RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• RNA, Bacterial metabolism   genetics   MeSH
• DNA-Directed RNA Polymerases metabolism   genetics   MeSH
• Endoribonucleases metabolism   genetics   MeSH
• Escherichia coli * genetics   metabolism   MeSH
• RNA, Messenger metabolism   genetics   MeSH
• NAD * metabolism   MeSH
• RNA Processing, Post-Transcriptional MeSH
• Escherichia coli Proteins metabolism   genetics   MeSH
• Protein Biosynthesis MeSH
• RNA Caps * metabolism   MeSH
• Stochastic Processes MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• RNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Endoribonucleases MeSH
• RNA, Messenger MeSH
• NAD * MeSH
• Escherichia coli Proteins MeSH
• ribonuclease E MeSH Browser
• RNA Caps * MeSH

Sigma factors bind and direct the RNA polymerase core to specific promoter sequences, and alternative sigma factors direct transcription of different regulons of genes. Here, we study the pBS32 plasmid-encoded sigma factor SigN of Bacillus subtilis to determine how it contributes to DNA damage-induced cell death. We find that SigN causes cell death when expressed at high levels and does so in the absence of its regulon suggesting it is intrinsically toxic. One way toxicity was relieved was by curing the pBS32 plasmid, which eliminated a positive feedback loop that led to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally encoded transcriptional repressor protein AbrB, thereby derepressing a potent antisense transcript that antagonized SigN expression. SigN efficiently competed with the vegetative sigma factor SigA in vitro, and SigN accumulation in the absence of positive feedback reduced SigA-dependent transcription suggesting that toxicity may be due to competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a toxic sigma factor is unclear but SigN may function in host-inhibition during lytic conversion, as phage lysogen genes are also encoded on pBS32. IMPORTANCE Alternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded alternative sigma factor SigN of Bacillus subtilis however, is activated by the DNA damage response and leads to cellular demise. Here we find that SigN impairs viability by hyper-accumulating and outcompeting the vegetative sigma factor for the RNA polymerase core. Why B. subtilis retains a plasmid with a deleterious alternative sigma factor is unknown.

• Keywords
• AbrB, SigN, cell death, pBS32, plasmid, prophage,
• MeSH
• Bacillus subtilis * genetics   metabolism   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases genetics   metabolism   MeSH
• Transcription, Genetic MeSH
• Immunoglobulin A, Secretory genetics   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Sigma Factor * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA-Directed RNA Polymerases MeSH
• Immunoglobulin A, Secretory MeSH
• Sigma Factor * MeSH