The ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) decorates various RNAs in different organisms. In the proteobacterium Escherichia coli, the NAD-cap confers stability against RNA degradation. To date, NAD-RNAs have not been identified in any other bacterial microorganism. Here, we report the identification of NAD-RNA in the firmicute Bacillus subtilis. In the late exponential growth phase, predominantly mRNAs are NAD modified. NAD is incorporated de novo into RNA by the cellular RNA polymerase using non-canonical transcription initiation. The incorporation efficiency depends on the -1 position of the promoter but is independent of sigma factors or mutations in the rifampicin binding pocket. RNA pyrophosphohydrolase BsRppH is found to decap NAD-RNA. In vitro, the decapping activity is facilitated by manganese ions and single-stranded RNA 5' ends. Depletion of BsRppH influences the gene expression of ∼13% of transcripts in B. subtilis. The NAD-cap stabilizes RNA against 5'-to-3'-exonucleolytic decay by RNase J1.

Efficient and selective methyltransferase-catalyzed transfer of photolabile groups onto DNA enables photoregulation of gene expression and can be performed even in the presence of AdoMet.

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Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. Engineered proteases with redesigned substrate specificities have the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering‐based screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of tobacco etch virus (TEV) protease. The target substrate EKLVFQA differs at three out of seven positions from the TEV consensus substrate sequence. Flow cytometric sorting of a semi‐rational TEV protease library, consisting of focused mutations of the substrate binding pockets as well as random mutations throughout the enzyme, led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate.