Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.
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.
Fu
D J
DJ
Department of Chemistry, Boston College, Chestnut Hill, MA 02167.
McLaughlin
L W
LW
D001483:Base Sequence; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D016337:RNA, Catalytic