Nucleic Acids Research Advance Access published online on December 19, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl686
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Nucleic Acid Enzymes |
Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases
1 Service de Biochimie & Génétique Moléculaire. Bâtiment 144 CEA-Saclay, F-91191, Gif-sur-Yvette, CEDEX, France 2 Department Biología Experimental—Area de Genética (ED.B3) Universidad de Jaén Paraje las Lagunillas E-23071 Jaén, SPAIN
*To whom correspondence should be addressed. Tel: +33 1 69 08 35 86; Fax: +33 1 69 08 47 12; Email: pierre.thuriaux{at}cea.fr
Received May 3, 2006. Revised July 30, 2006. Accepted September 7, 2006.
Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-ß25/26 (prolonging the Bridge helix) and Rpb1-
44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9
or its RNA polymerase I rpa12 counterpart. Rpb1-ß25/26, Rpb1-44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5–Rpb1 binding and the Rpb9–Rpb1 interaction is to hold the Bridge helix, the Rpb1-44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.
*Correspondence may also be addressed to Francisco Navarro. Tel: 34 9 53 21 27 71; Fax: 34 9 53 21 18 75; Email: fngomez{at}ujaen.es