The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization, translation efficiency1,2, and has been proposed to provide a layer of “epitranscriptomic” gene regulation3. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate “cap” in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria3–6. It has been proposed that NAD+, reduced NAD+ (NADH), and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps6–8. Here, we show instead that NAD+, NADH, and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated “ab initio capping” may occur in all organisms

Nucleoside-containing metabolites such as NAD can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD capping and define a consensus promoter sequence for NAD capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD and provide a general method for analysis of NCIN capping in vitro and in vivo.

dRNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the ␦ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of ␦ from Bacillus subtilis. We demonstrate that ␦ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that ␦ is essential for cell survival when facing a competing strain in a changing environment. Hence, although ␦ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis. RNA polymerase (RNAP) is the key enzyme responsible for transcription of DNA into RNA. Bacterial RNAP core enzyme consists of several subunits: the ␣ dimer that holds together ␤ and ␤=, which form the catalytic center, and the subunit that binds to ␤=. This core enzyme, ␣ 2 ␤␤=, is capable of elongating but not initiating transcription. To initiate transcription, the core enzyme must associate with a subunit that allows the holoenzyme to recognize specific sequences in the DNA, i.e., promoters. Typically, several different subunits are present in the cell and direct the expression of different subsets of genes (1, 2).While the ␣ 2 ␤␤= composition is conserved across the bacterial kingdom, Gram-positive Firmicutes contain an additional subunit, ␦, which is encoded by the rpoE gene in the model bacterium Bacillus subtilis. The ␦ subunit was first reported as an endogenous protein present in RNAP from phage SP01-infected Bacillus subtilis cells, which was required for its accurate middle gene transcription (3, 4). The rpoE gene specifies a protein of 173 amino acids (aa) with a molecular mass of ϳ20.5 kDa. The protein is highly acidic (pI, 3.6) (5). As determined by circular dichroism (CD) spectroscopy, it consists of two domains: (i) the N-terminal domain (NTD), which is structured; and (ii) the C-terminal domain, which is unstructured and whose amino acid composition-stretches of glutamic and aspartic acid residues-makes it virtually a polyanion (6). The structure of the ordered N-terminal domain was recently solved based on a truncated construct consisting of the N-terminal domain containing a His tag. The NTD contains four ␣-helices and an antiparallel ␤-sheet (7). Delta binds to RNAP in vivo (8), but the binding site is unknown.The in vitro effects of ␦ on transcription were previously examined in detail in the B. subtilis system. ␦ was reported to destabilize complexes between RNAP and DNA in vitro, thus increasing RNAP's specificity for good consensus promoter sequences (9, 10). Despite this inhibitory effect, ␦ was shown to stimulate transcription on some temp...