Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme.
A new approach for modification interference studies is presented. It involves the use of phosphorothioates as a handle to analyze any desired base or sugar modification. This method was applied to identify ribose and phosphate moieties which could be important in the pre-tRNA recognition of E. coli RNase P RNA (M1 RNA). The utility of this technique was confirmed by detecting the inhibitory effect of a deoxyribose in the 5'-flank (position-1). This site was already known to interfere with RNase P cleavage, if modified. We have analyzed pre-tRNA(Tyr) and pre-tRNA(Phe) and found different interference patterns for both tRNAs. Two unpaired regions were involved in both pre-tRNAs. Phosphorothioates interfered at the transition between acceptor- and D-arms. The results with deoxythymidines in the T-loop indicated that deoxyribose moieties or the extra methyl group in thymidine could interfere with RNAse P cleavage. These data suggest that even in complete pre-tRNAs, only a few intact ribonucleotides are important in the substrate recognition by RNase P. We have demonstrated the potential of this new approach which offers many future applications in all fields involving nucleic acids, for example RNA processing, action of ribozymes, tRNA charging and studies related to DNA promoter recognition.
Gaur
R K
RK
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany.
Krupp
G
G
D001483:Base Sequence; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000378:metabolism*; D043262:Ribonuclease P; D012266:Ribose / Q000032:analysis*; D013873:Thionucleotides / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D029968:Escherichia coli Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D012365:RNA, Transfer, Tyr; D013873:Thionucleotides; D013942:Thymine Nucleotides; C041133:thymidine 5'-O-(1-thiotriphosphate); C015109:2'-deoxyadenosine 5'-O-(1-thiotriphosphate); D012266:Ribose; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli