A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches.

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating in vitro and in vivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.

NusG prevents transcriptional invasion of H-NS-silenced genes.

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.

RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination.

RNA-binding protein CsrA is a key regulator of a variety of cellular processes in bacteria, including carbon and stationary phase metabolism, biofilm formation, quorum sensing, and virulence gene expression in pathogens. CsrA binds to bipartite sequence elements at or near the ribosome loading site in messenger RNA (mRNA), most often inhibiting translation initiation. Here we describe an alternative novel mechanism through which CsrA achieves negative regulation. We show that CsrA binding to the upstream portion of the 5' untranslated region of Escherichia coli pgaA mRNA-encoding a polysaccharide adhesin export protein-unfolds a secondary structure that sequesters an entry site for transcription termination factor Rho, resulting in the premature stop of transcription. These findings establish a new paradigm for bacterial gene regulation in which remodeling of the nascent transcript by a regulatory protein promotes Rho-dependent transcription attenuation.

A multivariate prediction model for Rho-dependent termination of transcription.

Bacterial transcription termination proceeds via two main mechanisms triggered either by simple, well-conserved (intrinsic) nucleic acid motifs or by the motor protein Rho. Although bacterial genomes can harbor hundreds of termination signals of either type, only intrinsic terminators are reliably predicted. Computational tools to detect the more complex and diversiform Rho-dependent terminators are lacking. To tackle this issue, we devised a prediction method based on Orthogonal Projections to Latent Structures Discriminant Analysis [OPLS-DA] of a large set of in vitro termination data. Using previously uncharacterized genomic sequences for biochemical evaluation and OPLS-DA, we identified new Rho-dependent signals and quantitative sequence descriptors with significant predictive value. Most relevant descriptors specify features of transcript C>G skewness, secondary structure, and richness in regularly-spaced 5'CC/UC dinucleotides that are consistent with known principles for Rho-RNA interaction. Descriptors collectively warrant OPLS-DA predictions of Rho-dependent termination with a ∼85% success rate. Scanning of the Escherichia coli genome with the OPLS-DA model identifies significantly more termination-competent regions than anticipated from transcriptomics and predicts that regions intrinsically refractory to Rho are primarily located in open reading frames. Altogether, this work delineates features important for Rho activity and describes the first method able to predict Rho-dependent terminators in bacterial genomes.

A role for Rho-dependent polarity in gene regulation by a noncoding small RNA.

Gene regulation by bacterial trans-encoded small RNAs (sRNAs) is generally regarded as a post-transcriptional process bearing exclusively on the translation and/or the stability of target messenger RNA (mRNA). The work presented here revealed the existence of a transcriptional component in the regulation of a bicistronic operon-the chiPQ locus-by the ChiX sRNA in Salmonella. By studying the mechanism by which ChiX, upon pairing near the 5' end of the transcript, represses the distal gene in the operon, we discovered that the action of the sRNA induces Rho-dependent transcription termination within the chiP cistron. Apparently, by inhibiting chiP mRNA translation cotranscriptionally, ChiX uncouples translation from transcription, causing the nascent mRNA to become susceptible to Rho action. A Rho utilization (rut) site was identified in vivo through mutational analysis, and the termination pattern was characterized in vitro with a purified system. Remarkably, Rho activity at this site was found to be completely dependent on the function of the NusG protein both in vivo and in vitro. The recognition that trans-encoded sRNA act cotranscriptionally unveils a hitherto neglected aspect of sRNA function in bacteria.

A Fluorogenic Assay To Monitor Rho-Dependent Termination of Transcription.

Transcription termination mediated by the ring-shaped, ATP-dependent Rho motor is a multipurpose regulatory mechanism specific to bacteria and constitutes an interesting target for the development of new antibiotics. Although Rho-dependent termination can punctuate gene expression or contribute to the protection of the genome at hundreds of sites within a given bacterium, its exact perimeter and site- or species-specific features remain insufficiently characterized. New advanced approaches are required to explore thoroughly the diversity of Rho-dependent terminators and the complexity of associated mechanisms. Current in vitro analyses of Rho-dependent termination rely on radiolabeling, gel electrophoresis, and phosphorimaging of transcription reaction products and are thus hazardous, inconvenient, and low-throughput. To address these limitations, we have developed the first in vitro assay using a fluorescence detection modality to study Rho-dependent transcription termination. This powerful experimental tool accurately estimates terminator strengths in a matter of minutes and is optimized for a microplate reader format allowing multiplexed characterization of putative terminator sequences and mechanisms or high-throughput screening of new drugs targeting Rho-dependent termination.

Transcription Termination: Variations on Common Themes.

Transcription initiates pervasively in all organisms, which challenges the notion that the information to be expressed is selected mainly based on mechanisms defining where and when transcription is started. Together with post-transcriptional events, termination of transcription is essential for sorting out the functional RNAs from a plethora of transcriptional products that seemingly have no use in the cell. But terminating transcription is not that easy, given the high robustness of the elongation process. We review here many of the strategies that prokaryotic and eukaryotic cells have adopted to dismantle the elongation complex in a timely and efficient manner. We highlight similarities and diversity, underlying the existence of common principles in a diverse set of functionally convergent solutions.

Direct observation of the translocation mechanism of transcription termination factor Rho.

Rho is a ring-shaped, ATP-fueled motor essential for remodeling transcriptional complexes and R-loops in bacteria. Despite years of research on this fundamental model helicase, key aspects of its mechanism of translocation remain largely unknown. Here, we used single-molecule manipulation and fluorescence methods to directly monitor the dynamics of RNA translocation by Rho. We show that the efficiency of Rho activation is strongly dependent on the force applied on the RNA but that, once active, Rho is able to translocate against a large opposing force (at least 7 pN) by a mechanism involving 'tethered tracking'. Importantly, the ability to directly measure dynamics at the single-molecule level allowed us to determine essential motor properties of Rho. Hence, Rho translocates at a rate of ∼56 nt per second under our experimental conditions, which is 2-5 times faster than velocities measured for RNA polymerase under similar conditions. Moreover, the processivity of Rho (∼62 nt at a 7 pN opposing force) is large enough for Rho to reach termination sites without dissociating from its RNA loading site, potentially increasing the efficiency of transcription termination. Our findings unambiguously establish 'tethered tracking' as the main pathway for Rho translocation, support 'kinetic coupling' between Rho and RNA polymerase during Rho-dependent termination, and suggest that forces applied on the nascent RNA transcript by cellular substructures could have important implications for the regulation of transcription and its coupling to translation in vivo.

ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis.

The bacterial transcription termination factor Rho-a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity-is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal 'RNA binding' domain and by a non-canonical R-loop residue in its C-terminal 'motor' domain. We also show that the 'motor' domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems.

Transcription termination controls prophage maintenance in Escherichia coli genomes.

Prophages represent a large fraction of prokaryotic genomes and often provide new functions to their hosts, in particular virulence and fitness. How prokaryotic cells maintain such gene providers is central for understanding bacterial genome evolution by horizontal transfer. Prophage excision occurs through site-specific recombination mediated by a prophage-encoded integrase. In addition, a recombination directionality factor (or excisionase) directs the reaction toward excision and prevents the phage genome from being reintegrated. In this work, we describe the role of the transcription termination factor Rho in prophage maintenance through control of the synthesis of transcripts that mediate recombination directionality factor expression and, thus, excisive recombination. We show that Rho inhibition by bicyclomycin allows for the expression of prophage genes that lead to excisive recombination. Thus, besides its role in the silencing of horizontally acquired genes, Rho also maintains lysogeny of defective and functional prophages.

The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators.

In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.

Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains.

The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1.

Evolution of pH-sensitive transcription termination during adaptation to repeated long-term starvation.

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in E. coli populations experimentally evolved under repeated long-term starvation conditions, during which feast and famine result in drastic environmental pH fluctuations. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI, which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers a plastic alkaline-induced reduction of Rho function that, when found in tandem with a ΔydcI allele, leads to intracellular alkalinization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species originating from fluctuating alkaline environments. Our results suggest that Arg to His substitutions in global regulators of gene expression can serve to rapidly coordinate complex responses through pH sensing and shed light on how cellular populations across the tree of life use environmental cues to coordinate rapid responses to complex, fluctuating environments.

Phyletic distribution and conservation of the bacterial transcription termination factor Rho.

