Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.
Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.
Bevers
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Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA.
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McLaughlin
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D001483:Base Sequence; D003853:Deoxyribonucleosides / Q000138:chemical synthesis / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011728:Pyridones / Q000737:chemistry*; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000138:chemical synthesis / Q000378:metabolism; D014529:Uridine / Q000737:chemistry* / Q000378:metabolism
D003853:Deoxyribonucleosides; D009841:Oligonucleotides; D011728:Pyridones; D016337:RNA, Catalytic; D013873:Thionucleotides; C017679:2-hydroxypyridine; D014529:Uridine