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-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.
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.
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.
Purpose : To asses the role of large b-value diffusion weighted imaging (DWI) in the characterization of the physicochemical properties ot the water in the brain edema under expeirmental and clinical conditions. Materials and Methods : Vasogenic brain edema was induced in mice by means of cold injury. A total of 17 patients wtih extensive peritumoral brain edema were also investigated. The longitudinal relaxation time(T-1) and apparent diffusion coefficient (D) were measured in the edematous area both in humans and in mice. D was calculated by using both mono- (D-mono) and biexponential (D-fast an D-slow) approaches in the low and overall range of b-values, respectively. The D values were correlated with the T-1 values. Results : A strong linear correlation was found between T-1 and D-mono in vasogenic brain edema, both in humans and in mice. After breakdown of D-mono into fast and slow diffusing components, only Dfast exhibited a strong correlation with T-1 : D-slow was unchanged in vasogenic brain edema. Conclusion : Large b-value DWI can furnish a detailed characterization of vasogenic brain edema, and may provide a quantitative approach for the differentiation of edema types on the basis of the physicochemical properties of the water molecules. Application of the DWI method may permit prediction and follow-up of the effects of antiedematous therapy.
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 Mg(2+) effectors are replaced by the more "relaxing" Mn(2+) ions, leading to transcript elongation beyond the termination point.
We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross-linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans-[PtCl2(NH3)2] (trans-DDP) by dimethylamine and isopropylamine. The data show that trans-[PtCl2(NH(CH)2)(NHCH(CH3)2)] is able to circumvent resistance to cis-[PtCl2(NH3)2] (cis-DDP, cisplatin) in A2780cisR cells. In fact, trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] shows a cytotoxic potency higher than that of cis-DDP and trans-DDP, with the mean IC50 values being 11, 58, and 300 microM, respectively. In addition, at equitoxic doses (concentrations of the platinum drugs equal to their IC50 values) and after 24 hours of drug treatment, the level of induction of apoptosis by trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is twice that produced by cis-DDP.
Ingénieur d’études , Biologie de l’ARN et ARN thérapeutiques