Transient ADP-ribosylation of a 2′-Phosphate Implicated in Its Removal from Ligated tRNA during Splicing in Yeast*
- From the ‡Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, the §Department of Chemistry, University of Rochester, Rochester, New York 14627, and the ¶Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Abstract
The last step of tRNA splicing in yeast is catalyzed by Tpt1 protein, which transfers the 2′-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2"-cyclic phosphate (Appr>p). Structural and functional TPT1 homologs are found widely in eukaryotes and, surprisingly, also in Escherichia coli, which does not have this class of tRNA splicing. To understand the possible roles of the Tpt1 enzymes as well as the unusual use of NAD, the reaction mechanism of the E. colihomolog KptA was investigated. We show here that KptA protein removes the 2′-phosphate from RNA via an intermediate in which the phosphate is ADP-ribosylated followed by a presumed transesterification to release the RNA and generate Appr>p. The intermediate was characterized by analysis of its components and their linkages, using various labeled substrates and cofactors. Because the yeast and mouse Tpt1 proteins, like KptA protein, can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate, these enzymes likely use the same reaction mechanism. Step 1 of this reaction is strikingly similar to the ADP-ribosylation of proteins catalyzed by a number of bacterial toxins.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM52347 (to E. M. P.) and GM22939 (to D. H. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel: 716-275-7268; Fax: 716-271-2683; E-mail: eric_phizicky{at}urmc.rochester.edu.
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↵2 M. A. Steiger, R. Kierzek, D. H. Turner, and E. M. Phizicky, unpublished observations.
- Abbreviations:
- Appr>p
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1"-2"-cyclic phosphate
- AppN
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NAD
- PEI
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polyethyleneimine
- p*ApApA
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p*ApAPpA, pApAPpA, p*ApApAP POCH3, superscript P is a 2-phosphate.
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- Received September 15, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
