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Molecular and Cellular Biology, August 2001, p. 5408-5416, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5408-5416.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

Shlomit Yehudai-Resheff, Merav Hirsh, and Gadi Schuster*

Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel

Received 22 February 2001/Returned for modification 17 April 2001/Accepted 15 May 2001

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of Pi and ADP. As expected of a phosphorylase, Pi enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

* Corresponding author. Mailing address: Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. Phone: 972-4-8293171. Fax: 972-4-8225153. E-mail: gadis{at}tx.technion.ac.il.

Molecular and Cellular Biology, August 2001, p. 5408-5416, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5408-5416.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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