Protection against 3'-to-5' RNA decay in Bacillus subtilis.
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.
Farr
G A
GA
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA.
Oussenko
I A
IA
Bechhofer
D H
DH
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D017353:Gene Deletion; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics*; D012329:RNA, Bacterial / Q000378:metabolism*; D013997:Time Factors
D012329:RNA, Bacterial; D011117:Polyribonucleotide Nucleotidyltransferase