Four 3-to-5 exoribonucleases have been identified in Bacillus subtilis: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5 ends were at the transcriptional start site and whose 3 ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of rpsO mRNA decay showed that decay intermediates accumulated as the result of a block to 3-to-5 processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA trans translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.The steady-state amount of a particular mRNA in a cell is a function of its synthesis and degradation. Thus, it is understood that mRNA decay is an important factor in setting levels of gene expression. In a generally accepted model for Escherichia coli (4, 18, 21), mRNA decay initiates with endonuclease cleavage by RNase E, a 5Ј-end-dependent endoribonuclease (23). Such cleavage generates an upstream fragment with an unprotected 3Ј end, which is rapidly degraded by the 3Ј-to-5Ј exonucleases, RNase II or polynucleotide phosphorylase (PNPase), and a downstream fragment that begins with a monophosphate nucleoside, which is a much better substrate for subsequent binding and cleavage by RNase E than the 5Ј-terminal triphosphate nucleoside of the initial transcription product. Additional cycles of rapid endonucleolytic cleavage and 3Ј-to-5Ј degradation result in the observed all-or-nothing pattern of decay, i.e., Northern blot analysis of a specific mRNA generally shows the full-length mRNA but few or no mRNA decay fragments. Rapid decay of mRNA seems to be an essential function, as either RNase II or PNPase is required for viability; inactivation of both RNase II and PNPase is lethal (11). E. coli contains six other 3Ј-to-5Ј exoribonucleases (39). Very recently, one of these, RNase R, was reported by several groups to be involved in mRNA decay (1).We have been studying mRNA decay in Bacillus subtilis. It was shown years ago by Duffy and colleagues that mRNA decay in B. subtilis occurs primarily phosphorolytically, rather than...

SummarySpore formation in Bacillus subtilis is significantly impaired by the deletion of the gene for tmRNA (ssrA), which facilitates the trans-translation reaction that rescues stalled ribosomes and degrades incompletely synthesized peptides. Microscopic analysis revealed that the sporulation of most DssrA cells is blocked after forespore formation. Expression analysis of lacZ-fused genes directed by several RNA polymerase s factors showed that the synthesis of active s K , encoded by the sigK gene, is predominantly inhibited in DssrA cells. The defect in s K synthesis is attributable to a defect in the skin element excision, which generates the sigK gene, caused in turn by reduced expression of SpoIVCA (recombinase) in DssrA cells.