DESCRIPTIF DU TRAVAIL DE RECHERCHE
Nous voulons créer une dynamique forte entre recherche académique sur le métabolisme de l’ARN et des applications biotechnologiques en recherche translationnelle dans le domaine de l’ARN thérapeutique.
Biologie de l’ARN
Pour le volet recherche fondamentale, nous poursuivons nos études sur les mécanismes co-transcriptionnels de biogénèse et contrôle qualité (QC) des particules ribonucléoprotéiques (mRNPs) dans la levure et les cellules de mammifères. Ces travaux sont réalisés en utilisant notre approche basée sur la perturbation de la biogénèse des mRNPs par le facteur bactérien Rho. Par des analyses globales en transcriptomique (ChIP-Seq et RNA-Seq) et protéomique (pulldown des mRNPs et analyse des protéines par Mass Spec) nous cherchons à comprendre les spécificités vis à vis des mRNPs aberrantes de deux voies de QC et dégradation que nous avons découvertes précédemment. Ce travail devrait nous permettre de mettre en évidence les déterminants structuraux des mRNPs impliqués dans la reconnaissance par chaque voie de QC et dégradation. Un autre aspect consiste à caractériser les défauts induits par Rho sur les mRNPs pendant leur biogénèse et qui sont responsables du déclanchement du système de dégradation par le QC. Ceci devrait nous mener à comprendre les spécificités qui différencient les mRNA qui sont compactés en mRNP et exportés vers le cytoplasme pour la traduction et des RNA noncodants (antisens et intergéniques) qui sont très instables alors qu’ils possèdent les mêmes attributs que les mRNA (coiffe en 5’ et queue polyA en 3’). Avec ce travail, nous pensons faire des contributions décisives dans le domaine en pleine expansion des RNA noncodants comme acteurs de la régulation des gènes au niveau de la transcription, dégradation des mRNA et traduction. Enfin, nous voulons déterminer le rôle de Rna15 dans le processus de détection des mRNPs aberrantes par le QC. Rna15 fait partie d’un complexe très conservé (Rna14-Rna15-Clp1-Pcf11) impliqué dans le processus de terminaison et polyadénylation des transcrits et qui est recruté par le complexe transcriptionnel à la fin des gènes. Cependant, Il a été très récemment montré que Rna15 co-localise aussi avec le complexe Nrd1-Nab3 responsable d’une voie du QC pour une partie des transcrits. En parallèle, nous utilisons notre approche basée sur la perturbation de la biogénèse des mRNPs chez la levure et le phénotype de retard de croissance qu’elle produit pour mettre au point des cribles pour rechercher des antibiotiques qui ciblent spécifiquement Rho.
Ces dernières années, nous assistons à l’émergence de produits biopharmaceutiques à base d’ARN comme une nouvelle classe de traitement et de prophylaxie des maladies chroniques et rares, y compris le cancer. Les ARN thérapeutiques sont reconnus comme étant plus prometteurs que les ADN thérapeutiques. Il est maintenant bien admis que l’avenir des thérapies à base d’ARN dans le domaine clinique repose sur l’amélioration de leur production, leur stabilité et leur délivrance cellulaire.
Dans le projet "Biomédicaments" nous avons l’intention de mettre en place une nouvelle technologie pour produire dans la levure de grandes quantités d’ARNm pouvant servir comme vaccin de qualité. Nous cherchons aussi à améliorer l’activité biologique des ARNm thérapeutiques en menant des études sur le devenir intracellulaire de l’ARN exogène.
to study mRNP biogenesis and quality control (QC) in S. cerevisiae
- Mosrin-Huaman C., Hervouet-Coste N. and Rahmouni A. R.
Co-transcriptional degradation by the 5’-3’ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae. (2016) RNA Biol. 13, 582–592.
- Stuparevic I., Mosrin-Huaman C., Hervouet-Coste N., Remenaric M. and Rahmouni A. R.
Co-transcriptional Recruitment of RNA Exosome Cofactors Rrp47p and Mpp6p and Two Distinct TRAMP Complexes Assists the Exonuclease Rrp6p in the Targeting and Degradation of an Aberrant mRNP in Yeast (2013) J. Biol. Chem. 288, 31816-31829.
- Honorine R., Mosrin-Huaman C., Hervouet-Coste N., Libri D. and Rahmouni A. R.
Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment (2011) Nucleic Acids Res. 39, 2809-2820.
- Proshkin S., Rahmouni A. R., Mironov A. and Nudler E.
Cooperation between Translating Ribosomes and RNA Polymerase in Transcription Elongation. (2010) Science. 328, 504-508.
Nehme, R., Atieh, C., Fayad, S., Claude, B., Chartier, A., Tannoury, M., Elleuch, F., Abdelkafi, S., Pichon, C. and Morin, P. (2017)
Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser-induced fluorescence detectionJournal of Separation Science (2017) 40 (2) 558-566
Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80 degrees C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM ; pH 9.4) and -cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.
Mosrin-Huaman C., Hervouet-Coste N. and Rahmouni A. R. (2016)
Co-transcriptional degradation by the 5’-3’ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiaeRNA Biology (2016) 13 (6) 582-592 - doi : 10.1080/15476286.2016.1181255
The co-transcriptional biogenesis of export-competent messenger ribonucleoprotein particles (mRNPs) in yeast is under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from inappropriate or inefficient processing and packaging reactions are detected by the QC system and retained in the nucleus with ensuing elimination of their mRNA component by a mechanism that requires the catalytic activity of Rrp6p, a 3’-5’ exonuclease associated with the RNA exosome. In previous studies, we implemented a new experimental approach in which the production of aberrant mRNPs is massively increased upon perturbation of mRNP biogenesis by the RNA-dependent helicase/translocase activity of the bacterial Rho factor expressed in S. cerevisiae. The analyses of a subset of transcripts such as PMA1 led us to substantiate the essential role of Rrp6p in the nuclear mRNP QC and to reveal a functional coordination of the process by Nrd1p. Here, we extended those results by showing that, in contrast to PMA1, Rho-induced aberrant HXK1 mRNPs are targeted for destruction by an Nrd1p- and Rrp6p-independent alternative QC pathway that relies on the 5’-3’ exonuclease activity of Rat1p. We show that the degradation of aberrant HXK1 mRNPs by Rat1p occurs co-transcriptionally following decapping by Dcp2p and leads to premature transcription termination. We discuss the possibility that this alternative QC pathway might be linked to the well-known specific features of the HXK1 gene transcription such as its localization at the nuclear periphery and gene loop formation.
Rahmouni A. R., Mosrin-Huaman C. (2015)
The structure and dynamics of Escherichia coli transcription elongation complex are now well documented. However, most of the studies have been conducted in vitro and frequently under artificial conditions that facilitate the biochemical characterization of the complex. Thus, little is known about relevance of these results for the regulatory aspects of transcription elongation inside the cell. Here, we describe the use of a single-strand-specific probe chloroacetaldehyde for in situ footprinting of E. coli elongation complex temporarily halted by a protein roadblock. The method provides valuable information on the dynamic features of transcriptionally engaged RNA polymerase within the cellular environment
Mosrin-Huaman, C., Hervouet-Coste, N., Le Dantec, A., Stuparevic, I., Rahmouni, A. R. (2014)
In eukaryotic cells, the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from faulty events are detected by the QC apparatus and retained in the nucleus with ensuing elimination of their mRNA component by the RNA degradation machinery. A decade of biochemical and genetic experiments in yeast allowed the identification of the nuclear degradation machinery including the core exosome and its two associated catalytic subunits Rrp6p and Rrp44p, its cofactors Rrp47p and Mpp6p as well as the activator complex TRAMP. Similarly, studies of the THO-Sub2 complex of the mRNP assembly and export apparatus have provided valuable information on the nuclear retention and degradation of a particular class of aberrant mRNPs. However, a unifying mechanism of action underlying the QC process remains elusive. Here, we review the implementation of a new experimental approach whereby the production of aberrant mRNPs is massively increased upon heterologous expression of the bacterial Rho helicase in yeast. Using this methodology, we have shown that the QC process is coordinated by Nrd1p (a component of the early termination complex) whose increased co-transcriptional recruitment promotes the attachment of the 3’-5’ exonuclease Rrp6p along with the co-factors Rrp47p and Mpp6p. Interestingly, we established that Rrp6p functions independently from the core exosome, yet is stimulated by two forms of the TRAMP complex that include Trf4p or Trf5p and Air2p but not Air1p. The results suggest that specific substrates could be primed for decay via various QC pathways owing to the versatility of the mRNA degradation apparatus. In this context, the bacterial Rho helicase provides a valuable tool to decipher the QC molecular process in yeast and possibly the homologous process in mammalian cells.
