INTRODUCTION

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Structural similarities shared by the RNA polymerases (Pols) of bacteria, archaea, and eukarya reflect a deep-rooted common ancestry of transcription systems in all organisms on earth. Archaea and eukaryotes exhibit greater similarity to each other in their Pol subunits, accessory transcription factors (TFs), and promoter elements than either does to bacteria (36). In eukaryotes, