Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3′ ends

Nucleic Acids Research, Apr 2016

Timely turnover of RNA is an important element in the control of bacterial gene expression, but relatively few specific targets of RNA turnover regulation are known. Deletion of the Bacillus subtilis pnpA gene, encoding the major 3′ exonuclease turnover enzyme, polynucleotide phosphorylase (PNPase), was shown previously to cause a motility defect correlated with a reduced level of the 32-gene fla/che flagellar biosynthesis operon transcript. fla/che operon transcript abundance has been shown to be inhibited by an excess of the small regulatory protein, SlrA, and here we find that slrA mRNA accumulated in the pnpA-deletion mutant. Mutation of slrA was epistatic to mutation of pnpA for the motility-related phenotype. Further, Rho-dependent termination was required for PNPase turnover of slrA mRNA. When the slrA gene was provided with a Rho-independent transcription terminator, gene regulation was no longer PNPase-dependent. Thus we show that the slrA transcript is a direct target of PNPase and that regulation of RNA turnover is a major determinant of motility gene expression. The interplay of specific transcription termination and mRNA decay mechanisms suggests selection for fine-tuning of gene expression.

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Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3′ ends

Nucleic Acids Research Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3 ends Bo Liu 1 Daniel B. Kearns 0 David H. Bechhofer 1 0 Department of Biology, Indiana University , Bloomington, IN 47405 , USA 1 Department of Pharmacology and Systems Therapeutics , Box 1603 , Icahn School of Medicine at Mount Sinai , New York, NY 10029 , USA Timely turnover of RNA is an important element in the control of bacterial gene expression, but relatively few specific targets of RNA turnover regulation are known. Deletion of the Bacillus subtilis pnpA gene, encoding the major 3 exonuclease turnover enzyme, polynucleotide phosphorylase (PNPase), was shown previously to cause a motility defect correlated with a reduced level of the 32-gene fla/che flagellar biosynthesis operon transcript. fla/che operon transcript abundance has been shown to be inhibited by an excess of the small regulatory protein, SlrA, and here we find that slrA mRNA accumulated in the pnpAdeletion mutant. Mutation of slrA was epistatic to mutation of pnpA for the motility-related phenotype. Further, Rho-dependent termination was required for PNPase turnover of slrA mRNA. When the slrA gene was provided with a Rho-independent transcription terminator, gene regulation was no longer PNPasedependent. Thus we show that the slrA transcript is a direct target of PNPase and that regulation of RNA turnover is a major determinant of motility gene expression. The interplay of specific transcription termination and mRNA decay mechanisms suggests selection for fine-tuning of gene expression. INTRODUCTION Levels of bacterial gene expression depend on the rate of transcription initiation, translation initiation, as well as the rate of messenger RNA decay. In Bacillus subtilis –– the best-studied Gram-positive species in terms of mRNA turnover –– initiation of mRNA decay is thought to begin most often with endonucleolytic cleavage catalyzed by RNase Y ( 1–3 ). Intra-transcript cleavage generates an upstream fragment that is degraded by polynucleotide phosphorylase (PNPase) or another 3 exonuclease ( 4 ), and a downstream fragment that is subject to additional RNase Y-mediated cleavages or processive decay by RNase J1, a 5 exonuclease ( 5,6 ). Decay from a transcript’s 5 end can also occur by the action of RNase J1, provided the 5 triphosphorylated end has been converted to a monophosphorylated form by an RNA pyrophosphohydrolase (7). Exonucleolytic decay from a transcript’s 3 end is normally hindered by the strong secondary structure that is part of the Rho-independent transcription termination mechanism. Rho-dependent termination, which could generate 3 ends without this strong structure, is not thought to play a significant role in B. subtilis transcription termination ( 8 ). Unlike in Escherichia coli, where about half of the transcription terminators are Rho-dependent and the rho gene is essential ( 9 ), the B. subtilis rho gene is not essential ( 10 ). Biochemical evidence suggests that PNPase is the major mRNA turnover enzyme in B. subtilis ( 11,12 ). A strain that is deleted for the gene encoding PNPase, the pnpA strain, shows several interesting phenotypes, including growth as non-motile chains of cells in liquid culture, competence deficiency and tetracycline sensitivity ( 12,13 ). However, at least in laboratory conditions, the pnpA strain grows only slightly slower than the wild-type, perhaps suggesting that other exonuclease activities compensate in the mRNA turnover process when PNPase is absent. A recent RNASeq study analyzed the pattern of decay intermediates in B. subtilis strains that were either wild-type or deleted for the PNPase gene, and found altered levels for many mRNAs in the pnpA strain ( 14 ). While many of the changes in gene expression in the pnpA strain are likely due to indirect effects, a direct effect of the lack of PNPase was observed for about 10% of expressed genes, for which there was a significant increase in the level of 5 -proximal reads relative to the level of 3 -proximal reads. Based on data from the RNA-Seq study, we were able to explain the chain growth phenotype of the pnpA strain by an effect on the fla/che operon, a 27-kb operon containing 32 genes, of which the Sigma D transcription factor gene is the penultimate gene. RNA-Seq analysis revealed a 2-4-fold decrease in fla/che operon read levels overall, with a 3-fold decrease in sigD expression. This, in turn, caused depression of the sigD regulon, including the autolysis genes that are required for separation of daughter cells upon cell division ( 15,16 ). We have now examined the ultimate cause of decreased fla/che operon expression in the pnpA strain. An earlier report demonstrated that fla/che operon RNA levels are controlled by SlrA, a small, 52-amino-acid protein. Insertion in the chromosome of a single extra copy of the slrA gene caused a severe decrease in expression of the fla/che oper (...truncated)


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Bo Liu, Daniel B. Kearns, David H. Bechhofer. Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3′ ends, Nucleic Acids Research, 2016, pp. 3364-3372, 44/7, DOI: 10.1093/nar/gkw069