We have sequenced and annotated the genome of ®ssion yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly re¯ecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have signi®cant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identi®ed, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.We report here the completion of the fully annotated genome sequence of the simple eukaryote Schizosaccharomyces pombe, a ®ssion yeast. It becomes the sixth eukaryotic genome to be sequenced, following Saccharomyces cerevisiae 1 , Caenorhabditis elegans 2 , Drosophila melanogaster 3 , Arabidopsis thaliana 4 and Homo sapiens 5,6 . The entire sequence of the unique regions of the three chromosomes is complete, with gaps in the centromeric regions of about 40 kb, and about 260 kb in the telomeric regions. The completion of this sequence, the availability of sophisticated research methodologies, and the expanding community working on S. pombe, will accelerate the use of S. pombe for functional and comparative studies of eukaryotic cell processes.

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10␣, Hs10␤, Hs8, and Hs6, respectively), homologous to the ABC10␣, ABC10␤, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10␣, Sc10␤, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Eukaryotic mRNAs are synthesized by large transcription complexes formed by RNA polymerase II and a number of protein cofactors controlling the selectivity and efficiency of transcriptional initiation, elongation, and termination (14, 36). Purified preparations of RNA polymerase II were obtained for several eukaryotes (references 26, 37, 38, and 54 and references therein) and were found to consist of at least 10 distinct polypeptides ranging from 220 to less than 10 kDa. Their subunit structure is thus much more complex than is that of the three-component bacterial core enzyme ␣ 2 ␤␤Ј. Archaeal RNA polymerases also contain a large number of polypeptides, and most of them are related to eukaryotic subunits (21,(23)(24)(25)38).The genes encoding the 12 subunits of the yeast enzyme have all been cloned and sequenced (see Table 1). The three largest subunits, Sc1, Sc2, and Sc3, are homologous to the ␤Ј, ␤, and ␣ components of the bacterial core enzyme (28, 38, 54). Sc11 (50) is homologous to AC19, a subunit which is shared by yeast RNA polymerases I and III (15). Sc7 is similar to what is most probably the C25 subunit of RNA polymerase III (39). Sc7 was initially believed to be nonessential for mRNA synthesis in vivo (54), but further studies indicated that deletion of the corresponding gene is lethal (29). Five small subunits, Sc5, Sc6, Sc8, Sc10␣, and Sc10␤ (22,44,49,52), are present in all three nuclear RNA polymerases (11,12,37,46). These 10 specific or common subunits are essential components of the transcription apparatus, as strains carrying the corresponding null alleles are nonviable. In contrast, the deletion of the genes encoding the RNA polymerase II-specific Sc4 and Sc9 subunits leads to slowly growing but viable mutants (48, 51).The human RNA polymerase II, although less extensively characterized, contains at least 10 distinct subunits (19,26). The yeast and animal enzymes are closely related antigenically (18), indicating a strong evolutionary conservation. This was directly established by cloning and sequencing seven human cDNAs encoding RNA polymerase II subunits Hs1, Hs2, Hs3, Hs5, Hs6, Hs9,[32][33][34]47), which all showed significant homology to the corresponding yeast subunits (see Table 1). However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the human enzyme (26) failed to reveal small polypeptides of less than 10 kDa that would correspond to the Sc10␣ and Sc10␤ subunits shared by all three yeast RNA polymerases (12).It was recently demonstrated that the common subunit Sc6 of Saccharomyces cerevisiae can be functionally replaced in vivo by homologs from Schizosaccharomyces pombe and ha...

ABC10␤, a small polypeptide common to the three yeast RNA polymerases, has close homology to the N subunit of the archaeal enzyme and is remotely related to the smallest subunit of vaccinial RNA polymerase. The eucaryotic, archaeal, and viral polypeptides share an invariant motif CX 2 C. . . CC that is strictly essential for yeast growth, as shown by site-directed mutagenesis, whereas the rest of the ABC10␤ sequence is fairly tolerant to amino acid replacements. ABC10␤ has Zn 2؉ binding properties in vitro, and the CX 2 C . . . CC motif may therefore define an atypical metal-chelating site. Hybrid subunits that derive most of their amino acids from the archaeal subunit are functional in yeast, indicating that the archaeal and eucaryotic polypeptides have a largely equivalent role in the organization of their respective transcription complexes. However, all eucaryotic forms of ABC10␤ harbor a HVDLIEK motif that, when mutated or replaced by its archaeal counterpart, leads to a polymerase I-specific lethal defect in vivo. This is accompanied by a specific lack in the largest subunit of RNA polymerase I (A190) in cell-free extracts, showing that the mutant enzyme is not properly assembled in vivo.The nuclear genome of eucaryotes is transcribed by three heteromultimeric RNA polymerases that respectively contain 14, 12, and 17 distinct subunits in Saccharomyces cerevisiae. The two largest subunits are related to the ␤Ј and ␤ components of the ␣ 2 ␤␤Ј bacterial core enzyme and to the equivalent subunits of the archaeal and vaccinial RNA polymerases. Biochemical and genetic studies have established that they harbor the active site of the yeast (1, 2) and bacterial (3) enzymes. Homology to the bacterial ␣ subunit, although less pregnant, was also observed with the eucaryotic and archaeal enzymes (4, 5). The ␣ 2 ␤␤Ј core polymerase structure is therefore preserved in all eucaryotic, archaeal, bacterial, and viral forms of RNA polymerases. A number of additional subunits are structurally conserved or even strictly identical from one to another polymerases (6 -8) and are functionally conserved from yeast to man (9, 10). Most of them are structurally related to known components of the archaeal polymerase, indicating the existence of an extended core enzyme form that is common to the archaeal and eucaryotic lineages (5).The present work deals with ABC10␤, a small polypeptide of 70 amino acids that is shared by all three yeast RNA polymerases, is able to bind Zn 2ϩ in vitro (11) and is essential for growth (12). We have previously determined the amino acid sequence of that polypeptide (13), which has a close homology to the N subunit of the archaeal enzyme (5,10,14) and is remotely related to the smallest subunit of vaccinial RNA polymerase (15). We report here that the yeast subunit and its archaeal homolog are largely interchangeable in vivo and that the eucaryotic, archaeal, and viral polypeptides have in common an invariant metal binding domain (CX 2 C . . . CC) critical for its biological activity in S. cerevisiae. Moreov...

Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP..LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP..LW.GKQ provokes a temperaturesensitive defect. Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-⌬ null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan gene YGR089w. A ygr089-⌬ null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.