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Journal of Bacteriology, December 1999, p. 7323-7330, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Protection against 3'-to-5' RNA Decay in Bacillus subtilis

Glen A. Farr,dagger Irina A. Oussenko, and David H. Bechhofer*

Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine of New York University, New York, New York 10029

Received 18 August 1999/Accepted 24 September 1999

A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5'-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3'-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3' end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3'-to-5' exonuclease. The finding of impediments to 3'-to-5' degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine of New York University, New York, NY 10029. Phone: (212) 241-5628. Fax: (212) 996-7214. E-mail: dbechho{at}smtplink.mssm.edu.

dagger Present address: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536.

Journal of Bacteriology, December 1999, p. 7323-7330, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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