Nucleoside tetraphosphate (Np4) caps have heretofore been reported in only one bacterial species: the model Gram-negative organism Escherichia coli, where RNAs acquire this epitranscriptomic modification during disulfide stress or when the dinucleoside tetraphosphate (Np4A) hydrolase ApaH is eliminated by mutation. Here, we show that caps of this kind also appear on RNA 5' ends in the prototypical Gram-positive organism Bacillus subtilis upon deleting the yqeK gene, which encodes an unrelated Np4A hydrolase. Present on diverse mRNAs irrespective of the identity of their 5'-terminal nucleotide, these caps are acquired in B. subtilis at least in part by Np4A incorporation at the 5' end of nascent transcripts during transcription initiation. YqeK suppresses Np4 capping by functioning both as an Np4A hydrolase that reduces the availability of Np4As for incorporation into RNA and as a decapping enzyme that can deprotect Np4-capped mRNA in B. subtilis by converting those caps to 5'-terminal diphosphates, thereby accelerating capped mRNA degradation. Disulfide stress increases Np4 capping in B. subtilis ∆yqeK mutants. Moreover, the elevated level of Np4 capping in these mutants is accompanied by an increased ability of B. subtilis to survive disulfide stress, suggesting a physiologically protective impact of Np4As and/or Np4 capping under these conditions.
Basu et al. (2026) studied this question.