title: Crucial role of a dicarboxylic motif in the catalytic center of yeast RNA polymerases.
abstract: The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic motif reveals its strictly essential character in RNA polymerase III, with a slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other substitutions completely prevent growth, whereas rpb2-E836A combines a dominant growth defect with severe lethal sectoring. A mild but systematic increase in RNA polymerase occupancy and a strict dependency on the transcript cleavage factor TFIIS (Dst1) also suggest a slower rate of translocation or higher probability of transcriptional stalling in this mutation. A conserved nucleotide triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found among the RNA-dependent eukaryotic RNA polymerases and may thus represent a specific adaptation to double-stranded DNA templates.
authors.affiliation: Service de Biochimie et Génétique Moléculaire, CEA-Saclay, Bâtiment 144, 91191 Gif-sur-Yvette Cedex, France.
authors.forename: Gwenaël
authors.initials: G
authors.lastname: Ruprich-Robert
authors.forename: Maxime
authors.initials: M
authors.lastname: Wery
authors.forename: Daphné
authors.initials: D
authors.lastname: Després
authors.forename: Yves
authors.initials: Y
authors.lastname: Boulard
authors.forename: Pierre
authors.initials: P
authors.lastname: Thuriaux
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D020134:Catalytic Domain; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012320:RNA Polymerase III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C068394:DST1 protein, S cerevisiae; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II; D012324:RNA-Dependent RNA Polymerase; D012320:RNA Polymerase III