Site-specific cleavage of natural mRNA sequences by newly designed hairpin catalytic RNAs.
The negative strand of tobacco ringspot virus satellite RNA is a self-cleaving RNA. Its catalytic domain and substrate domain have been identified, and the catalytic domain has been named hairpin catalytic RNA. Here we report the construction of a plasmid containing a modified hairpin catalytic RNA sequence that can be transcribed in vitro. Because this plasmid has two specific restriction enzyme recognition sites at both ends of the substrate binding site in the catalytic RNA sequence, it is possible to construct new plasmids by substituting different sequences in the substrate binding site. Using this plasmid, synthetic DNA, and in vitro transcription, we obtained three ribozymes designed to cleave Escherichia coli prolipoprotein signal peptidase (lsp) mRNA at specific sites. All three ribozymes cleaved the lsp mRNA sequence in vitro at the specific sites, and two of them cleaved it efficiently. Kinetic analyses showed that one had a higher kcat/Km value than that of the well-known hammerhead ribozyme. Problems associated with attaining the goal of expressing these ribozymes in vivo also are discussed.
Kikuchi
Y
Y
Department of Molecular Biology, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Sasaki
N
N
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008074:Lipoproteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010942:Plant Viruses / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012697:Serine Endopeptidases
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D008565:Membrane Proteins; D011498:Protein Precursors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase