5'-NADylation of RNA is well-tolerated by bacterial cell

Recently discovered ability of various RNA polymerases (RNAPs) of bacteria and eukaryotes to initiate RNA synthesis with cofactors such as NAD+/NADH (nicotinamide adenine dinucleotide). This phenomenon was quickly dubbed "non-canonical capping" as the extra 5 moieties of RNA resemble eukaryotic m7G cap superficially. NAD must outcompete abundant ATP, to serve as initiating nucleotide. It is still unclear which physiological conditions permit significant RNA NADylation and to what degree NADylation serves typical functions of a canonical cap in bacteria. Here we found that the extent of NADyation depends primarily on fidelity of transcription initiation -NAD concentration, specific contacts of RNAP active site with NAD/ATP alternative substrates, and genomic DNA supercoiling. It is much less affected by posttranscriptional processes such as de-capping, processing by main 5-dependent ribonucleases RNaseE and oligoribonuclease. 5-NAD inhibits neither translation of a leaderless 5-NAD-RNA nor a physiological base-pairing of small regulatory NADylated RNA with its antisense target RNA. Translation exposes 5-NAD for de-capping by NudC enzyme.