Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification ofAp4A hydrolase from S. pombe [Robinson, de la Pefia and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aphl, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aphl gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA f the aphl gene from a S. pombe cDNA library. The deduced open reading frame of the aphl gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Seraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn2+-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aphl gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

Multi-subunit transcription factors (TF) direct RNA polymerase (pol) III to synthesize a variety of essential small transcripts such as tRNAs, 5S rRNA and U6 snRNA. Use by pol III of both TATA-less and TATAcontaining promoters, together with progress in the Saccharomyces cerevisiae and human systems towards elucidating the mechanisms of actions of the pol III TFs, provides a paradigm for eukaryotic gene transcription. Human and S. cerevisiae pol III components reveal good general agreement in the arrangement of orthologous TFs that are distributed along tRNA gene control elements, beginning upstream of the transcription initiation site and extending through the 3′terminator element, although some TF subunits have diverged beyond recognition. For this review we have surveyed the Schizosaccharomyces pombe database and identified 26 subunits of pol III and associated TFs that would appear to represent the complete core set of the pol III machinery. We also compile data that indicate in vivo expression and/or function of 18 of the fission yeast proteins. A high degree of homology occurs in pol III, TFIIIB, TFIIIA and the three initiationrelated subunits of TFIIIC that are associated with the proximal promoter element, while markedly less homology is apparent in the downstream TFIIIC subunits. The idea that the divergence in downstream TFIIIC subunits is associated with differences in pol III termination-related mechanisms that have been noted in the yeast and human systems but not reviewed previously is also considered.

In addition to directing transcription initiation, core promoters integrate input from distal regulatory elements. Except for rare exceptions, it has been generally found that eukaryotic tRNA and rRNA genes do not contain TATA promoter elements and instead use protein-protein interactions to bring the TATA-binding protein (TBP), to the core promoter. Genomewide analysis revealed TATA elements in the core promoters of tRNA and 5S rRNA (Pol III), U1 to U5 snRNA (Pol II), and 37S rRNA (Pol I) genes in Schizosaccharomyces pombe. Using tRNA-dependent suppression and other in vivo assays, as well as in vitro transcription, we demonstrated an obligatory requirement for upstream TATA elements for tRNA and 5S rRNA expression in S. pombe. The Pol III initiation factor Brf is found in complexes with TFIIIC and Pol III in S. pombe, while TBP is not, consistent with independent recruitment of TBP by TATA. Template commitment assays are consistent with this and confirm that the mechanisms of transcription complex assembly and initiation by Pol III in S. pombe differ substantially from those in other model organisms. The results were extended to large-rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolishes rRNA expression in fission yeast. A survey of other organisms’ genomes reveals that a substantial number of eukaryotes may use widespread TATAs for transcription. These results indicate the presence of TATA-unified transcription systems in contemporary eukaryotes and provide insight into the residual need for TBP by all three Pols in other eukaryotes despite a lack of TATA elements in their promoters.

Eukaryotic tRNA genes are controlled by proximal and downstream elements that direct transcription by RNA polymerase (pol) III. Transcription factors (TFs) that reside near the initiation site are related in Saccharomyces cerevisiae and humans, while those that reside at or downstream of the B box share no recognizable sequence relatedness. Human TFIIICb is a transcriptional regulator that exhibits no homology to S. cerevisiae sequences on its own. We cloned an essential Schizosaccharomyces pombe gene that encodes a protein, Sfc6p, with homology to the S. cerevisiae TFIIIC subunit, TFC6p, that extends to human TFIIICb. We also isolated and cloned S. pombe homologs of three other TFIIIC subunits, Sfc3p, Sfc4p, and Sfc1p, the latter two of which are conserved from S. cerevisiae to humans, while the former shares homology with the S. cerevisiae B boxbinding homolog only. Sfc6p is a component of a sequence- specific DNA-binding complex that also contains the B box-binding homolog, Sfc3p. Immunoprecipitation of Sfc3p further revealed that Sfc1p, Sfc3p, Sfc4p, and Sfc6p are associated in vivo and that the isolated Sfc3p complex is active for pol III-mediated transcription of a S. pombe tRNA gene in vitro. These results establish a link between the downstream pol III TFs in yeast and humans.