Efficient cleavage of pre-tRNAs by E.coli RNAse P RNA requires the 2′-hydroxyl of the ribose at the cleavage site

RNase P cleaves pre-tRNAs to liberate 5′-flanks and 5′-matured, 5′-phosphorylated tRNAs. It is not evident if the 2′-hydroxyls of the ribose moieties in the substrate are involved in the reaction. To study their influence in two different pre-tRNAs, we have modified specifically the 2′-hydroxyl groups at the cleavage site and in neighbouring positions. We have shown that these hydroxyls are important but not essential for the processing of these substrates by E.coli RNase P RNA (M1 RNA). The reduction in the catalytic efficiency was moderate for 2′-deoxy and severe for 2′-methoxy substitutions at the cleavage site. Additional effects of modifications in neighbouring positions were smaller. Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.