Stuparevic, I., Mosrin-Huaman, C., Hervouet-Coste, N., Remenaric, M. and Rahmouni, A. R. (2013)
Co-transcriptional recruitment of the RNA exosome cofactors Rrp47p, Mpp6p and two distinct TRAMP complexes assists the exonuclease Rrp6p in the targeting and degradation of an aberrant mRNP in yeastJournal of Biological Chemistry 288 (44) 31816-31829 - doi : 10.1074/jbc.M113.491290
The co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent mRNPs are under the surveillance of quality control steps. Aberrant mRNPs resulting from faulty events are retained in the nucleus with ensuing elimination of their mRNA component. The molecular mechanisms by which the surveillance system recognizes defective mRNPs and stimulates their destruction by the RNA degradation machinery are still not completely elucidated. Using an experimental approach in which mRNP formation in yeast is disturbed by the action of the bacterial Rho helicase, we have shown previously that the targeting of Rho-induced aberrant mRNPs is mediated by Rrp6p which is recruited co-transcriptionally in association with Nrd1p following Rho action. Here, we investigated the specific involvement in this quality control process of different cofactors associated with the nuclear RNA degradation machinery. We show that, in addition to the main hydrolytic action of the exonuclease Rrp6p, the cofactors Rrp47p and Mpp6p as well as the TRAMP components Trf4p, Trf5p and Air2p contribute significantly by stimulating the degradation process upon their co-transcriptional recruitment. Trf4p and Trf5p are apparently recruited in two distinct TRAMP complexes that both contain Air2p as component. Surprisingly, Rrp47p appears to play an important role in mutual protein stabilization with Rrp6p which highlights a close association between the two partners. Together, our results provide an integrated view of how different cofactors of the RNA degradation machinery cooperate to target and eliminate aberrant mRNPs.
Brack, A. & Schwartz, A. (2011)
Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment,as revealed by the targeting of Rho-induced aberrant transcripts.Nucleic Acids Res. 39 (7) 2809-2820
The production of mature export-competent transcripts is under the surveillance of quality control steps where aberrant mRNP molecules resulting from inappropriate or inefficient processing and packaging reactions are subject to exosome-mediated degradation. Previously, we have shown that the heterologous expression of bacterial Rho factor in yeast interferes in normal mRNP biogenesis leading to the production of full-length yet aberrant transcripts that are degraded by the nuclear exosome with ensuing growth defect. Here, we took advantage of this new tool to investigate the molecular mechanisms by which an integrated system recognizes aberrancies at each step of mRNP biogenesis and targets the defective molecules for destruction. We show that the targeting and degradation of Rho-induced aberrant transcripts is associated with a large increase of Nrd1 recruitment to the transcription complex via its CID and RRM domains and a concomitant enrichment of exosome component Rrp6 association. The targeting and degradation of the aberrant transcripts is suppressed by the overproduction of Pcf11 or its isolated CID domain, through a competition with Nrd1 for recruitment by the transcription complex. Altogether, our results support a model in which a stimulation of Nrd1 co-transcriptional recruitment coordinates the recognition and removal of aberrant transcripts by promoting the attachment of the nuclear mRNA degradation machinery.
Proshkin, S., Rahmouni, A.R., Mironov, A. & Nudler, E. (2010)
During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that is engaged in translation of the newly synthesized transcript. Here we show that as a result of translation-transcription coupling the overall elongation rate of transcription is tightly controlled by translation. Acceleration and deceleration of a ribosome results in corresponding changes in the speed of RNAP. Consistently, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. We further show that the stimulating effect of a ribosome on RNAP is achieved by preventing RNAP from adopting non-productive states. The moving ribosome inhibits spontaneous backtracking of RNAP, thereby enhancing its pace and also facilitating read-through of roadblocks in vivo. Such a cooperative mechanism ensures the two gene expression machineries match precisely each other rates, so that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions.
