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Institutes/ Institute of Biology and Technology Saclay (iBiTec-S)/ Units/ Integrative Biology and Molecular Genetics (SBiGeM)/ Gene expression regulation and epigenetics laboratory (LREGE)/ Mechanics and regulation of eukaryotic RNA polymerases (C. Carles)

Mechanics and regulation of eukaryotic RNA polymerases

CEA
Christophe CARLES & Michel RIVA
CEA Saclay/Bât. 144

Tél: 33 1 69 08 84 17
E-mail: christophe.carles@cea.fr/michel.riva@cea.fr


iBiTec-S / SBIGeM / LREGE


Human resources
Christophe CARLES, Group Leader
Michel RIVA, Group Leader
Cécile DUCROT, Research Engineer
Gwenaëlle LE ROUX, Research Technician


Research Programs

Since its creation, our group has been interested in structure-function relationships of RNA polymerase from the yeast Saccharomyces cerevisiae. The approaches we use reflect our expertise in the fields of protein purification and characterization. We have made a significant contribution to the characterization of the genes coding the Pol I and Pol III subunits (Carles et Riva, 1996) and to the determination of the role of different subunits in transcriptional Pol I and Pol III mechanisms via a combination of molecular biology, reverse genetics and protein biochemistry (Lanzendörfer et al, 1997 ; Chédin et al, 1998 ; Peyroche et al, 2000 ; Landrieux et al, 2006). These studies, which are a continuation of the “historical “ interests of the group, are done in collaboration with the group of Terence Stick (Institut Jacques Monot Paris VII) on isolated molecules.

In parallel, the group has recently become interested in RNA polymerase regulation, in particular Pol I. This enzyme synthesizes the precursors of the large ribosomal RNAs and its activity represents more than 60% of overall cellular transcriptional activity. Based on our work on the function of a specific Pol I subunit (Peyroche et al, 2000), we have engineered a mutant yeast strain in which Pol I transcription under stress conditions is positively deregulated (Laferté et al, 2006; Chédin et al, 2007). This tool has enabled us to show that cellular concentrations of mRNAs coding ribosomal proteins and 5S rRNA depend on 35S rRNA synthesis by Pol I. This result suggests that Pol I transcriptional activity is a determining element in controlling the rate of ribosome synthesis. Our current work focuses on understanding cellular mechanisms that measure the level of Pol I activity, the transmission of this information to Pol II and Pol III and how specific regulation of the genes coding ribosomal proteins and 5S RNA is achieved.  This work is also carried out on higher eukaryotes in collaboration with the groups of Nouria Hernandez (Lausanne) and Ingrid Grummt (DKFZ, Heidelberg).

Furthermore, our group is studying the tri-dimensional structure of Pol I and Pol III RNAs and their transcriptional complexes. We have established collaborations with experienced groups in the field (P. Schultz, IGBMC-Strasbourg and Christoph Müller, EMBL-Heidelberg). Unlike Pol II RNA for which an atomic-level crystallographic structure is known, the other two enzyme forms have not yet been crystallized. The tri-dimensional low resolution envelopes of Pol I and Pol III have been determined by electron microscopy by the Strasburg and Heidelberg groups, respectively (Bischler et al, 2002 ; Fernandez-Tornero et al, 2007).  These studies confirm a significant structural conservation between the two enzymes. Our current objective is to crystallize these enzyme forms and their sub-complexes as well as to study the structure of the Pol I pre-initiation complex.

 Tri-dimensional envelope of yeast RNA Pol III obtained by electron-microscopy: The ADN path (blue) and the RNA path (red) are indicated.



Key words
Transcription, gene expression, gene expression regulation, multiple protein complexes, ribosome biosynthesis


Publications
Beckouët F, Mariotte-Labarre S, Peyroche G, Nogi Y, Thuriaux P. (2011). Rpa43 and its partners in the yeast RNA polymerase I transcription complex. FEBS Lett, 585, 3355-3359.

Dieci G, Ruotolo R, Braglia P, Carles C, Carpentieri A, Amoresano A, Ottonello S.  (2009). Positive modulation of RNA polymerase III transcription by ribosomal proteins. Biochem Biophys Res Commun. 379, 489-493.

Tavenet A, Suleau A, Dubreuil G, Ferrari R, Ducrot C, Michaut M, Aude J C, Dieci G, Lefebvre O, Conesa C, Acker J.  (2009). Genome-wide location analysis reveals a role for Sub1 in RNA polymerase III transcription. Proc Natl Acad Sci U S A. 106, 14265-14270.

Alic N, Ayoub N, Landrieux E, Favry F, Baudouin-Cornu P, Riva M,  Carles C. (2007). Selectivity and   proofreading both contribute significantly to the fidelity of RNA polymerase III transcription. Proc Natl Acad Sci USA. 104, 10400-10405

Chédin S, Laferté A, Hoang T, Lafontaine D, Riva M, Carles C.  (2007). Is Ribosome Synthesis Controlled by Pol I Transcription? Cell Cycle. 6, 11-15.

Fernandez-Tornero C, Böttcher B, Riva M, Carles C, Steuerwald U, Ruigrok RWH, Sentenac A, Müller CW, Schoehn G.  (2007). Insights into Transcription Initiation and Termination from the Electron Microscopy Structure of Yeast RNA Polymerase III. Mol Cell. 25, 813-823.

Oliva-Trastoy, M., Berthonaud, V., Chevalier, A., Ducrot, C., Marsolier-Kergoat, M.C., Mann, C., and Leteurtre, F. (2007). The Wip1 phosphatase (PPM1D) antagonizes activation of the Chk2 tumour suppressor kinase. Oncogene 26, 1449-1458.

