides by the 2',5'-oligoadenylate- dependent ribonuclease L.
RNase L, the 2',5' oligoadenylate-dependent ribonuclease, is one of the enzyme systems important in the cellular response to interferon. When activated in the presence of 2',5'-linked oligoadenylates, RNase L can catalyze the cleavage of synthetic oligoribonucleotides that contain dyad sequences of the forms UU, UA, AU, AA, and UG, but it cannot catalyze the cleavage of an oligoribonucleotide containing only cytosines. The primary site of the cleavage reaction with the substrate C11UUC7 has been defined to be 3' of the UU dyad by labeling either the 5' or the 3' end of the oligoribonucleotide and by examining the reaction products on polyacrylamide sequencing gels. Reaction time courses have been used to determine the kinetic parameters of the cleavage reactions. The effect of the overall length of the oligomeric substrate as well as the sequence of the bases around the position of the cleavage site on the kinetics of the cleavage reaction has been examined. The efficiency with which activated RNase L catalyzes the cleavage of the substrate C11UUC7 is 1.9 x 10(7) m-1 s-1. Because the cleavage of the synthetic oligoribonucleotide can be used to monitor the steady-state kinetics of catalysis by activated RNase L, this method offers an advantage over previous methods of assay for RNase L activity.
Carroll
S S
SS
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Chen
E
E
Viscount
T
T
Geib
J
J
Sardana
M K
MK
Gehman
J
J
Kuo
L C
LC
D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007313:Insecta; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D000227:Adenine Nucleotides; D017931:DNA Primers; D009843:Oligoribonucleotides; D011994:Recombinant Proteins; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease