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

Published online by Cambridge University Press:  01 April 2000

BRIAN C. THOMAS
Affiliation:
Department of Molecular Biosciences, The University of Kansas, 2045 Haworth Hall, Lawrence, Kansas 66045-2106, USA Molecular Genetics Program, The University of Kansas, 2045 Haworth Hall, Lawrence, Kansas 66045-2106, USA Present address: College of Natural Resources, University of California at Berkeley, Berkeley, California 94720, USA
JOEL CHAMBERLAIN
Affiliation:
Program in Cellular and Molecular Biology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA Present address: Department of Internal Medicine, Division of Rheumatology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0680, USA
DAVID R. ENGELKE
Affiliation:
Program in Cellular and Molecular Biology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA
PETER GEGENHEIMER
Affiliation:
Department of Molecular Biosciences, The University of Kansas, 2045 Haworth Hall, Lawrence, Kansas 66045-2106, USA Molecular Genetics Program, The University of Kansas, 2045 Haworth Hall, Lawrence, Kansas 66045-2106, USA
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Abstract

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.

Type
Research Article
Copyright
© 2000 RNA Society

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