Directeur de recherche, co-responsable du groupe thématique
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.
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.
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.
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-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.
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.
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 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.
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.
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.
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.
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.
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
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.
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.
To trigger transcription termination, the ring-shaped RNA-DNA helicase Rho from Escherichia coli chases the RNA polymerase along the nascent transcript, starting from a single-stranded C-rich Rut (Rho utilization) loading site. In some instances, a small hairpin structure divides harmlessly the C-rich loading region into two smaller Rut subsites, best exemplified by the tR1 terminator from phage lambda. Here, we show that the Rho helicase can also elude a RNA structural block located far downstream from the single-stranded C-rich region but upstream from a reporter RNA-DNA hybrid. In this process, Rho hexamers do not melt the intervening RNA motif but require single-stranded RNA segments on both of its sides.
Typical hexameric helicases form ring-shaped structures involved in DNA replication. These enzymes have been proposed to melt forked DNA substrates by binding to, and pulling, one strand within their central channel, while the other strand is forced outside of the hexamer by steric exclusion and specific contacts with the outer ring surface. Transcription termination factor Rho also assembles into ring-shaped hexamers that are capable to use NTP-derived energy to unwind RNA and RNA-DNA helices. To delineate the potential relationship between helicase structural organization and unwinding mechanism, we have performed in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. We show that a physical discontinuity (nick) inhibits RNA-DNA unwinding when present in the RNA but not in the DNA strand.
Transcription termination factor Rho forms ring-shaped hexameric structures that load onto segments of the nascent RNA transcript that are C-rich and mostly single-stranded. This interaction converts Rho hexamers into active molecular motors that use the energy resulting from their ATP hydrolase activity to move towards the transcript 3’-end. Upon translocation along the RNA chain, Rho can displace physical roadblocks, such as those formed by RNA-DNA helices, a feature that is likely central to the transcription termination mechanism. To study this translocase (helicase) activity, we have designed a collection of Rho substrate chimeras containing an RNA-DNA helix located at various positions with respect to a short (47 nucleotides) artificial loading site.
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.
Transcription terminators trigger the dissociation of RNA polymerase elongation complexes and the release of RNA products at specific DNA template positions. The mechanism by which these signals alter the catalytic properties of the highly processive elongation transcription complexes is unclear. Here, we propose that intrinsic terminators impede transcript elongation by promoting a misarrangement of reactants and catalytic effectors within the active site of T7 RNA polymerase. In effect, a productive catalytic coordination network can be readily restored when Mg2+ effectors are replaced by the more "relaxing" Mn2+ ions, leading to transcript elongation beyond the termination point.
The oligonucleotides 5’-d(TTTTCTTTTG) and 5’-d(AAAAGAAAAG) were cross-linked with a trans-[Pt(NH3)(2)](2+) entity via the N7 positions of the 3’-end guanine bases to give parallel-stranded (ps) DNA. At pH 4.2, CD and NMR spectroscopy indicate the presence of Hoogsteen base pairing. In addition, temperature-dependent UV spectroscopy shows an increase in melting temperature for the platinated duplex (35 degreesC) as compared to the non-platinated, antiparallel-stranded duplex formed from the same oligonucleotides (21 degreesC). A monomer-dimer equilibrium for the platinated 20mer is revealed by gel electrophoresis. At pH 4.2, addition of a third strand of composition 5’-d(AGCTTTTCTTTTAG) to the ps duplex leads to the formation of a triple helix with two distinct melting points at 38 degreesC (platinum crosslinked Hoogsteen part) and 21 degreesC (Watson-Crick part), respectively.
Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria(1,2). Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron(3). Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda’) links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5’ splice site. Highly conserved elements similar to components of the lambda-lambda’ interaction are found in the eukaryotic spliceosome.
The DNA duplex d(CTCTCG*AGTCTC)d(GAGACTC*GAGAG) containing a single trans-diammine-dichloroplatinum(ll) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR, All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5’/H5 ") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined.
Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity, By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2’-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron, The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5).
In the reaction between trans-diamminedichloroplatinum(II) and single-stranded oligo(2’-O-methyl ribonucleotide)s containing the sequence GNG (N being a nucleotide residue), the 1,3-trans-(Pt (NH3)(2)[GNG]} cross-links are formed. The 1,3-intrastrand cross-links are inert within the single-stranded oligonucleotides. By contrast, they rearrange into interstrand cross-links when the platinated oligonucleotides are paired with their complementary RNA strands. The rate of the interstrand cross-linking reaction depends upon the sequence facing the intrastrand cross-links.
In the context of developing an approach to irreversibly and specifically link oligonucleotides to RNA, the purpose of this work was to determine the factors interfering with the rate of the rearrangement of the transplatin 1,3-intrastrand crosslinks into interstrand crosslinks, rearrangement triggered by the formation of a double helix between platinated oligo-2’-O-methyl-ribonucleotides and their complementary strands, The rate of the rearrangement has been studied as a function of the length of the hybrids, the location of the intrastrand crosslinks, the nature of the oligonucleotide backbone, and the nature of the doublet replacing the triplet complementary to the intrastrand crosslinks, The thermal stability of the platinated hybrids has been determined in various salt conditions, The results are discussed in relation to the mechanism of the rearrangement, It is shown that the cellular proteins present weaker nonspecific interactions with single-stranded platinated oligo-2’-O-methyl-nucleotides than with the isosequential oligodeoxyribonucleotides.
A trans-diamminedichloroplatinum(II) (trans-DDP) intrastrand adduct within the sequence d(TCTG*TG*TC).d(GACACAGA) (where G* represents a platinated guanine) is modeled on the basis of qualitative experimental data concerning global unwinding and curvature as well as information on base pairing. Modeling is performed using the internal coordinate JUMNA program, specific to nucleic acids, and modified to include the possibility of covalently bound ligands. Calibration of the energy functions representing the Pt-N7 bond with guanine is described. The platinum atom and the platinum-nitrogen bonds are parameterized for use in the Huckel Del Re method to calculate monopoles at each atom. These monopoles are consistent with the Flex force field included in Jumna. By developing an appropriate minimization protocol we are able to generate stable, distorted three-dimensional structures compatible with the experimental data and including an unusually high global unwinding. No a priori geometric assumptions are made in generating these structures.
The first step of the reaction between DNA and the antitumor drug cisplatin or its clinically inactive isomer transplatin yields monofunctional adducts, Most of the cisplatin monofunctional adducts further react and rather rapidly (t(1/2) smaller than a few hours) to form intrastrand and interstrand crosslinks. It is generally accepted that the clinical activity of cisplatin is related to the formation of bifunctional lesions, As concerns transplatin, several studies disagree on the rate of closure of the monofunctional adducts and the nature of the bifunctional lesions, In order to explain these discrepancies, we have prepared several duplexes containing a single monofunctional trans-[Pt(NH3)(2)(dG)Cl](+) adduct and zero to two monofunctional [Pt(dien)(dG)](2+) adducts at defined positions.
The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans(Pt(NH3)(2) [d(GXG)-GN7,GN7]) intrastrand cross-links, These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes, The thermal stability is not much affected by the chemical nature of the X residue and its complementary base, By gel electrophoresis, it is shown that the trans- Pt(NH3)(2)[d(GTG)-GN7,GN7] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees), The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links.