Substitution of the 2'-hydroxyl group at position 2.1 by an amino group interferes with Mg(2+) binding and efficient cleavage by hammerhead ribozyme.
Recently we have demonstrated that hammerhead ribozymes can be fully substituted with 2'-amino pyrimidines without detriment to the catalytic activity, provided that positions 2.2 and/or 2.1 are not modified. We now report on the potential molecular mechanisms by which 2'-amino groups at these positions inhibit the ribozyme cleavage activity. In the presence of Mg(2+), the 2'-amino modification at positions 2.2 and/or 2.1 had no significant effect on substrate binding. Detailed analysis of the ribozyme initial cleavage rates in the presence of various Mg(2+) concentrations indicated that Mg(2+) binding is inhibited by the 2'-amino group at position 2.1. Furthermore, preannealed substrate molecules to the modified ribozyme are not effectively cleaved upon Mg(2+) addition, indicating an alteration of the ribozyme cleavage step. Surprisingly, the cleavage activity of the modified ribozymes was substantially increased when Mg(2+) ions were replaced by the thiophilic Mn(2+) ions, whereas only a moderate cleavage enhancement occurred with its unmodified version. Taken together, our findings indicate that changes in the sugar at position 2.1 alter Mg(2+)-promoting ribozyme cleavage.
Sioud
M
M
Department of Immunology, The Norwegian Radium Hospital, Oslo, Montebello, N-0310, Norway. mosioud@embnet.uio.no
Leirdal
M
M
D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011743:Pyrimidines / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D011743:Pyrimidines; D016337:RNA, Catalytic; D008345:Manganese; D008274:Magnesium