ARTIFICIAL SELF-CLEAVING MOLECULES CONSISTING OF A TRANSFER-RNA PRECURSOR AND THE CATALYTIC RNA OF RNASE-P

We synthesized two types of chimeric RNAs between the catalytic RNA subunit of RNase P from Escherichia coli (Ml RNA) and a tRNA precursor (pre-tRNA); one had pre-tRNA at the 3' side to the Ml RNA (M1 RNA-pre-tRNA). The second had pre-tRNA at the 5' side of the Ml RNA (pre-tRNA-M1 RNA). Both molecules were self-cleaving RNAs. The self-cleavage of Ml RNA-pre-tRNA occurred at the normal site (5'-end of mature tRNA sequence) and proceeded under the condition of 10 MM Mg2+ concentration. This reaction at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage), while, at 40 mM and 80 mM Mg2+, trans-cleavage partially occurred. The self-cleavage rate was strictly affected by the distance between the Ml RNA and the pre-tRNA in the molecule. The self-cleavage of pre-tRNA-M1 RNA occurred mainly at three sites within the mature tRNA sequence. This cleavage did not occur at 10 MM Mg2+. Use of Ml RNA-pre-tRNA molecule for the in vitro evolution of Ml RNA is discussed.