Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases

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Abstract
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-β25/26 (prolonging the Bridge helix) and Rpb1-α44/47 (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 rpb9Δ or its RNA polymerase I rpa12Δ counterpart. Rpb1-β25/26, Rpb1-α44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-α46/47 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-α44/47 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.