Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II, and III

Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved am ong eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all imp air a two-hybrid interaction with a fragment conserved in the largest subun its of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fra gment corresponds to the pore I module of the RNA polymerase II crystal str ucture and bears a highly conserved motif (P.I.KP..LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP..LW.GKQ provokes a temperature-sensitive defect. Increasi ng the gene dosage of another common subunit, Rpb6p, suppresses this phenot ype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally intera ct in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, no t included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still compl ements an rpb6-Delta null allele. A two-hybrid interaction also occurs betw een Rpb8p and the product of orphan gene YGR089w. A ygr089-Delta null mutan t has no detectable growth defect but aggravates the conditional growth def ect of rpb8 mutants, suggesting that the interaction with Rpb8p may be phys iologically relevant.