Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation.

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to the formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown.

Evolution of pH-sensitive transcription termination in 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.

Rho-dependent transcriptional switches regulate the bacterial response to cold shock.

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program.

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 as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization () sites.

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.

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.

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.

Regulatory interplay between small RNAs and transcription termination factor Rho.

The largest and best studied group of regulatory small RNAs (sRNAs) in bacteria act by modulating translation or turnover of messenger RNAs (mRNAs) through base-pairing interactions that typically take place near the 5' end of the mRNA. This allows the sRNA to bind the complementary target sequence while the remainder of the mRNA is still being made, creating conditions whereby the action of the sRNA can extend to transcriptional steps, most notably transcription termination. Increasing evidence corroborates the existence of a functional interplay between sRNAs and termination factor Rho.

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.

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.

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.

Evaluating the Effect of Small RNAs and Associated Chaperones on Rho-Dependent Termination of Transcription In Vitro.

Besides their well-known posttranscriptional effects on mRNA translation and decay, sRNAs and associated RNA chaperones (e.g., Hfq, CsrA) sometimes regulate gene expression at the transcriptional level.

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.

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.

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.

Monitoring RNA unwinding by the transcription termination factor Rho from Mycobacterium tuberculosis.

Transcription termination factor Rho is a ring-shaped, homo-hexamieric RNA translocase that dissociates transcription elongation complexes and transcriptional RNA-DNA duplexes (R-loops) in bacteria. The molecular mechanisms underlying these biological functions have been essentially studied with Rho enzymes from Escherichia coli or close Gram-negative relatives. However, phylo-divergent Rho factors may have distinct properties.

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.

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.

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.

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.

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.

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.

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.