# NAD+ capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp

## TL;DR

This study shows that NAD+ capping of RNA in E. coli is controlled by a specific promoter and enhanced by a bacterial alarmone called ppGpp.

## Contribution

The study identifies the minimal promoter of sibD as sufficient for NAD capping and reveals that ppGpp and DksA enhance this process.

## Key findings

- The 35-bp minimal promoter of sibD is sufficient for NAD capping of transcripts.
- ppGpp and DksA synergistically enhance transcription of NAD-capped and uncapped RNAs from sibD and related genes.
- A ppGpp⁰ mutant shows reduced NAD-RNA production from sibD and homologous genes.

## Abstract

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.

Graphical Abstract