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RfaH, member of the NusG/Spt5 family, activates virulence genes in Gram-negative pathogens. RfaH exists in two states, with its C-terminal domain (CTD) folded either as α-helical hairpin or β-barrel. In free RfaH, the α-helical CTD interacts with, and masks the RNA polymerase binding site on, the N-terminal domain, autoinhibiting RfaH and restricting its recruitment to ops DNA sequences. Upon activation, the domains separate and the CTD refolds into the β-barrel, which recruits a ribosome, activating translation. Using NMR spectroscopy, we show that only a complete ops -paused transcription elongation complex activates RfaH, probably via a transient encounter complex, allowing the refolded CTD to bind ribosomal protein S10. We also demonstrate that upon release from the elongation complex, the CTD transforms back into the autoinhibitory α-state, resetting the cycle. Transformation-coupled autoinhibition allows RfaH to achieve high specificity and potent activation of gene expression. The antitermination factor RfaH adopts two functional states where its C-terminal domain is folded either as an α-helical hairpin or β-barrel. Here the authors employ solution state NMR measurements to show that the C-terminal domain transforms into the β-barrel only upon binding to the elongation complex and refolds back after dissociation.

Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis , the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD) 2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues. The bacterial helicase-like transcription factor HelD interacts with the RNA polymerase (RNAP) and together with the RNAP δ subunit enhances RNAP cycling. Here, the authors present the cryo-EM structures of the monomeric and dimeric Bacillus subtilis RNAP-δ-HelD complexes and suggest a model for HelD/δ-mediated RNAP recycling and putative hibernation.

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

Key Points The transcription elongation complex is composed of a multisubunit RNA polymerase (RNAP), a DNA template and a nascent RNA. This complex is normally extremely stable but can be efficiently disassembled by the action of select proteins, called termination factors, or specific sequences, called intrinsic terminators.Regulated transcription termination is the underlying mechanism of attenuation, riboswitch function and polar repression of downstream expression. Efficient termination is also necessary to halt transcription elongation complexes at the end of genes in order to block unproductive or aberrant expression of downstream genes.Expression of many genes, most notably those encoding ribosomal RNA, is critically dependent on antitermination mechanisms that modify RNAP into a termination-resistant state. Antitermination factors can modulate or block the activity of termination factors, override intrinsic terminators in leader sequences under conditions necessitating downstream expression, and modify the activity of RNAP to provide expression of select genes (for example, upon phage infection). Many antitermination factors act just once during mRNA synthesis by blocking the formation of an RNA hairpin structure that is necessary for intrinsic termination. These factors usually employ direct binding to RNA, and structures as large as translating ribosomes are used to determine the RNA fold. Processive antitermination factors bind to, and modify the activity of, elongating RNAP such that the modified complex is resistant to multiple or tandem downstream termination signals. Studies of antitermination factors provide important insights into mechanisms of processive RNA synthesis in all domains. These factors provide stability to the transcription complex, either by shielding the encapsulated nucleic acids or preventing structural changes in RNAP necessary for RNA release and complex disassembly. Termination is an important way of regulating transcription and requires stringent control. Here, Santangelo and Artsimovitch discuss the different mechanisms of antitermination in bacteria and phages. Termination signals induce rapid and irreversible dissociation of the nascent transcript from RNA polymerase. Terminators at the end of genes prevent unintended transcription into the downstream genes, whereas terminators in the upstream regulatory leader regions adjust expression of the structural genes in response to metabolic and environmental signals. Premature termination within an operon leads to potentially deleterious defects in the expression of the downstream genes, but also provides an important surveillance mechanism. This Review discusses the actions of bacterial and phage antiterminators that allow RNA polymerase to override a terminator when the circumstances demand it.

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli ( E. coli ) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 ( σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α -C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ -independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation. Tiny forces cause E. coli RNA polymerase to slide along DNA after transcription termination at up to 700 bp/s. In this paper, the authors use magnetic tweezers to show that sliding complexes containing complete alpha C-terminal domains repeat transcription from protein roadblocks sites and promoters in either orientation.

Fidaxomicin was recently approved for the treatment of Clostridium difficile infection. It inhibits transcription by bacterial RNA polymerase. Because transcription is a multistep process, experiments were conducted in which fidaxomicin was added at different stages of transcriptional initiation to identify the blocked step. DNA footprinting experiments were also conducted to further elucidate the stage inhibited. Fidaxomicin blocks initiation only if added before the formation of the \"open promoter complex,\" in which the template DNA strands have separated but RNA synthesis has not yet begun. Binding of fidaxomicin precludes the initial separation of DNA strands that is prerequisite to RNA synthesis. These studies show that it has a mechanism distinct from that of elongation inhibitors, such as streptolydigin, and from the transcription initiation inhibitors myxopyronin and the rifamycins.

