Importance of exocyclic base functional groups of central core guanosines for hammerhead ribozyme activity.
The three guanosines of the central core of a hammerhead ribozyme were replaced by 2-aminopurine ribonucleoside, xanthosine, isoguanosine, inosine, and deoxyguanosine. These analogues were incorporated by automated solid-phase synthesis, with the exception of isoguanosine. This was introduced by ligating a donor, which carried the isoguanosine at its 5'-end, and an acceptor oligoribonucleotide by a T4 DNA ligase-catalyzed reaction. Most of these modifications lowered the rate constant of cleavage by the hammerhead ribozyme drastically. Inspection of the possible hydrogen-bonding interactions disturbed by these modifications suggests that there is no G12A9 or A13G8 mismatched base pair in the central region. Increasing the Mg2+ concentration from 10 to 50 mM did not enhance these rates appreciably. This makes it improbable that the guanosines, including their 2'-hydroxyl groups, are involved in the binding of the catalytically active Mg2+. Transition-state destabilizing energies of 0.6-4.7 kcal mol-1 suggest that essentially all guanosines are involved in a hydrogen-bonding network.
Tuschl
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Max-Planck-Institut für experimentelle Medizin, Göttingen, Germany.
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Pieken
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Benseler
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Eckstein
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D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D006151:Guanosine / Q000378:metabolism; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D006151:Guanosine; D011088:DNA Ligases