Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria

We have identified a mutant in RPB3, the third-largest subunit of yeast RNA polymerase II, that is defective in activator-dependent transcription, but not defective inactivator-independent, basal transcription. The mutant con tains two amino-acid substitutions, C92R and A159G, that are both required for pronounced defects in activator-dependent transcription. Synthetic enha ncement of phenotypes of C92R and A159G, and of several other pairs of subs titutions, is consistent with a functional relationship between residues 92 -95 and 159-161. Homology modeling of RPB3 on the basis of the crystallogra phic structure of alpha NTD indicates that residues 92-95 and 159-162 are l ikely to be adjacent within the structure of RPB3. In addition, homology mo deling: indicates that the location of residues 159-162 within RPB3 corresp onds to the location of an activation target within alpha NTD (the target o f activating region 2 of catabolite activator protein, an activation target involved in a protein-protein interaction that facilitates isomerization o f the RNA polymerase promoter closed complex to the RNA polymerase promoter open complex). The apparent finding of a conserved surface required for ac tivation in eukaryotes and bacteria raises the possibility of conserved mec hanisms of activation in eukaryotes and bacteria.