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 not known, however, which subunits participate directly
in phosphodiester-bond hydrolysis. The RNA subunit of nuclear
RNase P is evolutionarily related to its catalytically
active bacterial counterpart, prompting speculation that
in eukaryotes the RNA may be the catalytic component. In
the bacterial RNase P reaction, Mg(II) is required to coordinate
the nonbridging phosphodiester oxygen(s) of the scissile
bond. As a consequence, bacterial RNase P cannot cleave
pre-tRNA in which the pro-RP nonbridging
oxygen of the scissile bond is replaced by sulfur. In contrast,
the RNase P reaction in plant chloroplasts is catalyzed
by a protein enzyme whose mechanism does not involve Mg(II)
coordinated by the pro-RP oxygen. To
determine whether the mechanism of nuclear RNase P resembles
more closely an RNA- or a protein-catalyzed reaction, we
analyzed the ability of Saccharomyces cerevisiae
nuclear RNase P to cleave pre-tRNA containing a sulfur
substitution of the pro-RP oxygen at
the cleavage site. Sulfur substitution at this position
prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic
RNase P thus depends on the presence of a thio-sensitive
ligand to the pro-RP oxygen of the
scissile bond, and is consistent with a common, RNA-based
mechanism for the bacterial and eukaryal enzymes.