Boudvillain, M., Walmacq, C., Schwartz, A. and Jacquinot, F. (2009)
Simple enzymatic assays for the in vitro motor activity of transcription termination factor Rho from Escherichia coliin "Helicases : Methods and Protocols", Ed. Abdelhaleem, M.M.M., Humana Press, vol. 587, chap.10, 137-154 Mosrin-Huaman, C., Honorine, R. & Rahmouni, A.R. (2009)
Expression of Bacterial Rho Factor in Yeast Identifies New Factors Involved in the Functional Interplay between Transcription and mRNP Biogenesis.Mol. Cell. Biol. 29, 4033-4044.
In eukaryotic cells, the nascent pre-mRNA molecule is coated sequentially with a large set of processing and binding proteins that mediate its transformation into an export-competent ribonucleoprotein particle (mRNP) that is ready for translation in the cytoplasm. We have implemented an original assay that monitors the dynamic interplay between transcription and mRNP biogenesis and that allows the screening for new factors linking mRNA synthesis to translation in Saccharomyces cerevisiae. The assay is based on the perturbation of gene expression induced by the bacterial Rho factor, an RNA-dependent helicase/translocase that acts as a competitor at one or several steps of mRNP biogenesis in yeast. We show that the expression of Rho in yeast leads to a dose-dependent growth defect that stems from its action on RNA polymerase II-mediated transcription. Rho expression induces the production of aberrant transcripts that are degraded by the nuclear exosome. A screen for dosage suppressors of the Rho-induced growth defect identified several genes that are involved in the different steps of mRNP biogenesis and export, as well as other genes with both known functions in transcription regulation and unknown functions. Our results provide evidence for an extensive cross talk between transcription, mRNP biogenesis, and export. They also uncover new factors that potentially are involved in these interconnected events.
Schwartz, A ; Margeat, E ; Rahmouni, AR ; Boudvillain, M (2007)
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.
Non-canonical interactions in the management of RNA structural blocks by the transcription termination Rho helicaseBiochemistry 46 9366-9379
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.
H-NS inhibits transcription by forming repressing nucleoprotein complexes next to promoters. We investigated repression by binding of H-NS within the transcription unit using the bgl and proU operons. Repression of both operons requires a downstream regulatory element (DRE) in addition to an upstream element (URE). In bgl, H-NS binds to a region located between 600 to 700 bp downstream of the transcription start site, whereas in proU the DRE extends up to position +270. We show that binding of H-NS to the bgl-DRE inhibits transcription initiation at a step before open complex formation, as shown before for proU. This was shown by determining the occupancy of the bgl transcription unit by RNA polymerases, expression analysis of bgl and proU reporter constructs, and chloroacetaldehyde footprinting of RNA polymerase promoter complexes.
Noncanonical interactions in the management of RNA structural blocks by the transcription termination Rho helicaseBiochemistry 46 (33) 9366-9379
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.
Spatial distribution and hormonal regulation of gene products from methyl erythritol phosphate and monoterpene-secoiridoid pathways in Catharanthus roseusPlant Molecular Biology 65 (1-2) 13-30
The monoterpene indole alkaloids (MIAs) from Madagascar periwinkle (Catharanthus roseus) are secondary metabolites of high interest due to their therapeutical values. Secologanin, the monoterpenoid moiety incorporated into MIAs, is derived from the plastidial methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have cloned a cDNA encoding hydroxymethylbutenyl diphosphate synthase (HDS), a MEP pathway enzyme, and generated antibodies to investigate the distribution of transcripts and protein in MIA-producing aerial tissues. Consistent with our earlier work, transcripts for the genes encoding the so-called early steps in monoterpenoid biosynthesis (ESMB) enzymes (HDS, others MEP pathway enzymes and geraniol 10-hydroxylase) were preferentially co-localized to internal phloem associated parenchyma (IPAP) cells.
Walmacq, C ; Rahmouni, AR ; Boudvillain, M (2006)
Testing the steric exclusion model for hexameric helicases : Substrate features that alter RNA-DNA unwinding by the transcription termination factor RhoBiochemistry 45 (18) 5885-5895
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.
