universally conserved region of the largest subunit participates in the active site of RNA polymerase III

The largest subunits of the three eukaryotic nuclear RNA polymerases present extensive sequence homology with the beta' subunit of the bacterial enzymes over five major co-linear regions. Region d is the most highly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which is invariant in all multimeric RNA polymerases. An extensive mutagenesis of that region in yeast RNA polymerase III led to a vast majority (16/22) of lethal single-site substitutions. A few conditional mutations were also obtained. One of them, rpc160-112, corresponds to a double substitution (T506I, N509Y) and has a slow growth phenotype at 25 degrees C. RNA polymerase III from the mutant rpc160-112 was severely impaired in its ability to transcribe a tRNA gene in vitro. The transcription defect did not originate from a deficiency in transcription complex formation and RNA chain initiation, but was mainly due to a reduced elongation rate. Under conditions of substrate limitation, the mutant enzyme showed increased pausing at the intrinsic pause sites of the SUP4 tRNA gene and an increased rate of slippage of nascent RNA, as compared with the wild-type enzyme. The enzyme defect was also detectable with poly[d(A-T)] as template, in the presence of saturating DNA, ATP and UTP concentrations. The mutant enzyme behavior is best explained by a distortion of the active site near the growing point of the RNA product.