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Accueil > Thèmes de recherche > Aspects moléculaires du vivant > Remodelage de l’ARN : Facteurs et Mécanismes

Remodelage de l’ARN : Facteurs et Mécanismes

par Frapart - publié le , mis à jour le

DESCRIPTIF DU TRAVAIL DE RECHERCHE

Notre groupe étudie les mécanismes de remodelage de la structure des ARN et des complexes ribonucléoprotéiques (RNP). Cette structure, souvent dynamique, peut évoluer au cours de la vie cellulaire et gouverne le rôle et le devenir des ARN. Deux familles principales de protéines ubiquitaires –ARN chaperones et ARN hélicases- assurent la structuration et le remodelage des ARN et des complexes RNP. Ces protéines sont impliquées dans diverses pathologies dont certains cancers. Nous étudions comment ARN hélicases et ARN chaperones fonctionnent et coopèrent pour moduler les fonctions biologiques des ARN.

La fixation des ARN chaperones à l’ARN provoque généralement la destruction de structures secondaires et la formation de motifs ou assemblages structuraux alternatifs. Les ARN hélicases, elles, utilisent l’énergie dérivée de leur activité NTPase pour détruire des régions ARN en double-brin, des hétéroduplexes ARN-ADN et/ou des complexes RNP. Ces propriétés résultent parfois d’une activité de translocation directionnelle le long de l’ARN.

Pour étudier les mécanismes de remodelage de l’ARN, nous utilisons des approches complémentaires de biologie, de biochimie et de biophysique. Nous concentrons nos efforts sur l’étude d’un système modèle complexe au cœur duquel se trouve Rho, un moteur moléculaire ‘six-cylindres’ à activité ARN hélicase impliqué dans la terminaison de la transcription et le maintien de l’intégrité génomique chez les bactéries. Nous étudions les mécanismes moléculaires gouvernant l’activité de Rho, son rôle dans les mécanismes de régulation génique impliquant ARN non-codants et ARN chaperones (Hfq, CsrA), son importance pour la pathogénicité et l’adaptabilité bactérienne, la conservation de ses fonctions et mode(s) d’action au sein de la biodiversité, etc.

Nous étudions également d’autres ARN hélicases, en particulier celles qui, a priori, remodèlent l’ARN via des mécanismes distincts de ceux de Rho. De cette façon, nous espérons appréhender dans leur ensemble les mécanismes fondamentaux qui gouvernent ces enzymes, déterminer les points communs ou les différences qui les caractérisent au niveau moléculaire et identifier les facteurs qui contribuent à leur régulation. L’objectif, à terme, est d’exploiter cette information à des fins de ciblage thérapeutique ou de conception de bio-senseurs et de circuits géniques inductibles intéressants pour la biologie synthétique.

ILLUSTRATIONS




Principales publications

  • D’Heygère, F., Coste, F., Schwartz, A., Castaing, B. & Boudvillain, M. 
    ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis
    Nucleic Acids Res. (2015) 43, 6099-6111.
  • Figueroa-Bossi, N., Schwartz, A., Gillemardet, B., D’Heygère, F., Bossi, L. & Boudvillain, M.
    RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination
    Genes Dev. (2014) 28, 1239-51.
  • Soaeres, E., Schwartz, A., Nollmann,M., Margeat, E., & Boudvillain, M.
    The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved Nucleotide Analog Interference Probing (trNAIP)
    Nucleic Acids Res. (2014) 42, 9270-84.
  • Bossi, L., Schwartz, A., Gillemardet, B., Boudvillain, M. & Figueroa-Bossi, N.
    A role for Rho-dependent polarity in gene regulation by a noncoding small RNA
    Genes Dev. (2012) 26 (16) 1864-73.
  • Rabhi, M., Espéli, O., M., Schwartz, A., Cayrol, B., Rahmouni, R., Arluison, V. & Boudvillain, M.
    The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators
    EMBO J. (2011) 14, 2805-162.

Membres du groupe thématique

BOUDVILLAIN Marc Directeur de recherche CNRS, Responsable du groupe thématique   @

EVENO Eric Ingénieur de Recherche CNRS @

SCHWARTZ Annie Ingénieur d’études CNRS   @

SOAERES Emilie Assistant ingénieur CDD  @

NADIRAS Cédric Doctorant   @


Publications

2016   Références trouvées : 1

Porrua O., Boudvillain M. and Libri D.  (2016)

Transcription Termination : Variations on Common Themes

Trends in genetics : TIG (2016) 32 (8) 508-522 - doi : 10.1016/j.tig.2016.05.007
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.

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.


