Origins and Molecular Evolution of the NusG Paralog RfaH.
ABSTRACT: The only universally conserved family of transcription factors comprises housekeeping regulators and their specialized paralogs, represented by well-studied NusG and RfaH. Despite their ubiquity, little information is available on the evolutionary origins, functions, and gene targets of the NusG family members. We built a hidden Markov model profile of RfaH and identified its homologs in sequenced genomes. While NusG is widespread among bacterial phyla and coresides with genes encoding RNA polymerase and ribosome in all except extremely reduced genomes, RfaH is mostly limited to Proteobacteria and lacks common gene neighbors. RfaH activates only a few xenogeneic operons that are otherwise silenced by NusG and Rho. Phylogenetic reconstructions reveal extensive duplications and horizontal transfer of rfaH genes, including those borne by plasmids, and the molecular evolution pathway of RfaH, from "early" exclusion of the Rho terminator and tightened RNA polymerase binding to "late" interactions with the ops DNA element and autoinhibition, which together define the RfaH regulon. Remarkably, NusG is not only ubiquitous in Bacteria but also common in plants, where it likely modulates the transcription of plastid genes.IMPORTANCE In all domains of life, NusG-like proteins make contacts similar to those of RNA polymerase and promote pause-free transcription yet may play different roles, defined by their divergent interactions with nucleic acids and accessory proteins, in the same cell. This duality is illustrated by Escherichia coli NusG and RfaH, which silence and activate xenogenes, respectively. We combined sequence analysis and recent functional and structural insights to envision the evolutionary transformation of NusG, a core regulator that we show is present in all cells using bacterial RNA polymerase, into a virulence factor, RfaH. Our results suggest a stepwise conversion of a NusG duplicate copy into a sequence-specific regulator which excludes NusG from its targets but does not compromise the regulation of housekeeping genes. We find that gene duplication and lateral transfer give rise to a surprising diversity within the only ubiquitous family of transcription factors.
Project description:NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a ? barrel in NusG and an ? hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-? to an all-? conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:
Project description:In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the ? gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the ?' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.
Project description: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 ?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.
Project description:Elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase (RNAP) to modulate transcription. However, although NusG associates with RNAP transcribing most Escherichia coli genes, RfaH regulates just a few operons containing ops, a DNA sequence that mediates RfaH recruitment. Here, we describe the mechanism by which this specificity is maintained. We observe that RfaH action is indeed restricted to those several operons that are devoid of NusG in vivo. We also show that RfaH and NusG compete for their effects on transcript elongation and termination in vitro. Our data argue that RfaH recognizes its DNA target even in the presence of NusG. Once recruited, RfaH remains stably associated with RNAP, thereby precluding NusG binding. We envision a pathway by which a specialized regulator has evolved in the background of its ubiquitous paralogue. We propose that RfaH and NusG may have opposite regulatory functions: although NusG appears to function in concert with Rho, RfaH inhibits Rho action and activates the expression of poorly translated, frequently foreign genes.
Project description:NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ?-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.
Project description:Timely and accurate RNA synthesis depends on accessory proteins that instruct RNA polymerase (RNAP) where and when to start and stop transcription. Among thousands of transcription factors, NusG/Spt5 stand out as the only universally conserved family of regulators. These proteins interact with RNAP to promote uninterrupted RNA synthesis and with diverse cellular partners to couple transcription to RNA processing, modification or translation, or to trigger premature termination of aberrant transcription. NusG homologs are present in all cells that utilize bacterial-type RNAP, from endosymbionts to plants, underscoring their ancient and essential function. Yet, in stark contrast to other core RNAP components, NusG family is actively evolving: horizontal gene transfer and sub-functionalization drive emergence of NusG paralogs, such as bacterial LoaP, RfaH, and UpxY. These specialized regulators activate a few (or just one) operons required for expression of antibiotics, capsules, secretion systems, toxins, and other niche-specific macromolecules. Despite their common origin and binding site on the RNAP, NusG homologs differ in their target selection, interacting partners and effects on RNA synthesis. Even among housekeeping NusGs from diverse bacteria, some factors promote pause-free transcription while others slow the RNAP down. Here, we discuss structure, function, and evolution of NusG proteins, focusing on unique mechanisms that determine their effects on gene expression and enable bacterial adaptation to diverse ecological niches.
Project description: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 opsDNA 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.
Project description: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.
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.
Project description:Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho's primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface.