Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P

Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is n ot known, however, which subunits participate directly in phosphodiester-bo nd hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation t hat in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphod iester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-R-P nonbridging oxygen of the sciss ile bond is replaced by sulfur, In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not invo lve Mg(II) coordinated by the pro-R-P oxygen. To determine whether the mech anism of nuclear RNase P resembles more closely an RNA- or a protein-cataly zed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear R Nase P to cleave pre-tRNA containing a sulfur substitution of the pro-R-P o xygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends o n the presence of a thio-sensitive ligand to the pro-R-P oxygen of the scis sile bond, and is consistent with a common, RNA-based mechanism for the bac terial and eukaryal enzymes.