2015   Références trouvées : 4

Kondakova T., D’Heygere F., Feuilloley M. J., Orange N., Heipieper H. J. and Poc C. D.  (2015)

Glycerophospholipid synthesis and functions in Pseudomonas

Chemistry and Physics of Lipids (2015) 190, 27-42 - doi : 10.1016/j.chemphyslip.2015.06.006
The genus Pseudomonas is one of the most heterogeneous groups of eubacteria, presents in all major natural environments and in wide range of associations with plants and animals. The wide distribution of these bacteria is due to the use of specific mechanisms to adapt to environmental modifications. Generally, bacterial adaptation is only considered under the aspect of genes and protein expression, but lipids also play a pivotal role in bacterial functioning and homeostasis. This review resumes the mechanisms and regulations of pseudomonal glycerophospholipid synthesis, and the roles of glycerophospholipids in bacterial metabolism and homeostasis. Recently discovered specific pathways of P. aeruginosa lipid synthesis indicate the lineage dependent mechanisms of fatty acids homeostasis. Pseudomonas glycerophospholipids ensure structure functions and play important roles in bacterial adaptation to environmental modifications. The lipidome of Pseudomonas contains a typical eukaryotic glycerophospholipid phosphatidylcholine, which is involved in bacteria host interactions. The ability of Pseudomonas to exploit eukaryotic lipids shows specific and original strategies developed by these microorganisms to succeed in their infectious process. All compiled data provide the demonstration of the importance of studying the Pseudomonas lipidome to inhibit the infectious potential of these highly versatile germs.

The genus Pseudomonas is one of the most heterogeneous groups of eubacteria, presents in all major natural environments and in wide range of associations with plants and animals. The wide distribution of these bacteria is due to the use of specific mechanisms to adapt to environmental modifications. Generally, bacterial adaptation is only considered under the aspect of genes and protein expression, but lipids also play a pivotal role in bacterial functioning and homeostasis. This review resumes the mechanisms and regulations of pseudomonal glycerophospholipid synthesis, and the roles of glycerophospholipids in bacterial metabolism and homeostasis. Recently discovered specific pathways of P. aeruginosa lipid synthesis indicate the lineage dependent mechanisms of fatty acids homeostasis. Pseudomonas glycerophospholipids ensure structure functions and play important roles in bacterial adaptation to environmental modifications. The lipidome of Pseudomonas contains a typical eukaryotic glycerophospholipid phosphatidylcholine, which is involved in bacteria host interactions. The ability of Pseudomonas to exploit eukaryotic lipids shows specific and original strategies developed by these microorganisms to succeed in their infectious process. All compiled data provide the demonstration of the importance of studying the Pseudomonas lipidome to inhibit the infectious potential of these highly versatile germs.

D’Heygère F., Schwartz A., Coste F., Castaing B., Boudvillain M.  (2015)

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

Nucleic Acids Research (2015) First published online : May 20, 2015 - doi : 10.1093/nar/gkv505
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 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.

D’Heygère F., Schwartz A., Coste F., Castaing B., Boudvillain M.  (2015)

Monitoring RNA Unwinding by the Transcription Termination Factor Rho from Mycobacterium tuberculosis

In "RNA Remodeling Proteins" (2015) vol. 1259, chap 18, 293-311 - doi : 10.1007/978-1-4939-2214-7_18
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. Here, we describe methods for the preparation and in vitro characterization (ATPase and helicase activities) of the Rho factor from Mycobacterium tuberculosis, a specimen with uncharacteristic molecular and enzymatic features. These methods set the stage for future studies aimed at better defining the diversity of enzymatic properties of Rho across the bacterial kingdom.

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. Here, we describe methods for the preparation and in vitro characterization (ATPase and helicase activities) of the Rho factor from Mycobacterium tuberculosis, a specimen with uncharacteristic molecular and enzymatic features. These methods set the stage for future studies aimed at better defining the diversity of enzymatic properties of Rho across the bacterial kingdom.

Gocheva V., Le Gall A., Boudvillain, M., Margeat, E., Nollmann M.  (2015)

Direct observation of the translocation mechanism of transcription termination factor Rho

Nucleic Acids Research (2015) 43 (4) 2367-2377 - doi : 10.1093/nar/gkv085
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.

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.


2014   Références trouvées : 2

Soares, E., Schwartz, A., Nollmann, M., Margeat, M., Boudvillain, M.  (2014)

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved Nucleotide Analog Interference Probing (trNAIP)

Nucleic Acids Research (2014) 42 (14) 9270-9284 - doi : 10.1093/nar/gku595
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.

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.

Figueroa-Bossi, N., Schwartz, A., Guillemardet, B., D’Heygère, F., Bossi, L. and Boudvillain, M.  (2014)

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

Genes & Development (2014) 28 (11) 1239-1251 - doi : 10.1101/gad.240192.114
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.

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.


