Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

AUTOR(ES)

Fu, D J

RESUMO

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A11, G13, or A14. Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in Km and a decrease in kcat. These results suggest that the exocyclic amino group at-G10 and the hydroxyls at G10 and G13 are important both for ribozyme-substrate binding and for the Mg(2+)-catalyzed cleavage reaction.

Documentos Relacionados

• Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.
• Intracellular RNA cleavage by the hairpin ribozyme.
• Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.
• Specificity of hammerhead ribozyme cleavage.
• Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme.