We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13°C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDCSS, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIUIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase.

In Saccharomyces cerevisiae, expression of the ADH2 gene is undetectable during growth on glucose. The transcription factor ADR1 is required to fuly activate expression when glucose becomes depleted. Partial activation during growth on

Unlike the majority of genes encoding small nuclear RNAs, which are transcribed by RNA polymerase B, the U6 gene contains features found in both class B and class C genes, indicating the involvement of a combination of transcription factors normally specific to each class of genes. We present direct genetic and biochemical evidence that the U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C in vivo as well as in vitro. A mutant strain with a temperature‐sensitive defect in the large subunit of RNA polymerase C that results in defective transcription of tRNA and 5S RNA genes shows a corresponding defect in U6 RNA levels. Also, purified RNA polymerase C transcribes the U6 gene when supplemented with partially purified TFIIIB. The other class C transcription factors, TFIIIA and Tau (TFIIIC), are not required in this system.

Yeast transcription factor tau forms a stable complex with tRNA genes. Using this property, the factor could be highly purified on a specific tDNA column. The purified factor was found by DNA footprinting to protect the whole yeast tRNA3Glu gene from position ‐8 to +81. A DNase‐sensitive site was retained in the middle of the gene on both strands. The 3′ border of the complex was mapped by exonuclease digestion at +88, just downstream of the termination signal. The 5′ limit of the complex was found at position ‐11. However, upon prolonged incubation with exonuclease, the ‐11 blockage disappeared and the DNA molecules were digested to position +30 to 38 in the middle of the gene. Contact points at guanine residues were identified by dimethyl sulphate protection experiments. Reduced methylation of G residues in the presence of factor was found solely within the A block and in the B block region. All six invariant GC pairs (i.e., G10, G18, G19 and G53, C56 and C61) were found to have strong contacts with the factor. These results show that tau factor interacts with both the 5′ and 3′ half of the tRNA3Glu gene, with the B block region being the predominant binding site. The presence of this dual binding site suggests a model in which the factor would bind alternately at the A and B block regions to allow transcription of the internal promoter by RNA polymerase C.

A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin‐agarose and DEAE‐Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor‐DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis.