Marc Boudvillain

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.

Terminator still moving forward: expanding roles for Rho factor.

Rho factor is a molecular motor that translocates along nascent RNA and acts on the transcription elongation complex to promote termination. Besides contributing to transcriptional punctuation of the bacterial genome, Rho can act intragenically under conditions that perturb coupling of translation and transcription. Recent advances have shed new light onto several aspects of Rho function, including the translocation mechanism, the avoidance of potential conflicts between DNA replication and transcription, suppression of pervasive antisense transcription and recruitment in riboswitch and small RNA-dependent regulation. Altogether, these findings further highlight the relevance of Rho factor, both as a multi-task housekeeper and gene regulator.

A stepwise 2'-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase.

The bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho–RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large (∼7-nucleotide-long) discrete steps triggered by 2′-hydroxyl activation events. This periodic 2′-OH–dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.

Transcription Termination Factor Rho Can Displace Streptavidin from Biotinylated RNA

In Escherichia coli, binding of the hexameric Rho protein to naked C-rich Rut (Rho utilization) regions of nascent RNA transcripts initiates Rho-dependent termination of transcription. Although the ring-shaped Rho factor exhibits in vitro RNA-dependent ATPase and directional RNA-DNA helicase activities, the actual molecular mechanisms used by Rho to disrupt the intricate network of interactions that cement the ternary transcription complex remain elusive. Here, we show that Rho is a molecular motor that can apply significant disruptive forces on heterologous nucleoprotein assemblies such as streptavidin bound to biotinylated RNA molecules. ATP-dependent disruption of the biotin-streptavidin interaction demonstrates that Rho is not mechanistically limited to the melting of nucleic acid base pairs within molecular complexes and confirms that specific interactions with the roadblock target are not required for Rho to operate properly. We also show that Rho-induced streptavidin displacement depends significantly on the identity of the biotinylated transcript as well as on the position, nature, and length of the biotin link to the RNA chain. Altogether, our data are consistent with a “snow plough” type of mechanism of action whereby an early rearrangement of the Rho-substrate complex (activation) is rate-limiting, physical force (pulling) is exerted on the RNA chain by residues of the central Rho channel, and removal of structural obstacles from the RNA track stems from their nonspecific steric exclusion from the hexamer central hole. In this context, a simple model for the regulation of Rho-dependent termination based on the modulation of disruptive dynamic loading by secondary factors is proposed.

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.

Keeping up to speed with the transcription termination factor rho motor

In bacteria, a subset of transcription termination events requires the participation of the transcription termination factor Rho. Rho is a homo-hexameric, ring-shaped, motor protein that uses the energy derived from its RNA-dependent ATPase activity to directionally unwind RNA and RNA-DNA helices and to dissociate transcription elongation complexes. Despite a wealth of structural, biochemical and genetic data, the molecular mechanisms used by Rho to carry out its biological functions remain poorly understood. Here, we briefly discuss the most recent findings on Rho mechanisms and function and highlight important questions that remain to be addressed.

The functional anatomy of an intrinsic transcription terminator

To induce dissociation of the transcription elongation complex, a typical intrinsic terminator forms a G·C‐rich hairpin structure upstream from a U‐rich run of approximately eight nucleotides that define the transcript 3′ end. Here, we have adapted the nucleotide analog interference mapping (NAIM) approach to identify the critical RNA atoms and functional groups of an intrinsic terminator during transcription with T7 RNA polymerase. The results show that discrete components within the lower half of the hairpin stem form transient termination‐specific contacts with the RNA polymerase. Moreover, disruption of interactions with backbone components of the transcript region hybridized to the DNA template favors termination. Importantly, comparative NAIM of termination events occurring at consecutive positions revealed overlapping but distinct sets of functionally important residues. Altogether, the data identify a collection of RNA terminator components, interactions and spacing constraints that govern efficient transcript release. The results also suggest specific architectural rearrangements of the transcription complex that may participate in allosteric control of intrinsic transcription termination.

Chapter 10:Transcription Termination Factor Rho: A Ring-Shaped RNA Helicase from Bacteria

Many NTP-dependent protein machines form oligomeric structures resembling doughnouts.1–3 Such ring-shaped machines include DNA translocases such as viral DNA packaging motors4 or chromosomal segregation enzymes5 as well as numerous DNA helicases involved in recombination and replication.1,6–8 In con...

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.

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.