Importance of exocyclic base functional groups of central core guanosines for hammerhead ribozyme activity

• Published in: Biochemistry (Easton) 1993-11, Vol.32 (43), p.11658-11668
• Main Authors: Tuschl, Thomas, Ng, Mabel M. P., Pieken, Wolfgang, Benseler, Fritz, Eckstein, Fritz
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Bacteriophage T4 - enzymology; Base Sequence; Biological and medical sciences; DNA Ligases - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Guanosine - metabolism; Hydrogen Bonding; Miscellaneous; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides - chemical synthesis; Oligoribonucleotides - metabolism; RNA, Catalytic - chemistry; RNA, Catalytic - metabolism; Structure-Activity Relationship; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00094a023

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.