Functional characterization of ABC10 alpha, an essential polypeptide shared by all three forms of eukaryotic DNA-dependent RNA polymerases

ABC10 alpha is a small polypeptide shared by the three yeast RNA polymerase s, Homologous polypeptides in higher eukaryotes have a zinc-binding CX2C .. . CX2C motif and a conserved basic C-terminal end. These features are also found in archaeal gene products that may encode an RNA polymerase subunit, The CX2C ... CX2C motif is partly dispensable, since only its first cystein e is essential for growth, whereas the basic C-terminal end is critical in vivo, A mutant in the latter domain has an RNA polymerase III-specific defe ct and, in vitro, impairs RNA polymerase III assembly. Polymerase activity was, however, not affected in various faithful transcription assays. The mu tant is suppressed by a high gene dosage of the second largest subunit of R NA polymerase III, whereas the homologous subunits of RNA polymerase I and II have aggravating effects. In a two-hybrid assay, ABC10 alpha binds to th e C-terminal half of the second largest subunit of RNA polymerase I, in a w ay that requires the integrity of the CX2C ... CX2C motif Thus, ABC10 alpha appears to interact directly with the second largest subunit during polyme rase assembly. This interaction is presumably a major rate-limiting step in assembly, since diploid cells containing only one functional gene copy for ABC10 alpha have a partial growth defect.