NAD+ capping of <i>sibD</i> transcripts in <i>E. coli</i> is mediated by its minimal promoter and enhanced by ppGpp

Recently, nicotinamide adenine dinucleotide (NAD+) and other nucleotide analogs have been identified as non-canonical RNA caps in both prokaryotes and eukaryotes. In Escherichia coli, NAD capping has been shown to be influenced by environmental conditions in a gene-specific manner, yet its regulatory mechanisms remain poorly understood. We previously reported that most transcripts produced by sibD are NAD-capped during the stationary phase. In this study, we found that the 35-bp minimal promoter of sibD is sufficient for its NAD capping. When this minimal promoter was applied to express genes not typically producing NAD-RNAs, their transcripts could also be NAD-capped. These findings strongly support that NAD capping in E. coli occurs during transcription initiation mediated by the promoter and RNA polymerase. Additionally, the bacterial alarmone ppGpp and the small protein DksA, both transcription initiation regulators, synergistically enhance transcription of both NAD-capped and uncapped RNAs from sibD and its homologous genes, sibC and sibE, in vitro. In contrast, the ppGpp⁰ mutant, deficient in ppGpp synthesis, showed a significant reduction in NAD-RNA production from these genes. Our findings elucidate the cis-elements and trans-acting factors mediating NAD capping during transcription initiation, offering novel insight into this regulatory process.