Bacterial RNA helicase ρ is a genome sentinel that terminates the synthesis of damaged and junk RNAs that are not translated by the ribosome. It is unclear how ρ is regulated during dormancy or stress, when translation is inefficient and RNAs are vulnerable to ρ-mediated release. We use cryogenic electron microscopy, biochemical, and genetic approaches to show that substitutions of residues in the connector between two ρ domains or ADP promote the formation of extended Escherichia coli ρ filaments. By contrast, (p)ppGpp induces the formation of transient ρ dodecamers. Our results demonstrate that ADP and (p)ppGpp nucleotides bound at subunit interfaces inhibit ρ ring closure that underpins the hexamer activation, thus favoring the assembly of inactive higher-order oligomers. Connector substitutions and antibiotics that inhibit RNA and protein syntheses trigger ρ aggregation in the cell. These and other recent data implicate aggregation as a widespread strategy to tune ρ activity. Bacterial transcription factor Rho (ρ) is a key regulator of transcription termination. Here, the authors show that Escherichia coli ρ forms inactive oligomers and filaments in the presence of stress-associated nucleotides (p)ppGpp and ADP.

Many bacteriophages modulate host transcription to favor expression of their own genomes. Phage satellite P4 polarity suppression protein, Psu, a building block of the viral capsid, inhibits hexameric transcription termination factor, ρ, by presently unknown mechanisms. Our cryogenic electron microscopy structures of ρ-Psu complexes show that Psu dimers clamp two inactive, open ρ rings and promote their expansion to higher-oligomeric states. ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders ρ ring closure and traps nucleotides in their binding pockets on ρ. Structure-guided mutagenesis in combination with growth, pull-down, and termination assays further delineated the functional ρ-Psu interfaces in vivo. Bioinformatic analyses revealed that Psu is associated with a wide variety of phage defense systems across Enterobacteriaceae , suggesting that Psu may regulate expression of anti-phage genes. Our findings show that modulation of the ρ oligomeric state via diverse strategies is a pervasive gene regulatory principle in bacteria. Many bacteriophages can modulate host transcription. Here, the authors uncover the anti-termination mechanism of phage satellite P4 protein, Psu. Psu dimers clamp two open rings of the termination factor ρ, promote ρ hyper-oligomerization, and trap nucleotides in ρ’s binding pockets.

Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits ρ-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Here, our cryo-EM structures of ρ-Rof and ρ-RNA complexes show that Rof undergoes pronounced conformational changes to bind ρ at the protomer interfaces, undercutting ρ conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between ρ and RNA polymerase. Consistently, Rof impedes ρ ring closure, ρ-RNA interactions and ρ association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirms that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination in vitro. Bioinformatic analyses reveal that Rof is restricted to Pseudomonadota and that the ρ-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence ρ under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by ρ may be detrimental. Said et al. used cryoEM, biochemistry and bioinformatics to uncover how the Sm-like protein Rof regulates transcription termination. Rof binds termination factor ρ, inhibiting ρ ring closure and its association with RNA or transcription complexes.

RfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an elongating RNA polymerase (RNAP) and a specific DNA element called ops to modify RNAP into a pause- and NusG-resistant state. rfaH null mutations confer hypersensitivity to antibiotics and detergents, altered susceptibility to bacteriophages, and defects in virulence. Here, we carried out a selection for suppressors that restore the ability of a Δ rfaH mutant Escherichia coli strain to grow in the presence of sodium dodecyl sulfate. We isolated rho , rpoC , and hns suppressor mutants with changes in regions previously shown to be important for their function. In addition, we identified mutants with changes in an unstructured region that connects the primary RNA-binding and helicase domains of Rho. The connector mutants display strong defects in vivo , consistent with their ability to compensate for the loss of RfaH, and act synergistically with bicyclomycin (BCM), which has been recently shown to inhibit Rho transformation into a translocation-competent state. We hypothesize that the flexible connector permits the reorientation of Rho domains and serves as a target for factors that control the motor function of Rho allosterically. Our results, together with the existing data, support a model in which the connector segment plays a hitherto overlooked role in the regulation of Rho-dependent termination. IMPORTANCE The transcription termination factor Rho silences foreign DNA, reduces antisense transcription, mediates surveillance of mRNA quality, and maintains genome integrity by resolving transcription-replication collisions and deleterious R loops. Upon binding to RNA, Rho undergoes a rate-limiting transition from an open “lock washer” state to a closed ring capable of processive translocation on, and eventually the release of, the nascent transcript. Recent studies revealed that Rho ligands, including its cofactor NusG and inhibitor bicyclomycin, control the ring dynamics allosterically. In this work, we used a genetic selection for suppressors of RfaH, a potent inhibitor of Rho, to isolate a new class of mutations in a flexible region that connects the primary RNA-binding and ATPase/translocase domains of Rho. We propose that the connector is essential for the modulation of Rho activity by different RNA sequences and accessory proteins. The transcription termination factor Rho silences foreign DNA, reduces antisense transcription, mediates surveillance of mRNA quality, and maintains genome integrity by resolving transcription-replication collisions and deleterious R loops. Upon binding to RNA, Rho undergoes a rate-limiting transition from an open “lock washer” state to a closed ring capable of processive translocation on, and eventually the release of, the nascent transcript. Recent studies revealed that Rho ligands, including its cofactor NusG and inhibitor bicyclomycin, control the ring dynamics allosterically. In this work, we used a genetic selection for suppressors of RfaH, a potent inhibitor of Rho, to isolate a new class of mutations in a flexible region that connects the primary RNA-binding and ATPase/translocase domains of Rho. We propose that the connector is essential for the modulation of Rho activity by different RNA sequences and accessory proteins.