Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae

ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA poly merases. In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown. We have cloned and charact erised the Schizosaccharomyces pombe rpb8(+) cDNA. We found that S.pombe rp b8, unlike the similarly diverged human orthologue, cannot substitute for S .cerevisiae ABC14.5 in vivo. To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally alte red molecule in heterologous expression assays in S.cerevisiae. Amino acid residue differences within the 67 N-terminal residues contribute to the fun ctional distinction of the two yeast orthologues in S,cerevisiae. Overexpre ssion of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S,cerevisiae, sugge sting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8 ) and C160 subunits, We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNA polymerase III but not RNA polymerase I complex assembly, We a lso report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.