Identification of a region within M1 RNA of Escherichia coli RNase P important for the location of the cleavage site on a wild-type tRNA precursor.

To investigate the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, we studied cleavage by different M1 RNA mutants of wild-type precursors to tRNA(TyrSu3), tRNA(His) and tRNA(SerSu1). We showed that deletion or substitution of the conserved nucleotides G291, G292, U294 and A295 in M1 RNA resulted in a shift of the cleavage site for the tRNA(SerSu1) precursor, whereas the other two tRNA precursors were cleaved at the normal position. By using chimeric tRNA precursors in which the acceptor-stem of the tRNA(TyrSu3) precursor was replaced by the acceptor-stem derived from the tRNA(SerSu1) precursor, we showed that the aberrant cleavage by M1 RNA mutants could be reversed by substituting the nucleotide at position -2 in one of the chimeric precursors. These results suggest, in support of our previous findings, that different tRNA precursors are processed differently and that the primary structure of the amino acid acceptor-stem of a tRNA precursor plays a significant role in the RNase P cleavage reaction. Furthermore, in agreement with a previous report, a truncated tRNA(TyrSu3) precursor was cleaved aberrantly by a mutant M1 RNA in which the nucleotide at position 92 had been deleted. In contrast, a corresponding truncated tRNA(SerSu1) precursor was cleaved at the same position both by the wild-type and by this mutant M1 RNA. We conclude that not only the primary structures of the acceptor-stems of tRNA precursors, but also the primary structures in different regions of M1 RNA determine the location of the cleavage site on various tRNA precursors. Here we have identified the region G291 to A295 to be important for the selection of the cleavage site on the tRNA(SerSu1) precursor. We discuss the possibility that the conformation of M1 RNA in the enzyme-substrate complex is dependent on the identity of the substrate.