The catalytic center of yeast RNA polymerase II and III contains an acidic loop 
borne by their second largest subunit (Rpb2-(832)GYNQED(837), 
Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral 
DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic 
motif reveals its strictly essential character in RNA polymerase III, with a 
slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other 
substitutions completely prevent growth, whereas rpb2-E836A combines a dominant 
growth defect with severe lethal sectoring. A mild but systematic increase in 
RNA polymerase occupancy and a strict dependency on the transcript cleavage 
factor TFIIS (Dst1) also suggest a slower rate of translocation or higher 
probability of transcriptional stalling in this mutation. A conserved nucleotide 
triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an 
Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a 
second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role 
of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found 
among the RNA-dependent eukaryotic RNA polymerases and may thus represent a 
specific adaptation to double-stranded DNA templates.