We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI(+)] and [psi(-)] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.

The largest subunits of the three eukaryotic nuclear RNA polymerase 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.

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.RNA polymerases belong to two different classes. One is represented by the RNA polymerases of SP6, T-uneven phages, and mitochondria that have polymerizing activity associated with a single polypeptide. The second class is made up of multisubunit enzymes found in eubacterial, archaebacterial, and eukaryotic cells (see references 41 and 47 for reviews). The two largest subunits of these enzymes are well conserved in all cellular enzymes, as indicated initially by immunological studies (reviewed in reference 40) and by subsequent comparison of the amino acid sequences deduced from the DNA sequences of the cloned genes (see references 41 and 47 for reviews). Comparison of the largest subunits of eukaryotic RNA polymerases A, B, and C and of the archaebacterial enzymes disclosed eight regions of homology, which have been termed domain a to domain h (20,41,47). Similarly, the second-largest subunit can be divided into a succession of conserved domains (41, 46). Most but not all of these domains are also present in the cognate eubacterial large subunits P' and ,B. Since the two largest subunits account for around 70% of the molecular mass of RNA polymerases, it is reasonable to propose that conserved domains participate in the catalytic functions of these enzymes. Some biochemical and genetic evidence supports this view. For instance, mutations affecting the nucleotidebinding site (34) and transcription termination (18,22) are located within conserved domains of the P-like subunit.Little is known of the structural or catalytic roles played by the largest subunit of RNA polymerases. Several genetic analyses have shown that RNA polymerase conditional mutations in the largest subunit tend to map in the conserved domains (14,38,53). Moreover, the fact that all a-amanitinresistant mutations affect the largest subunit of the B enzyme (see reference 37 for a review) suggests that the largest subunit is implicated in chain elongation. In the mouse, a-amanitin resistance results from an amino acid change in the strongly conserved domain f (3).The two large subunits are also involved in zinc binding.