Six human RNA polymerase subunits functionally substitute for their yeast counterparts

Abstract

To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.