Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3 '-S-phosphorothiolate internucleotide linkage

Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3' -OH and 5'-phosphate termini, and at least two metal ions are thought to be essential for catalysis, To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3'-oxyanion leav ing group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3'-S-phosphorothiolate linkages at the RNase P cleav age site were synthesized. With a 3'-S-phosphorothiolate-modified ptRNA car rying a 7 nt 5'-flank, a complete shift of the cleavage site to the next un modified phosphodiester in the 5'-direction was observed. Cleavage at the m odified linkage was not restored in the presence of thiophilic metal ions, such as Mn2+ or Cd2+. To suppress aberrant cleavage, we also constructed a 3'-S-phosphorothiolate-modified ptRNA with a 1 nt 5'-flank. Mo detectable c leavage of this substrate was seen in reactions catalyzed by RNase P RNAs f rom Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impa ired, suggesting that the 3'-S-phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.