Toulme, F ; Mosrin-Huaman, C ; Artsimovitch, I ; Rahmouni, AR (2005)
Transcriptional pausing in vivo : A nascent RNA hairpin restricts lateral movements of RNA polymerase in both forward and reverse directionsJournal of Molecular Biology 351 (1) 39-51
Transcriptional pausing by RNA polymerase has been the subject of extensive investigations in vitro, yet little is known about its occurrence and significance in vivo. The transient nature of the pausing events makes them difficult to observe inside the cell, whereas their studies in vitro by classical biochemical methods are usually conducted under non-physiological conditions that increase the pause duration. Here, we have used an Escherichia coli system in which several RNA polymerase molecules transcribing in tandem traverse a pausing sequence while approaching a protein roadblock. The in vivo DNA footprinting and RNA 3’ end mapping of the elongation complexes are consistent with a dynamic view of the pausing event, during which RNA polymerase first loses its lateral stability and slides backward, and is subsequently rescued from extended backtracking and stabilized at the pause site by a nascent RNA hairpin. Our results show also that the folding of the hairpin provides an assisting force that promotes forward translocation of a trailing polymerase under a strained configuration by balancing the opposing force created by a steric clash with a leading elongation complex. (c) 2005 Elsevier Ltd. All rights reserved.
Topological insulators inhibit diffusion of transcription-induced positive supercoils in the chromosome of Escherichia coliMolecular Microbiology 55 (2) 601-610
The double helical nature of DNA implies that progression of transcription machinery that cannot rotate easily around the DNA axis creates waves of positive supercoils ahead of it and negative supercoils behind it. Using topological reporters that detect local variations in DNA supercoiling, we have characterized the diffusion of transcription-induced (TI) positive supercoils in plasmids or in the chromosome of wild type Escherichia coli cells. Transcription-induced positive supercoils were able to diffuse and affect local supercoiling several kilobases away from the site of origin. By testing the effect of various DNA sequences, these reporters enabled us to identify elements that impede supercoil diffusion, i.e. behave as topological insulators. All the elements tested correspond to DNA gyrase catalytic targets. These results correlate the ability of a DNA sequence to be cleaved by DNA gyrase with topological insulator activity. Implications of the asymmetry in supercoil diffusion for the control of DNA topology are discussed.
Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alpha beta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.
Mosrin-Huaman, C ; Turnbough, CL ; Rahmouni, AR (2004)
Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongationMolecular Microbiology 51 (5) 1471-1481
Current models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre- and post-translocated states. Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor. Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e. low K-m). The addition of an extra nucleotide destabilizes the repressor-operator roadblock leading to an increase in transcriptional readthrough. Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e. high K(m)s) by specifically increasing their intracellular concentrations. Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding. They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level.
Influence of substrate composition on the helicase activity of transcription termination factor Rho : Reduced processivity of unwinding of RNA-DNA Rho hexamers during hybrid regionsJournal of Molecular Biology 342 (2) 403-420
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.
Dual role of boxB RNA motif in the mechanisms of termination/antitermination at the lambda tR1 terminator revealed in vivoJournal of Molecular Biology 339 (5) 1077-1087
Rho-dependent transcription termination at the phage lambda tR1 terminator is governed primarily by the upstream rut element that encodes two RNA regions rutA and rutB. The two regions are separated by the boxB RNA motif, which is believed to be dispensable for Rho activity but serves as a binding site for lambda N protein in the antitermination process. By using a minimal in vivo termination system, we show that the intervening boxB RNA motif has a double function in the mechanisms of termination/antitermination at lambdatR1. As a folded hairpin structure, it acts as a clamp that holds rutA and rutB side by side for optimal interactions with Rho leading to efficient termination. Conversely, the binding of N protein to boxB induces antitermination at lambdatR1 by preventing access of Rho to the rut sequences. This dual role was clearly shown in vivo by studying the effects of multiple mutations within the boxB hairpin stem on transcription termination and by substituting the N/boxB couple with the unrelated coat protein of phage MS2 and its stem-loop RNA binding site. (C) 2004 Elsevier Ltd. All rights reserved.
Schwartz, A ; Rahmouni, AR ; Boudvillain, M (2003)
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.
During transcription, cellular RNA polymerases (RNAP) have to deal with numerous potential roadblocks imposed by various DNA binding proteins. Many such proteins partially or completely interrupt a single round of RNA chain elongation in vitro. Here we demonstrate that Escherichia coli RNAP can effectively read through the site-specific DNA-binding proteins in vitro and in vivo if more than one RNAP molecule is allowed to initiate from the same promoter. The anti-roadblock activity of the trailing RNAP does not require transcript cleavage activity but relies on forward translocation of roadblocked complexes. These results support a cooperation model of transcription whereby RNAP molecules behave as ’partners’ helping one another to traverse intrinsic and extrinsic obstacles.
Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins : Thermodynamics and structureJournal of Molecular Biology 331 (1) 101-121
The Escherichia coli histone-like HU protein pool is composed of three dimeric forms : two homodimers, EcHUa(2) and EcHUß(2), and a heterodimer, EcHUaß. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles.
Schmidt, KS ; Boudvillain, M ; Schwartz, A ; Van Der Marel, GA ; Van Boom, JH ; Reedijk, J ; Lippert, B (2002)
Monofunctionally trans-diammine platinum(II)-modified peptide nucleic acid oligomers : a new generation of potential antisense drugsChemistry 8 (24) 5566-5570 Boudvillain, M ; Schwartz, A ; Rahmouni, R (2002)
Limited topological alteration of the T7 RNA polymerase active-center at intrinsic termination sitesBiochemistry 41 3137-3146
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.
Apoptosis induction and DNA interstrand cross-link formation by cytotoxic trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2) : cross-linking between d(G) and complementary d(C) within oligonucleotide duplexes.Chembiochem 3 (1) 61-67
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.
Limited topological alteration of the T7 RNA polymerase active center at intrinsic termination sitesBiochemistry 41 (9) 3137-3146
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.
Crystallization and preliminary X-ray crystallographic studies of a complex between the Lactococcus lactis Fpg DNA-repair enzyme and an abasic site containing DNAActa Crystallographica Section D-Biological Crystallography 58 679-682 Part 4
For protein-DNA complex crystallization, the choice of the DNA fragment is crucial. With the aim of crystallizing the 31 kDa Fpg DNA-repair enzyme bound to DNA, oligonucleotide duplexes varying in length, sequence, end type and nature of the specific DNA target site were used. Crystals of several protein-DNA combinations grew from solutions containing both polyethylene glycol and salt. This systematic crystallization screening followed by optimization of the crystallization conditions by microseeding led to crystals of Fpg bound to a 13 base-pair duplex DNA carrying the 1,3-propanediol abasic site analogue which are suitable for crystallographic analysis. Complete native data sets have been collected to 2.1 Angstrom resolution.
Serre, L ; Verdon, G ; Choinowski, T ; Hervouet, N ; Risler, JL ; Zelwer, C (2001)
Amino acid selection by aminoacyl-tRNA synthetases requires efficient mechanisms to avoid incorrect charging of the cognate tRNAs. A proofreading mechanism prevents Escherichia coli methionyl-tRNA synthetase (EcMet-RS) from activating in vivo L-homocysteine, a natural competitor of L-methionine recognised by the enzyme. The crystal structure of the complex between EcMet-RS and L-methionine solved at 1.8 Angstrom resolution exhibits some conspicuous differences with the recently published free enzyme structure. Thus, the methionine delta -sulphur atom replaces a water molecule H-bonded to Leu13N and Tyr260O(eta) in the free enzyme. Rearrangements of aromatic residues enable the protein to form a hydrophobic pocket around the ligand side-chain. The subsequent formation of an extended water molecule network contributes to relative displacements, up to 3 Angstrom, of several domains of the protein. The structure of this complex supports a plausible mechanism for the selection of L-methionine versus L-homocysteine and suggests the possibility of information transfer between the different functional domains of the enzyme. (C) 2001 Academic Press.
Toulme, F ; Mosrin-Hauman, C ; Sparkowski, J ; Das, A ; Leng, M ; Rahmouni, AR (2000)
The GreA and GreB proteins of Escherichia coil show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock, When RNAP is stalled within an (ATC/TAG), sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3’ terminus and its re-synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB-induced ’cleavage-and-restart’ process to become catalytically competent. Clearly, by promoting transcript shortening and re-alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.
Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses ren amongst others, Recently, several proteins have been shown to assist Dorsal in the repression of ten, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of ten but not in the activation of twist.
Parallel-stranded DNA with Hoogsteen base pairing stabilized by a trans-[Pt(NH3)(2)](2+) cross-link : characterization and conversion into a homodimer and a triplexJournal of Biological Inorganic Chemistry 5 (5) 603-611
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.