A UNIVERSALLY CONSERVED REGION OF THE LARGEST SUBUNIT PARTICIPATES INTHE ACTIVE-SITE OF RNA-POLYMERASE-III

The largest subunits of the three eukaryotic nuclear RNA polymerases p resent extensive sequence homology with the beta' subunit of the bacte rial enzymes over five major co-linear regions. Region d is the most h ighly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which i s invariant in all multimeric RNA polymerases. An extensive mutagenesi s of that region in yeast RNA polymerase III led to a vast majority (1 6/22) 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 degr ees C. RNA polymerase III from the mutant rpc160-112 was severely impa ired in its ability to transcribe a tRNA gene in vitro. The transcript ion defect did not originate from a deficiency in transcription comple x 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 SU P4 tRNA gene and an increased rate of slippage of nascent RNA, as comp ared 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 b y a distortion of the active site near the growing point of the RNA pr oduct.