Abstract
The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-832GYNQED837, Rpc128-764GYDIED769) 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 Rpb2-832GYNQED837 loop by an Rpb2-R1020/Rpb2-D837 salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K752/Rpb2-E836), which may account for the critical role of the invariant Rpb2-E836. Rpb2-E836 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.
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Acknowledgments
We thank Maria-Pilar Gomez for her help in the Rpb2 mutagenesis and Sylvie Mariotte, Marta Kwapisz, Marina Pinskaya, Elena Shematorova, Julie Soutourina and Michel Werner for useful discussions. This work was supported by the French Agence Nationale de la Recherche (BLAN08-3-309259) and by a fellowship from the Association pour la Recherche sur le Cancer to D.D.
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Communicated by K. Breunig.
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Ruprich-Robert, G., Wery, M., Després, D. et al. Crucial role of a dicarboxylic motif in the catalytic center of yeast RNA polymerases. Curr Genet 57, 327–334 (2011). https://doi.org/10.1007/s00294-011-0350-6
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DOI: https://doi.org/10.1007/s00294-011-0350-6