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Rahmouni Rachid


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tél : 02.38.25.76.08 - fax : 02.38.63.15.17


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Publications

2016   Références trouvées : 1

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. cerevisiae

RNA 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.

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.


2015   Références trouvées : 1

Rahmouni A. R., Mosrin-Huaman C.  (2015)

In Situ Footprinting of E. coli Transcription Elongation Complex with Chloroacetaldehyde

In "Bacterial Transcriptional Control" (2015) 1276 229-240 - doi : 10.1007/978-1-4939-2392-2_13
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

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


2014   Références trouvées : 1

Mosrin-Huaman, C., Hervouet-Coste, N., Le Dantec, A., Stuparevic, I., Rahmouni, A. R.  (2014)

Bacterial Rho helicase : a new tool to dissect mRNP biogenesis and quality control in yeast

Trends in Cell & Molecular Biology (2014) 9, 79-93 - doi : 10.1128/MCB.00272-09
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.

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.


2013   Références trouvées : 1

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 yeast

Journal 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.

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.


2011   Références trouvées : 2

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

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

Embo Journal 30 (14) 2805-2816
In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq : Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (lambda tR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity. The EMBO Journal ( 2011) 30, 2805-2816. doi:10.1038/emboj.2011.192 ; Published online 14 June 2011

In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq : Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (lambda tR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity. The EMBO Journal ( 2011) 30, 2805-2816. doi:10.1038/emboj.2011.192 ; Published online 14 June 2011

Honorine, R., Mosrin-Huaman, C., Hervouet, N., Libri, D. & Rahmouni, A.R.  (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.

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.


2010   Références trouvées : 2

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

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

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

Proshkin, S., Rahmouni, A.R., Mironov, A. & Nudler, E.  (2010)

Cooperation between Translating Ribosomes and RNA Polymerase in Transcription Elongation.

Science 328, 504-508.
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.

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.


2009   Références trouvées : 2

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

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

Nat. Struct. Mol. Biol. 16, 1309-1316.
The bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho-RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large ( approximately 7-nucleotide-long) discrete steps triggered by 2'-hydroxyl activation events. This periodic 2'-OH-dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.

The bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho-RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large ( approximately 7-nucleotide-long) discrete steps triggered by 2’-hydroxyl activation events. This periodic 2’-OH-dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.

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.

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.


2007   Références trouvées : 3

Schwartz, A ; Margeat, E ; Rahmouni, AR ; Boudvillain, M  (2007)

Transcription termination factor Rho can displace streptavidin from biotinylated RNA

Journal of Biological Chemistry 282 31469-31476
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.

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.

Nagarajavel, V ; Madhusudan, S ; Dole, S ; Rahmouni, AR ; Schnetz, K  (2007)

Repression by binding of H-NS within the transcription unit

Journal of Biological Chemistry 282 23622-23630
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.

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.

Schwartz, A ; Walmacq, C ; Rahmouni, AR ; Boudvillain, M  (2007)

Noncanonical interactions in the management of RNA structural blocks by the transcription termination Rho helicase

Biochemistry 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.

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.


2006   Références trouvées : 1

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 Rho

Biochemistry 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.

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.


2005   Références trouvées : 2

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 directions

Journal 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.

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.

Moulin, L ; Rahmouni, AR ; Boccard, F  (2005)

Topological insulators inhibit diffusion of transcription-induced positive supercoils in the chromosome of Escherichia coli

Molecular 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.

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.


2004   Références trouvées : 3

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 elongation

Molecular 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.

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.

Walmacq, C ; Rahmouni, AR ; Boudvillain, M  (2004)

Influence of substrate composition on the helicase activity of transcription termination factor Rho : Reduced processivity of unwinding of RNA-DNA Rho hexamers during hybrid regions

Journal 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.

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.

Vieu, E ; Rahmouni, AR  (2004)

Dual role of boxB RNA motif in the mechanisms of termination/antitermination at the lambda tR1 terminator revealed in vivo

Journal 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.

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.


