Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.
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-beta25/26 (prolonging the Bridge helix) and Rpb1-alpha44/47 (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 rpb9Delta or its RNA polymerase I rpa12Delta counterpart. Rpb1-beta25/26, Rpb1-alpha44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-alpha46/47 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-alpha44/47 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.
Zaros
Cécile
C
Service de Biochimie & Génétique Moléculaire, Bâtiment 144 CEA-Saclay, F-91191, Gif-sur-Yvette, CEDEX, France.
Briand
Jean-François
JF
Boulard
Yves
Y
Labarre-Mariotte
Sylvie
S
Garcia-Lopez
M Carmen
MC
Thuriaux
Pierre
P
Navarro
Francisco
F
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae; C509257:Rpb9 protein, S cerevisiae; D012321:DNA-Directed RNA Polymerases; C517511:POLR2E protein, human; C517510:RPB5 protein, S cerevisiae