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...

SUMMARY

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3′-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved −5T (3 bp downstream from the −10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species.

Background: TRPA1 channel is modulated by Ca 2ϩ , but the molecular mechanisms are unclear. Results: Mutations in the distal C-terminal acidic domain altered Ca 2ϩ dependence of TRPA1. Conclusion: The C-terminal acidic cluster is involved in the Ca 2ϩ -induced potentiation and inactivation of TRPA1. Significance: Identification of the Ca 2ϩ -dependent domain is important for understanding the role of TRPA1 in chemical nociception.

RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

The grafting of cationic groups to synthetic oligonucleotides (ONs) in order to reduce the charge repulsion between the negatively charged strands of a duplex or triplex, and consequently to increase a complex's stability, has been extensively studied. Guanidinium groups, which are highly basic and positively charged over a wide pH range, could be an efficient ON modification to enhance their affinity for nucleic acid targets and to improve cellular uptake. A straightforward post-synthesis method to convert amino functions attached to ONs (on sugar, nucleobase or backbone) into guanidinium tethers has been perfected. In comparison to amino groups, such cationic groups anchored to alpha-oligonucleotide phosphoramidate backbones play important roles in duplex stability, particularly with RNA targets. This high affinity could be explained by dual recognition resulting from Watson-Crick or Hoogsteen base pairing combined with cationic/anionic backbone recognition between strands involving H-bond formation and salt bridging. Molecular-dynamics simulations corroborate interactions between the cationic backbones of the alpha-ONs and the anionic backbones of the nucleic acid targets. Moreover, ONs with guanidinium modification increased cellular uptake relative to negatively charged ONs. The cellular localization of these new cationic phosphoramidate ONs is mainly cytoplasmic. The uptake of these ON analogues might occur through endocytosis.