Transcription termination factor Rho is a ring-shaped, ATP-dependent molecular motor that targets hundreds of transcription units in Escherichia coli. Interest in Rho was renewed recently on the realization that this essential factor is involved in multiple interactions and cellular processes that protect the E. coli genome and regulate its expression on a global scale. Yet it is currently unknown if (and how) Rho-dependent mechanisms are conserved throughout the bacterial kingdom. Here, we mined public databases to assess the distribution, expression and structural conservation of Rho across bacterial phyla. We found that rho is present in more than 90 % of sequenced bacterial genomes, although Cyanobacteria, Mollicutes and a fraction of Firmicutes are totally devoid of rho. Genomes lacking rho tend to be small and AT-rich and often belong to species with parasitic/symbiotic lifestyles (such as Mollicutes). By contrast, large GC-rich genomes, such as those of Actinobacteria, often contain rho duplicates and/or encode Rho proteins that bear insertion domains of unknown function(s). Notwithstanding, most Rho sequences across taxa contain canonical RNA-binding and ATP hydrolysis signature motifs, a feature suggestive of largely conserved mechanism(s) of action. Mutations that impair binding of bicyclomycin are present in ~5 % of rho sequences, implying that species from diverse ecosystems have developed resistance against this natural antibiotic. Altogether, these findings assert that Rho function is widespread among bacteria and suggest that it plays a particularly relevant role in the expression of complex genomes and/or bacterial adaptation to changing environments.

Posttranscriptional Regulation by Copper with a New Upstream Open Reading Frame.

Copper is essential to most living beings but also highly toxic and as such is an important player at the host-pathogen interface. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. Known Cu export and import systems are under transcriptional control, whereas posttranscriptional regulatory mechanisms are yet to be characterized. We identified a three-gene operon, bp2923-bfrG-bp2921, downregulated by copper and notably encoding a TonB-dependent transporter in Bordetella pertussis. We show here that the protein encoded by the first gene, which is a member of the DUF2946 protein family, represents a new type of upstream Open Reading Frame (uORF) involved in posttranscriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologs of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has thus unveiled a new mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator. IMPORTANCE Copper is a transition metal necessary for living beings but also extremely toxic. Bacteria thus tightly control its homeostasis with transcriptional regulators. In this work, we have identified in the whooping cough agent Bordetella pertussis a new control mechanism mediated by a small protein called CruR, for copper-responsive upstream regulator. While being translated by the ribosome CruR is able to perceive intracellular copper, which shuts down the transcription of downstream genes of the same operon, coding for a copper uptake system. This mechanism limits the import of copper in conditions where it is abundant for the bacterium. This is the first report of "posttranscriptional regulation" in response to copper. Homologs of CruR genes head many operons harboring copper-related genes in various bacteria, and therefore the regulatory function unveiled here is likely a general property of this new protein family.

Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism.

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of MtbRho at 3.3 Šresolution. The MtbRho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of MtbRho and provides a framework for future antibiotic development.

A Simple Fluorescence Microplate Assay to Monitor RNA-DNA Hybrid Unwinding by the Bacterial Transcription Termination Factor Rho.

Transcription termination factor Rho contributes to shape the transcriptomes of many bacteria and is essential in a large subset of them. Although the transcription termination function of Rho is not always easy to reconstitute and to study in vitro, assays based on the ATP-dependent RNA-DNA hybrid unwinding activity of the factor can prove useful to dissect Rho mechanisms or to seek new antibiotics targeting Rho. However, current in vitro assays of Rho helicase activity are time-consuming, as they usually require radiolabeling of the hybrid substrates and analysis of reaction products by gel electrophoresis. Here, we describe a fluorescence-based microplate assay that informs on Rho helicase activity in a matter of minutes and allows the multiplexed analysis of conditions required for primary biochemical characterization or for drug screening.

A Large Insertion Domain in the Rho Factor From a Low G + C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity.

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli (EcRho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho (BfRho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (~40% of total mass) strongly increases BfRho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of ß-amyloids. Thus, despite its unusually large RNA binding domain, BfRho behaves more like the NID-less EcRho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho's N-terminal region and illustrate how RDTT is adapted to distinct genomic contents.

RNA remodeling by hexameric RNA helicases.

The unwinding of RNA helices and the disruption of RNA-protein complexes are critical steps of cellular metabolism that are carried out by ubiquitous NTP-dependent enzymes named RNA helicases. Here, we review the structures, mechanisms, and biochemical properties of two RNA helicases known to adopt a homo-hexameric ring architecture: the P4 packaging motor of bacteriophage φ8, a Super-Family 4 helicase, and Escherichia coli's transcription termination factor Rho from the Super-Family 5 of helicases. We emphasize the many similarities as well as key differences that characterize the Rho and P4 motor mechanisms and highlight important questions that remain to be addressed.