Ducrot, C., Lefebvre, O., Landrieux, E., Guirouilh-Barbat, J., Sentenac, A., and Acker, J. (2006). Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors. J Biol Chem  281, 11685-11692.

Laferté A, Favry E, Sentenac A, Riva M, Carles C, Chédin S.  (2006). The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes and Dev. 20, 2030-2040.

Landrieux E, Alic N, Ducrot C, Acker J, Riva M, Carles C.  (2006). A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation. EMBO J. 25, 118-128.

Soutourina, J., Bordas-Le Floch, V., Gendrel, G., Flores, A., Ducrot, C., Dumay-Odelot, H., Soularue, P., Navarro, F., Cairns, B.R., Lefebvre, O., and Werner, M. (2006). Rsc4 connects the chromatin remodeler RSC to RNA polymerases. Mol Cell Biol  26,  4920-4933.

Bouchoux C, Hautbergue G, Grenetier S, Carles C, Riva M, Goguel V.  (2004). CTD kinase I is involved in RNA polymerase I transcription. Nucleic Acids Res. 32, 5851-5860.

De Carlo S, Carles C, Riva M, Schultz P.  (2003). Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I. J Mol Biol. 329, 891-902.

Bischler N, Brino L, Carles C, Riva M, Tschochner H, Mallouh V, Schultz P.  (2002).  Localization of the yeast RNA polymerase I-specific subunits. EMBO J. 21,  4136-4144.

Peyroche G, Levillain E, Siaut M, Callebaut I, Schultz P, Sentenac A, Riva M, Carles C.  (2002). The A14-A43 heterodimer subunit in yeast RNA pol I and their relationship to Rpb4-Rpb7 pol II subunits. Proc Natl Acad Sci USA. 99, 14670-14675.

Fath S, Milkereit P, Peyroche G, Riva M, Carles C, Tschochner H.  (2001). Differential roles of phosphorylation in the formation of transcriptional active RNA polymerase I. Proc Natl Acad Sci USA. 98,  14334-14339.

Ferri ML, Peyroche G, Siaut M, Lefèbvre O, Carles C, Conesa C, Sentenac A.  (2000). A novel subunit of yeast RNA polymerase III interacts with the TFIIB-related domain of TFIIIB70. Mol Cell Biol. 20, 488-495.

Peyroche G, Milkereit P, Bischler N, Tschochner H, Schultz P, Sentenac A, Carles C, Riva M.  (2000). The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3. EMBO J. 19, 5473-5482.

Flores A, Briand JF, Gadal O, Andrau JC, Rubbi L, Van Mullem V, Boschiero C, Goussot M, Marck C, Carles C, Thuriaux P, Sentenac A, Werner M.  (1999). A protein-protein interaction map of yeast RNA polymerase III. Proc Natl Acad Sci USA. 96, 7815-7820.

Voutsina A, Riva M, Carles C, Alexandraki D.  (1999). Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae Nucleic Acids Res. 27, 1047-1055.

Carles C, Riva, M. (1998). Yeast RNA polymerase I subunits and genes, in Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I, M. R. Paule (ed.) R.G. Landes &Co., Austin, 9-38.

Arrebola R, Manaud N, Rozenfeld S, Marsolier MC, Lefèbvre O, Carles C, Thuriaux P, Conesa C, Sentenac A.  (1998). tau 91, an essential subunit of yeast transcription factor IIIC, cooperates with tau 138 in DNA binding. Mol Cell Biol. 18, 1-9.

Chédin S, Riva M, Schultz P, Sentenac A, Carles C.  (1998). The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination. Genes and Dev. 12, 3857-3871.

Chédin S, Ferri ML, Peyroche G, Andrau JC, Jourdain S, Lefèbvre O, Werner M, Carles C, Sentenac A.  (1998). The yeast RNA polymerase III transcription machinery : a paradigm for eukaryotic gene activation. Cold Spring Harb Symp Quant Biol. 63, 381-389.

Manaud N, Arrebola R, Buffin-Meyer B, Lefèbvre O, Voss H, Riva M, Conesa C, Sentenac A.  (1998). A chimeric subunit of yeast transcription factor IIIC forms a subcomplex with tauc 95. Mol Cell Biol. 18, 3191-3200.

Boullais C, Riva M, Noël JP.  (1997). New chemo-enzymatic synthesis of very high specific radioactivity [35S] (S) methionine [1]. J Labelled Compd Rad. 39, 621-624.

Gadal O, Mariotte-Labarre S, Chédin S, Quémeneur E, Carles C, Sentenac A, Thuriaux P.  (1997). A34.5, a nonessential component of yeast RNA polymerase I, cooperates with subunit A14 and DNA topoisomerase I to produce a functional rRNA synthesis machine Mol Cell Biol. 17, 1787-1795.

Huet J, Conesa C, Carles C, Sentenac A.  (1997). A cryptic DNA binding domain at the COOH terminus of TFIIIB70 affects formation, stability and function of preinitiation complexes J Biol Chem. 272, 18341-18349.

Lanzendorfer M, Smid A, Klinger C, Schultz P, Sentenac A, Carles C, Riva M.  (1997). A shared subunit belongs to the eukaryotic core RNA polymerase. Genes and Dev. 11, 1037-1047.


Thomas M, Chédin S, Carles C, Riva M, Famulok M, Sentenac A.  (1997). Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers. J Biol Chem. 272, 27980-27986.