We developed a guide DNA technique with which the cleavage efficiency of pre-tRNA substrate raised in the RNase P reaction. The 20-mer guide DNAs hybridizing to the upstream region of the cleaving site enhanced the cleavage reactions of RNA substrates by Escherichia coli RNase P. This guide DNA technique was also applicable to cleavage site selection by choosing the DNA-hybridizing site. Results showed that RNase P accepts DNA/RNA double-stranded 5′-leader region with high catalytic efficiency as well as single-stranded RNA region in pre-tRNAs as substrates, which suggests that the protein component of bacterial RNase P prefers bulky nucleotides. The protein component did not affect the normal 5′-processing reaction of pre-tRNAs, but enhanced the mis-cleaving (hyperprocessing) reactions of tRNA in non-cloverleaf folding. Our results suggested that the protein component of RNase P is a modifier for substrate recognition.
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