Mechanisms and regulation of an essential RNA helicase in E. coli : the bacterial transcription termination factor Rho

Chez E. coli, Rho est un facteur essentiel qui contrôle l’expression de multiples unités transcriptionnelles via le phénomène de terminaison de la transcription. Rho est un moteur moléculaire ATP-dépendant ayant une activité ARN hélicase caractéristique de sa capacité à dissocier des obstacles (comme l’ARN polymérase) lors de sa translocation le long de sa piste ARN. Il existe différentes structures de Rho en interaction avec l’ARN qui suggèrent des mécanismes de translocation contradictoires. Afin de mieux comprendre ces mécanismes, nous avons utilisé deux approches complémentaires pour identifier les fonctionnalités moléculaires importantes au sein de l’ARN et de Rho : l’approche NAIM (Nucleotide Analog Interference Mapping) développée au laboratoire et la mutagenèse dirigée. Nos résultats excluent une organisation de l’anneau hexamérique en «trimère de dimère» (ainsi que les mécanismes de translocation qui en découlent) mais sont compatibles avec différents aspects rencontrés dans une structure en anneau asymétrique plus récente. Toutefois, nos résultats ne supportent pas le mécanisme d’escorte nucléotide par nucléotide qui découle de cette structure asymétrique. Ainsi, nous montrons que Rho contacte la chaîne ARN de façon hétérogène et ne nécessite un groupement 2’-OH que tous les sept nucléotides en moyenne. Par ailleurs, nous avons exploré l’interactome d’E. coli dans le but d’identifier d’éventuels régulateurs de la fonction de Rho. Nous montrons que la protéine hexamèrique Hfq présente une similitude topologique avec les protéines endogènes NusG et YaeO et que, comme elles, Hfq s’associe à Rho pour en réguler la fonction. L’interaction Hfq:Rho inhibe les activités enzymatiques de Rho. Ces résultats révèlent un nouveau mécanisme d’anti-terminaison de la transcription avec diverses implications possibles dans le métabolisme bactérien et/ou la virulence de germes pathogènes.In E. coli, Rho is an essential factor that controls the expression of multiple transcriptional units via the phenomenon of transcription termination. Rho is an ATP-dependent molecular motor displaying RNA helicase activity, a feature typical of Rho’s ability to dissociate obstacles (such as RNA polymerase) during translocation along its RNA track. Different structures of the Rho-RNA complex have been published and suggest contradictory mechanisms of translocation. In order to understand these mechanisms, we have used two complementary approaches to identify functionality molecular comports in RNA and Rho : the NAIM (Nucleotide Analog Interference Mapping) approach developed in the laboratory and site-directed mutagenesis. Our results exclude that Rho forms a functional "trimer of dimer" ring (which rules out related translocation mechanisms) but are compatible with various aspects encountered in a recent asymmetric ring structure. However, our results do not support the "nucleotide by nucleotide" escort mechanism inferred from this asymmetric structure. Indeed, we show that Rho forms heterogonous contacts with the RNA chain and only requires a 2'-OH every seven nucleotides on average. Furthermore, we explored the interactome of E. coli in order to identify potential regulators of Rho function. We show that the hexameric protein Hfq displays topological similarity with the endogenous proteins NusG and YaeO and, that, like them, Hfq associates with Rho to regulate Rho function. The Hfq:Rho interaction inhibits the enzymatic activities of Rho. These results reveal a novel mechanism of transcription anti-termination with potentially important implications in bacterial metabolism and/or virulence of pathogens

Mechanisms and regulation of an essential RNA helicase in E. coli (the bacterial transcription termination factor Rho)

Chez E. coli, Rho est un facteur essentiel qui contrôle l expression de multiples unités transcriptionnelles via le phénomène de terminaison de la transcription. Rho est un moteur moléculaire ATP-dépendant ayant une activité ARN hélicase caractéristique de sa capacité à dissocier des obstacles (comme l ARN polymérase) lors de sa translocation le long de sa piste ARN. Il existe différentes structures de Rho en interaction avec l ARN qui suggèrent des mécanismes de translocation contradictoires. Afin de mieux comprendre ces mécanismes, nous avons utilisé deux approches complémentaires pour identifier les fonctionnalités moléculaires importantes au sein de l ARN et de Rho : l approche NAIM (Nucleotide Analog Interference Mapping) développée au laboratoire et la mutagenèse dirigée. Nos résultats excluent une organisation de l anneau hexamérique en trimère de dimère (ainsi que les mécanismes de translocation qui en découlent) mais sont compatibles avec différents aspects rencontrés dans une structure en anneau asymétrique plus récente. Toutefois, nos résultats ne supportent pas le mécanisme d escorte nucléotide par nucléotide qui découle de cette structure asymétrique. Ainsi, nous montrons que Rho contacte la chaîne ARN de façon hétérogène et ne nécessite un groupement 2 -OH que tous les sept nucléotides en moyenne. Par ailleurs, nous avons exploré l interactome d E. coli dans le but d identifier d éventuels régulateurs de la fonction de Rho. Nous montrons que la protéine hexamèrique Hfq présente une similitude topologique avec les protéines endogènes NusG et YaeO et que, comme elles, Hfq s associe à Rho pour en réguler la fonction. L interaction Hfq:Rho inhibe les activités enzymatiques de Rho. Ces résultats révèlent un nouveau mécanisme d anti-terminaison de la transcription avec diverses implications possibles dans le métabolisme bactérien et/ou la virulence de germes pathogènes.In E. coli, Rho is an essential factor that controls the expression of multiple transcriptional units via the phenomenon of transcription termination. Rho is an ATP-dependent molecular motor displaying RNA helicase activity, a feature typical of Rho s ability to dissociate obstacles (such as RNA polymerase) during translocation along its RNA track. Different structures of the Rho-RNA complex have been published and suggest contradictory mechanisms of translocation. In order to understand these mechanisms, we have used two complementary approaches to identify functionality molecular comports in RNA and Rho : the NAIM (Nucleotide Analog Interference Mapping) approach developed in the laboratory and site-directed mutagenesis. Our results exclude that Rho forms a functional "trimer of dimer" ring (which rules out related translocation mechanisms) but are compatible with various aspects encountered in a recent asymmetric ring structure. However, our results do not support the "nucleotide by nucleotide" escort mechanism inferred from this asymmetric structure. Indeed, we show that Rho forms heterogonous contacts with the RNA chain and only requires a 2'-OH every seven nucleotides on average. Furthermore, we explored the interactome of E. coli in order to identify potential regulators of Rho function. We show that the hexameric protein Hfq displays topological similarity with the endogenous proteins NusG and YaeO and, that, like them, Hfq associates with Rho to regulate Rho function. The Hfq:Rho interaction inhibits the enzymatic activities of Rho. These results reveal a novel mechanism of transcription anti-termination with potentially important implications in bacterial metabolism and/or virulence of pathogens.ORLEANS-SCD-Bib. electronique (452349901) / SudocSudocFranceF

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

International audienceTranscription 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

RNA remodeling by hexameric RNA helicases

International audienceThe 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.8, 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

Analysis of helicase-RNA interactions using nucleotide analog interference mapping.

International audienceNucleotide 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

Rm62, a DEAD box RNA helicase, complexes with DSP1 in Drosophila embryos

International audienceTwo main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB-like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD-box RNA helicase, Rm62, as partner of DSP1 in a 250-kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3- to 12-h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes

Mutagenesis-Based Evidence for an Asymmetric Configuration of the Ring-Shaped Transcription Termination Factor Rho

International audienceTranscription termination factor Rho is an ATP-dependent ring-shaped molecular motor that tracks along RNA to dissociate RNA DNA duplexes and transcription complexes in its path. The Rho hexamer contains two distinct sites for interaction with RNA. The primary binding site is composed of pyrimidine-specific binding clefts that are located in the N-terminal domains and anchor Rho to transcripts at C-rich Rut (Rho utilization) sites. Components of the secondary binding site (SBS) in the C-terminal domains directly couple RNA binding to ATP hydrolysis in order to translocate RNA through the Rho ring. Published crystal structures of RNA-bound Rho display distinct architectures ('trimer-of-dimers' or asymmetric hexamer) and SBS RNA interaction networks that suggested conflicting models of RNA "handoff" or "escort" by the Rho subunits. To probe the mechanism of mechanochemical transduction in Rho, we have mutated into alanines (or glycines) the residues that make SBS contacts with RNA in the 'trimer-of-dimers' structure supporting the "handoff" model. We find that the resulting single-point mutants have similar RNA binding affinities but exhibit significantly different ATP hydrolysis, transcription termination, and RNA DNA unwinding activities that are more compatible with the asymmetric Rho structure than with the 'trimer-of-dimers' structure and the resulting "handoff" model. We discuss our findings in connection with specific features of the asymmetric Rho structure yet argue that a simple RNA "escort" model is insufficient to account for all experimental evidence. (C) 2010 Elsevier Ltd. All rights reserved