2013   Références trouvées : 4

Menouni, R., Champ, S., Espinosa, L., Boudvillain, M., Ansaldi, M.  (2013)

Transcription termination controls prophage maintenance in Escherichia coli genomes

Proceedings of the National Academy of Sciences 110 (35) 14414-14419
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.

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.

Fiorini F., Boudvillain M., Le Hir H.  (2013)

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

Nucleic Acids Research 41 (4) 2404-2415
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.

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.

D’Heygère F., Rabhi M., Boudvillain M.  (2013)

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

Microbiology 159 (pt 7) 1423-1436
Transcription termination factor Rho is a ring-shaped, ATP-dependent molecular motor that targets hundreds of transcription units in Escherichia coli. Interest for Rho has been 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 have 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 taxons 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.

Transcription termination factor Rho is a ring-shaped, ATP-dependent molecular motor that targets hundreds of transcription units in Escherichia coli. Interest for Rho has been 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 have 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 taxons 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.

Boudvillain M, Figueroa-Bossi N. and Bossi L.  (2013)

Terminator still moving forward : expanding roles for Rho factor

Curr Opin Microbiol. 16 (2)118-124
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.

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.


2012   Références trouvées : 2

Bossi, L., Schwartz, A., Guillemardet, B., Boudvillain, M. and Figueroa-Bossi, N.  (2012)

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

Genes & Development 26 (16) 1864-1873
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.

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.

Schwartz, A., Rabhi, M., Margeat, E. and Boudvillain, M.  (2012)

Analysis of Helicase–RNA Interactions Using Nucleotide Analog Interference Mapping

Methods in Enzymology 511 - 7 - 149-169
Nucleotide analog interference mapping (NAIM) is a combinatorial approach that probes individual atoms and functional groups in an RNA molecule and identifies those that are important for a specific biochemical function. Here, we show how NAIM can be adapted to reveal functionally important atoms and groups on RNA substrates of helicases. We explain how NAIM can be used to investigate translocation and unwinding mechanisms of helicases and discuss the advantages and limitations of this powerful chemogenetic approach.

Nucleotide analog interference mapping (NAIM) is a combinatorial approach that probes individual atoms and functional groups in an RNA molecule and identifies those that are important for a specific biochemical function. Here, we show how NAIM can be adapted to reveal functionally important atoms and groups on RNA substrates of helicases. We explain how NAIM can be used to investigate translocation and unwinding mechanisms of helicases and discuss the advantages and limitations of this powerful chemogenetic approach.


2011   Références trouvées : 2

Rabhi, M., Espeli, O., Schwartz, A., Cayrol, B., Rahmouni, A. R., Arluison, V. & Boudvillain, M.  (2011)

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

Embo Journal 30 (14) 2805-2816
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 (lambda tR1) 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. The EMBO Journal ( 2011) 30, 2805-2816. doi:10.1038/emboj.2011.192 ; Published online 14 June 2011

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 (lambda tR1) 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. The EMBO Journal ( 2011) 30, 2805-2816. doi:10.1038/emboj.2011.192 ; Published online 14 June 2011

Rabhi, M., Gocheva, V., Jacquinot, F., Lee, A., Margeat, E. & Boudvillain, M.  (2011)

Mutagenesis-based evidence for an asymmetric configuration of the ring-shaped transcription termination Rho factor

J. Mol. Biol. (2011) 405 (2) 497-518


2010   Références trouvées : 3

Rabhi, M., Tuma, R. & Boudvillain, M.  (2010)

RNA remodeling by hexameric RNA helicases

RNA Biology (2010) 7 (6) 655-666

Boudvillain, M, Nollmann, M. & Margeat, E.  (2010)

Keeping up to speed with the transcription termination factor Rho motor

Transcription 1 (2) 70-75

Rabhi, M., Rahmouni, A. R. & M. Boudvillain, M.   (2010)

Transcription termination factor Rho : a ring-shaped RNA helicase from bacteria

In "RNA helicases" Eckhard Jankowsky (Ed.) RSC Publishing, Londres, 243-271


2009   Références trouvées : 1

Schwartz, A., Rabhi, M., Jacquinot, F., Margeat, E., Rahmouni, A.R. & Boudvillain, M.  (2009)

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

Nat. Struct. Mol. Biol. 16, 1309-1316.
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 ( approximately 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.

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 ( approximately 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.


2005   Références trouvées : 1

Fedorova, O., Boudvillain, M., Kawaoka, J. and Pyle, A. M.  (2005)

Nucleotide Analog Interference Mapping and Suppression : Specific Applications in Studies of RNA Tertiary Structure, Dynamic Helicase Mechanism and RNA-Protein Interactions

In "Handbook of RNA Biochemistry" (2005) p. 259-293 - doi : 10.1002/9783527619504.ch17