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 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.(Received July 12 1999)
(Revised September 20 1999)
(Accepted January 27 2000)
Key Words: catalytic mechanism; enzyme; magnesium ion; ribonuclease P; ribozyme; tRNA processing; thiosubstitution; yeast.
c1 Reprint requests to: Peter Gegenheimer, Department of Molecular Biosciences, The University of Kansas, 2045 Haworth Hall, Lawrence, Kansas 66045-2106, USA; e-mail: PGegen@UKans.edu.
p1 Present address: College of Natural Resources, University of California at Berkeley, Berkeley, California 94720, USA
p2 Present address: Department of Internal Medicine, Division of Rheumatology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0680, USA