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Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism.
Thomas, Brian C. Li, Xinqiang Gegenheimer, Peter Albert
Thomas, Brian C.
Li, Xinqiang
Gegenheimer, Peter Albert
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Abstract
The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P.
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This is the published version. Copyright 2000 by the RNA Society.
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2000
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Thomas, Brian C., Xinqiang Li, and Peter Gegenheimer. "Chloroplast Ribonuclease P Does Not Utilize the Ribozyme-type Pre-tRNA Cleavage Mechanism." Rna 6.4 (2000): 545-53. http://rnajournal.cshlp.org/content/6/4/545.