Binding and cleavage of nucleic acids by the "hairpin" ribozyme.
The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.
Chowrira
B M
BM
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Burke
J M
JM
D001483:Base Sequence; D001665:Binding Sites; D006868:Hydrolysis; D006878:Hydroxides / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010100:Oxygen / Q000737:chemistry; D010766:Phosphorylation; D010942:Plant Viruses / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*; D012266:Ribose / Q000737:chemistry; D013873:Thionucleotides / Q000737:chemistry
D006878:Hydroxides; D016337:RNA, Catalytic; D012367:RNA, Viral; D013873:Thionucleotides; D012266:Ribose; C031356:hydroxide ion; D010100:Oxygen