2003   Références trouvées : 2

Schwartz, A ; Rahmouni, AR ; Boudvillain, M  (2003)

The functional anatomy of an intrinsic transcription terminator

Embo Journal 22 3385-3394
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.

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.

Epshtein, V ; Toulme, F ; Rahmouni, AR ; Borukhov, S ; Nudler, E  (2003)

Transcription through the roadblocks : the role of RNA polymerase cooperation

Embo Journal 22 (18) 4719-4727
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.

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.


2002   Références trouvées : 1

Boudvillain, M ; Schwartz, A ; Rahmouni, AR  (2002)

Limited topological alteration of the T7 RNA polymerase active center at intrinsic termination sites

Biochemistry 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.

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.


2000   Références trouvées : 1

Toulme, F ; Mosrin-Hauman, C ; Sparkowski, J ; Das, A ; Leng, M ; Rahmouni, AR  (2000)

GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming

Embo Journal 19 (24) 6853-6859
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.

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.


1999   Références trouvées : 1

Toulme, F ; Guerin, M ; Robichon, N ; Leng, M ; Rahmouni, AR  (1999)

In vivo evidence for back and forth oscillations of the transcription elongation complex

Embo Journal 18 (18) 5052-5060
We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock, We show that the complex readblocked within an (ATC/TAG)(n), sequence is in a dynamic equilibrium between upstream- and downstream-translocated conformers. By increasing the strength of the putative RNA-DNA hybrid, the ternary complex is readily trapped in the downstream-translocated conformation, where the melted DNA region is limited to 8 bp, The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5' end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis-acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation.

We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock, We show that the complex readblocked within an (ATC/TAG)(n), sequence is in a dynamic equilibrium between upstream- and downstream-translocated conformers. By increasing the strength of the putative RNA-DNA hybrid, the ternary complex is readily trapped in the downstream-translocated conformation, where the melted DNA region is limited to 8 bp, The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5’ end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis-acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation.


1998   Références trouvées : 2

Figueroa-Bossi, N ; Guerin, M ; Rahmouni, R ; Leng, M ; Bossi, L  (1998)

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control

Embo Journal 17 (8) 2359-2367
In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich, Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and +1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich, Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and +1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter’s sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

Guerin, M ; Robichon, N ; Geiselmann, J ; Rahmouni, AR  (1998)

A simple polypyrimidine repeat acts as an artificial Rho-dependent terminator in vivo and in vitro

Nucleic Acids Research 26 (21) 4895-4900
In this paper, we present evidence that an efficient Rho-dependent terminator can be created by introducing a simple (AG/TC)(n) DNA repeat into a transcription unit, The Rho termination activity in vivo and in vitro is dependent on the length and the orientation of the insert. The transcription of at least 30 bp of the (AG/TC)(n) repeat in the orientation encoding the (rUrC)(n) sequence on the transcript leads to Rho-dependent termination at a downstream non-terminator site, Our results indicate that the high efficiency of this artificial Rho-dependent terminator is due to optimal interactions between the (rUrC), RNA sequence and Rho protein, Thus, our findings strongly suggest that an adequate loading site is the primary determinant for Rho termination activity and provide a more defined system for future investigations.

In this paper, we present evidence that an efficient Rho-dependent terminator can be created by introducing a simple (AG/TC)(n) DNA repeat into a transcription unit, The Rho termination activity in vivo and in vitro is dependent on the length and the orientation of the insert. The transcription of at least 30 bp of the (AG/TC)(n) repeat in the orientation encoding the (rUrC)(n) sequence on the transcript leads to Rho-dependent termination at a downstream non-terminator site, Our results indicate that the high efficiency of this artificial Rho-dependent terminator is due to optimal interactions between the (rUrC), RNA sequence and Rho protein, Thus, our findings strongly suggest that an adequate loading site is the primary determinant for Rho termination activity and provide a more defined system for future investigations.


