A CALCIUM-METALLORIBOZYME WITH AUTODECAPPING AND PYROPHOSPHATASE ACTIVITIES

A previously-isolated ribozyme with capping activity has self-decappin g activity, here characterized alongside its additional, somewhat para llel, pyrophosphatase reaction. Decapping is 10-50 times slower than t he pyrophosphatase activity, depending on pH. The RNA accelerates pyro phosphate release 170 000 times over a control composed of randomized pppRNA, and 5' capped RNA accelerates decapping 1000-fold over random capped RNA. Triphosphate-linked G(S')pppRNA also supports an unusual c ap-exchange reaction, exchanging its cap with guanosine 5'-tetraphosph ate to form pentaphosphate-linked G(S')pppppRNA. GDP, a capping reacta nt for the RNA, appears to suppress both decapping and pyrophosphatase activities. Autodecapping and pyrophosphatase activities have in comm on an unusual divalent metal ion requirement for Ca2+ or less effectiv ely Mn2+, and both are active over a broad pH range of 4.5-9.0. These characteristics resemble the capping activity of the same RNA. Kinetic analysis reveals a well-defined Ca2+-RNA complex, and Mg2+ and Sr2+ a ct as competitive inhibitors of Ca2+. A strong Ca2+-binding site is su ggested by a low K-M Of 40-60 mu M at pH greater than or equal to 7.0. The role of Ca2+ in these reactions can be surmized from literature d ata on reactivity of nucleotide phosphates. Pyrophosphatase, capping, and decapping activities of isolate 6 RNA are apparently carried out b y a single reaction center, whose rate of reaction with all nucleophil es sums to a constant total rate. This suggests a universal rate-limit ing step. Versatile activation of alpha-phosphate by this reaction cen ter raises the possibility of combinatorial ribozymes.