Isolation and characterization of temperature-sensitive mutations in the gene (rpb3) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the alph a subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both beta and beta' subunits. Previously we demonstrated that the Schizosaccharo myces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the b eta homologue) and Rpb11 (the second alpha homologue) subunits, as in the c ase of the prokaryotic alpha(2)beta complex. In order to obtain further ins ight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis, A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S, pombe mutants have been isolated, each (with the exc eption of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A-D) that are conserved between the homologues of eukaryotic subunit 3, The three Cs mutations were all located in region Al in agreement with the central role of the corresponding region in the asse mbly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were fou nd in all four regions. Growth of the Ts mutants was reduced to various ext ents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature: we predict that these mutant Rpb3 proteins are defective in polymerase ass embly or the mutant RNA polymerases containing mutant Rpb3 subunits are uns table. In accordance with this prediction, the Ts phenotype of all the muta nts was suppressed to varying extents by overexpression of Rpb11, the pairi ng partner of Rpb3 in the core subassembly. We conclude that the majority o f rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered.