Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• DNA-Directed RNA Polymerases * metabolism   MeSH
• Transcription, Genetic MeSH
• Transcription Initiation, Genetic * MeSH
• Mycobacterium smegmatis * metabolism   genetics   MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Rifampin * pharmacology   MeSH
• Sigma Factor * metabolism   genetics   MeSH
• Transcription Factors metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Bacterial Proteins * MeSH
• DNA-Directed RNA Polymerases * MeSH
• Rifampin * MeSH
• Sigma Factor * MeSH
• Transcription Factors MeSH

During the first step of gene expression, RNA polymerase (RNAP) engages DNA to transcribe RNA, forming highly stable complexes. These complexes need to be dissociated at the end of transcription units or when RNAP stalls during elongation and becomes an obstacle ('sitting duck') to further transcription or replication. In this review, we first outline the mechanisms involved in these processes. Then, we explore in detail the torpedo mechanism whereby a 5'-3' RNA exonuclease (torpedo) latches itself onto the 5' end of RNA protruding from RNAP, degrades it and upon contact with RNAP, induces dissociation of the complex. This mechanism, originally described in Eukaryotes and executed by Xrn-type 5'-3' exonucleases, was recently found in Bacteria and Archaea, mediated by β-CASP family exonucleases. We discuss the mechanistic aspects of this process across the three kingdoms of life and conclude that 5'-3' exoribonucleases (β-CASP and Xrn families) involved in the ancient torpedo mechanism have emerged at least twice during evolution.

• MeSH
• Archaea genetics   MeSH
• Bacteria genetics   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• DNA metabolism   MeSH
• Eukaryota genetics   MeSH
• Exoribonucleases metabolism   MeSH
• Transcription, Genetic MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• 5'-exoribonuclease MeSH Browser
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH
• Exoribonucleases MeSH

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.

• MeSH
• Bacterial Proteins chemistry   metabolism   ultrastructure   MeSH
• DNA, Bacterial chemistry   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   metabolism   ultrastructure   MeSH
• Cryoelectron Microscopy MeSH
• Catalytic Domain MeSH
• Models, Molecular MeSH
• Mycobacterium smegmatis enzymology   MeSH
• Nucleic Acids metabolism   MeSH
• Protein Domains MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Nucleic Acids MeSH

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.

• MeSH
• RNA, Bacterial genetics   MeSH
• Dinucleoside Phosphates genetics   MeSH
• DNA-Directed RNA Polymerases genetics   MeSH
• Escherichia coli genetics   MeSH
• Acid Anhydride Hydrolases metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Methylation MeSH
• Escherichia coli Proteins metabolism   MeSH
• RNA Caps genetics   MeSH
• RNA Stability MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ApaH protein, E coli MeSH Browser
• RNA, Bacterial MeSH
• Dinucleoside Phosphates MeSH
• DNA-Directed RNA Polymerases MeSH
• Acid Anhydride Hydrolases MeSH
• Escherichia coli Proteins MeSH
• RNA Caps MeSH
• RppH protein, E coli MeSH Browser

The σI sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI Further analysis revealed that the majority of these genes were affected indirectly by σI The σI regulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σI in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilisIMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σI regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σI affects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.

• Keywords
• RNA-seq, RNAP, iron metabolism, promoter, sigma factor,
• MeSH
• Bacillus subtilis genetics   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases genetics   MeSH
• Transcription, Genetic * MeSH
• Operon MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Regulon MeSH
• Sigma Factor genetics   MeSH
• Transcriptome MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA-Directed RNA Polymerases MeSH
• Sigma Factor MeSH
• Iron MeSH

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.

• Keywords
• NudC, RNA capping, RNA polymerase, RNA-seq, Rai1, nicotinamide adenine dinucleotide, non-canonical initiating nucleotide, transcription, transcription initiation, transcription start site,
• MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Endoribonucleases metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression genetics   MeSH
• Transcription, Genetic genetics   MeSH
• NAD metabolism   MeSH
• Nucleotides genetics   MeSH
• Transcription Initiation Site physiology   MeSH
• Promoter Regions, Genetic genetics   MeSH
• RNA Caps genetics   MeSH
• Transcriptome genetics   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Endoribonucleases MeSH
• mRNA decapping enzymes MeSH Browser
• NAD MeSH
• Nucleotides MeSH
• RNA Caps MeSH