1997   Références trouvées : 1

Rahmouni, AR ; Guérin, M ; Leng, M  (1997)

Structure dynamique du complexe d’élongation de la transcription

Les Cahiers Imabio 19 65-69


1996   Références trouvées : 2

Albert, AC ; Spirito, F ; FigueroaBossi, N ; Bossi, L ; Rahmouni, AR  (1996)

Hyper-negative template DNA supercoiling during transcription of the tetracycline-resistance gene in topA mutants is largely constrained in vivo

Nucleic Acids Research 24 (15) 3093-3099
The excess linking deficit of plasmid DNA from topoisomerase I-defective bacteria (topA mutants) results mainly from transcription and is commonly ascribed to unbalanced relaxation of transcription-induced twin-supercoiled domains. This defect is aggravated in genes for membrane-binding proteins (such as the tet gene) where anchoring of the transcription complex to the bacterial membrane is thought to enhance twin-domain partitioning. Thus, it is often assumed that the 'hyper-negative' linking difference of plasmid DNA from topA mutants reflects unconstrained, hyper-negative DNA supercoiling inside the cell.

The excess linking deficit of plasmid DNA from topoisomerase I-defective bacteria (topA mutants) results mainly from transcription and is commonly ascribed to unbalanced relaxation of transcription-induced twin-supercoiled domains. This defect is aggravated in genes for membrane-binding proteins (such as the tet gene) where anchoring of the transcription complex to the bacterial membrane is thought to enhance twin-domain partitioning. Thus, it is often assumed that the ’hyper-negative’ linking difference of plasmid DNA from topA mutants reflects unconstrained, hyper-negative DNA supercoiling inside the cell.

Guerin, M ; Leng, M ; Rahmouni, AR  (1996)

High resolution mapping of E-coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand

Embo Journal 15 (19) 5397-5407
We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at

We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at


1995   Références trouvées : 1

Albert, AC ; Leng, M ; Rahmouni, AR  (1995)

The size of the topological domain modulates the b-z transition of a (tg)(n) containing repeat

Journal of Biomolecular Structure & Dynamics 13 (1) 47-56
Under negative superhelical stress,long(TG), containing repeats experience a stepwise multiple B-Z transitions. We have investigated the effect of the plasmid size on this transitional behavior. A 66-bp (TG)(n) containing repeat from the 5'-untranscribed region of mouse ribosomal DNA was inserted in a 3-kb, a 6.5-kb and a 12.5-kb plasmids and its supercoil-driven B-Z transition was followed by OsO4 probing of topoisomer-populations. Our results show a clear correlation between the size of the topological domain and the extent of the region that converts cooperatively into Z-DNA at the initial transition.

Under negative superhelical stress,long(TG), containing repeats experience a stepwise multiple B-Z transitions. We have investigated the effect of the plasmid size on this transitional behavior. A 66-bp (TG)(n) containing repeat from the 5’-untranscribed region of mouse ribosomal DNA was inserted in a 3-kb, a 6.5-kb and a 12.5-kb plasmids and its supercoil-driven B-Z transition was followed by OsO4 probing of topoisomer-populations. Our results show a clear correlation between the size of the topological domain and the extent of the region that converts cooperatively into Z-DNA at the initial transition.


1994   Références trouvées : 2

Tartier, L ; Michalik, V ; Spotheim-Maurizot, M ; Rahmouni, AR ; Sabattier, R ; Charlier, M  (1994)

Radiolytic signature of z-DNA

Nucleic Acids Research 22 (25) 5565-5570
Ionizing radiations induce various damages in DNA via the hydroxyl radical OH. generated by the radiolysis of water. We compare here the radiosensitivity of BE and Z-DNA, by using a Z-prone stretch included in a plasmid. In the supercoiled plasmid, the stretch is in the Z-form, whereas it is in the B-form when the plasmid is relaxed. Frank strand breaks (FSB) and alkali-revealed breaks (ARE) were located and quantified using sequencing gel electrophoresis. We show that B- and Z-DNA have the same mean sensitivity towards radiolytic attack, for both FSB and ARE. Nevertheless, the guanine sites are more sensitive, and the cytosine sites less sensitive in Z- than in B-DNA, leading to a characteristic signature of the Z-form. The comparison of experiments with the outcome of a Monte Carlo simulation of OH. radical attack suggests that transfer of initial damage from a guanine base to its attached sugar or the adjacent 3' cytosine is more important in Z-DNA than in B-DNA.

Ionizing radiations induce various damages in DNA via the hydroxyl radical OH. generated by the radiolysis of water. We compare here the radiosensitivity of BE and Z-DNA, by using a Z-prone stretch included in a plasmid. In the supercoiled plasmid, the stretch is in the Z-form, whereas it is in the B-form when the plasmid is relaxed. Frank strand breaks (FSB) and alkali-revealed breaks (ARE) were located and quantified using sequencing gel electrophoresis. We show that B- and Z-DNA have the same mean sensitivity towards radiolytic attack, for both FSB and ARE. Nevertheless, the guanine sites are more sensitive, and the cytosine sites less sensitive in Z- than in B-DNA, leading to a characteristic signature of the Z-form. The comparison of experiments with the outcome of a Monte Carlo simulation of OH. radical attack suggests that transfer of initial damage from a guanine base to its attached sugar or the adjacent 3’ cytosine is more important in Z-DNA than in B-DNA.

Albert, AC ; Roman, AM ; Bouche, G ; Leng, M ; Rahmouni, AR  (1994)

Gradual and oriented b-z transition in 5’-untranscribed region of mouse ribosomal DNA

Journal of Biological Chemistry 269 (30) 19238-19244
The structural transition of an alternating purine-pyrimidine sequence ((CG)(5)(TG)(28)) from the 5'-untranscribed region of the mouse ribosomal DNA was analyzed by two-dimensional gel electrophoresis and chemical probes. The repeat undergoes a supercoil dependent gradual and oriented B-Z transition. At a threshold level of negative supercoiling, a limited region of the repeat encompassing the (CG)(5) motif converts cooperatively into Z-DNA. As the superhelical stress increases, the Z-structure propagates along the remaining part of the repeat by successive transitions until the full-length sequence is converted. By in situ OsO4 probing experiments, we show also that this (TG)(n)-containing repeat adopts the Z-structure in Escherichia coli.

The structural transition of an alternating purine-pyrimidine sequence ((CG)(5)(TG)(28)) from the 5’-untranscribed region of the mouse ribosomal DNA was analyzed by two-dimensional gel electrophoresis and chemical probes. The repeat undergoes a supercoil dependent gradual and oriented B-Z transition. At a threshold level of negative supercoiling, a limited region of the repeat encompassing the (CG)(5) motif converts cooperatively into Z-DNA. As the superhelical stress increases, the Z-structure propagates along the remaining part of the repeat by successive transitions until the full-length sequence is converted. By in situ OsO4 probing experiments, we show also that this (TG)(n)-containing repeat adopts the Z-structure in Escherichia coli.


1992   Références trouvées : 2

Rahmouni, AR  (1992)

Z-DNA as a probe for localized supercoiling invivo

Molecular Microbiology 6 (5) 569-572
Biological processes such as transcription are expected to generate local variations in DNA supercoiling. The existence of localized supercoiling was recently demonstrated in Escherichia coli by using the supercoil-driven B-to-Z transition as a superhelicity probe. This new methodology is described and its extension to other biological systems discussed.

Biological processes such as transcription are expected to generate local variations in DNA supercoiling. The existence of localized supercoiling was recently demonstrated in Escherichia coli by using the supercoil-driven B-to-Z transition as a superhelicity probe. This new methodology is described and its extension to other biological systems discussed.

Rahmouni, AR ; Wells, RD  (1992)

Direct evidence for the effect of transcription on local DNA supercoiling invivo

Journal of Molecular Biology 223 (1) 131-144


1991   Références trouvées : 1

Lemaire, M.-A., Schwartz, A., Rahmouni, A.R. and Leng, M.  (1991)

Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloro-platinum(II) and DNA.

