Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates.
Ribonuclease P (RNaseP) generates the mature 5' end of tRNAs by removing 5'leader sequences from pre-tRNAs. In vitro, the RNA subunit is sufficient to catalyze this reaction and is therefore a ribozyme. The kinetic analysis of RNase P-mediated catalysis is complicated because product release is normally rate-limiting. Furthermore, the intermolecular nature of the cleavage reaction precludes many applications of in vitro selection schemes to the analysis of RNaseP. To examine and manipulate the RNase P function more effectively, we designed a pair of ribozymes in which the RNase P RNA is covalently linked to a pre-tRNA substrate. To facilitate intramolecular cleavage, pre-tRNA molecules were tethered to circulatory permuted RNaseP RNA molecules at nucleotides implicated in substrate binding. These "active-site-tethered" pre-tRNA-RNaseP RNA conjugates undergo accurate and efficient self-cleavage in vitro, with first-order reaction rates equivalent to the rate of the chemical step of the native RNase P reaction. Unlike most ribozymes, RNase P recognizes its substrate through tertiary RNA-RNA interactions, rather than through extensive Watson-Crick base-pairing. However, the development of the active-site-tethered conjugates has led us to create a sequence-specific endonuclease, termed Endo.P. In the Endo.P configuration, the 3'half of the pre-tRNA acceptor stem binds exogenous RNA substrates via Watson-Crick base-pairing; the bound substrate is subsequently cleaved at the predicted site. The demonstration of sequence-specific cleavage by Endo.P expands the potential of RNase P and its derivatives as reagents in gene therapy.
Frank
D N
DN
Department of Biology, Indiana University, Bloomington 47405.
Harris
M E
ME
Pace
N R
NR
D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D016337:RNA, Catalytic; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P