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J. Biol. Chem., Vol. 278, Issue 28, 25832-25838, July 11, 2003

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Secondary Structures Common to Chloroplast mRNA 3'-Untranslated Regions Direct Cleavage by CSP41, an Endoribonuclease Belonging to the Short Chain Dehydrogenase/Reductase Superfamily^*

Thomas J. Bollenbach and David B. Stern 

From the Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853

CSP41 (chloroplast stem-loop-binding protein of 41 kDa), a chloroplast endonuclease belonging to the SDR superfamily, preferentially cleaves stem-loop-containing RNAs in vitro. This potentially directs it to the 3'-ends of mature chloroplast mRNAs, which generally possess such structures. To understand the basis for this discrimination, the RNA elements directing CSP41 cleavage of petD RNA in vitro were dissected. Substrates containing fully base-paired stem-loops were optimal substrates, whereas deletion of part of the stem-loop decreased activity by 100-fold, and deletion of the distal arm of the stem-loop abolished cleavage, even in substrates containing the primary CSP41 cleavage site. Competition assays showed that the decrease in activity resulted from decreased affinity for the RNA by CSP41. Mutations of the residues at the scissile bond and mutations and deletions at the terminal loop of the stem had a moderate effect on activity but no effect on cleavage site specificity, suggesting that CSP41 has no sequence specificity. Titration of ethidium bromide into the assay decreased activity to a basal level of 18%, and introduction of a single base bulge into either arm of the stem-loop decreased cleavage at the primary cleavage site by up to 70%. This suggests that changing the structure of the helical stem has a mild effect on activity. Deletion analysis of CSP41 suggests that the specificity domain lies in the first 73 amino acids of the protein, a domain that also contains a putative dehydrogenaselike mononucleotide binding motif. These results are consistent with a broad role for CSP41 in the degradation of stem-loop-containing mRNAs.

Received for publication, April 7, 2003 , and in revised form, May 5, 2003.

^* This work was supported by Department of Energy Biosciences Program Award DE-FG02-90ER20015. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Boyce Thompson Institute for Plant Research, Cornell University, Tower Rd., Ithaca, NY 14853. Tel.: 607-254-1306; Fax: 607-255-6695; E-mail: ds28{at}cornell.edu.

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