Proc. Natl. Acad. Sci. USA 88, 1982-1985


1990   Références trouvées : 4

Wells, RD ; Amirhaeri, S ; Blaho, JA ; Collier, DA ; Hanvey, JC ; Hsieh, WT ; Jaworski, A ; Larson, JE ; Rahmouni, AR ; Rajagopalan, M ; Shimizu, M ; Wohlrab, F ; Zacharias, W  (1990)

Z-DNA and Triplexes

Molecular Mechanisms in Dna Replication and Recombination 79-91 - Editor(s) : Richardson, CC ; Lehman, IR (Wiley-Liss, New York, NY)

Wells, RD ; Amirhaeri, S ; Blaho, JA ; Collier, DA ; Hanvey, JC ; Jaworski, A ; Larson, JE ; Rahmouni, AR ; Rajagopalan, M ; Shimizu, M ; Wohlrab, F ; Zacharias, W   (1990)

Left-Handed Z-DNA In Vivo

Structure and Methods Ii 25-31 - Editor(s) : Sarma, RH ; Sarma, MH (Adenine Press)

Rajagopalan, M ; Rahmouni, AR ; Wells, RD  (1990)

Flanking at-rich tracts cause a structural distortion in z-DNA in plasmids

Journal of Biological Chemistry 265 (28) 17294-17299

Wells, RD ; Amirhaeri, S ; Blaho, JA ; Collier, DA ; Dohrman, A ; Griffin, JA ; Hanvey, JC ; Hsieh, WT ; Jaworski, A ; Larson, JE ; McLean, MJ ; Rahmouni, AR ; Rajagopalan, M ; Shimizu, M ; Wohlrab, F ; Zacharias, W  (1990)

Biology and Chemistry of Z-DNA and Triplexes

The Bacterial Chromosome 187-194 - Editor(s) : Drlica, K ; Riley, M (American Society for Microbiology, Washington DC)


1989   Références trouvées : 4

Mazeau, K ; Vovelle, F ; Ptak, M ; Rahmouni, A ; Leng, M  (1989)

Structure of the Intrastrand cis-Pt d(GpCpG) adduct in a Dodecanucleotide Duplex : II. A Molecular Mechanics Modeling Study

Anti-Cancer Drug Design 4 (1) 63-78

Ptak, M ; Rahmouni, A ; Mazeau, K ; Thuong, NT ; Leng, M  (1989)

Structure of the Intrastrand cis-Pt d(GpCpG) adduct in a Dodecanucleotide Duplex : I. A 1H and 31P NMR Study

Anti-Cancer Drug Design 4 (1) 53-61

Rahmouni, AR ; Wells, RD  (1989)

Stabilization of z DNA invivo by localized supercoiling

Science 246 (4928) 358-363

Nandi, R ; Vogt, N ; Rahmouni, A ; Malfoy, B ; Leng, M  (1989)

Antibodies to unusual DNA structures

Journal of The Indian Institute of Science 69 3-11


1988   Références trouvées : 1

Rahmouni, A ; Schwartz, A ; Leng, M  (1988)

Importance of DNA Sequence in the Reaction of d(ApG) and d(GpA) with cis-diamminedichloroplatinum (II).

Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy, Martinus Nighoff-Publishing, Boston, MA 127-131


1987   Références trouvées : 1

Rahmouni, A ; Leng, M  (1987)

Reaction of Nucleic Acids with cis-diamminedichloroplatinum (II) : Interstrand Cross-Links

Biochemistry 26 (23) 7229-7234


1985   Références trouvées : 1

Rahmouni, A ; Malinge, JM ; Schwartz, A ; Leng, M  (1985)

Comparison Between poly(dG-dC).poly(dG-dC) and DNA modified by cis-diamminedichloroplatinium (II) : Immunological and Spectroscopic Studies

Journal of Biomolecular Structure & Dynamics 3 (2) 363-375


Mots-clés

Responsable de groupe thématique , Directeur de recherche , Biologie de l’ARN et ARN thérapeutiques