A calcium-metalloribozyme with autodecapping and pyrophosphatase activities.
A previously-isolated ribozyme with capping activity has self-decapping activity, here characterized alongside its additional, somewhat parallel, pyrophosphatase reaction. Decapping is 10-50 times slower than the pyrophosphatase activity, depending on pH. The RNA accelerates pyrophosphate 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(5')pppRNA also supports an unusual cap-exchange reaction, exchanging its cap with guanosine 5'-tetraphosphate to form pentaphosphate-linked G(5')pppppRNA. GDP, a capping reactant for the RNA, appears to suppress both decapping and pyrophosphatase activities. Autodecapping and pyrophosphatase activities have in common an unusual divalent metal ion requirement for Ca2+ or less effectively 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+ act as competitive inhibitors of Ca2+. A strong Ca2+-binding site is suggested by a low KM of 40-60 microM at pH >/= 7.0. The role of Ca2+ in these reactions can be surmized from literature data on reactivity of nucleotide phosphates. Pyrophosphatase, capping, and decapping activities of isolate 6 RNA are apparently carried out by a single reaction center, whose rate of reaction with all nucleophiles sums to a constant total rate. This suggests a universal rate-limiting step. Versatile activation of alpha-phosphate by this reaction center raises the possibility of combinatorial ribozymes.
Huang
F
F
Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309-0347, USA.
Yarus
M
M
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011755:Pyrophosphatases / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*
D012315:RNA Caps; D016337:RNA, Catalytic; D011755:Pyrophosphatases; D002118:Calcium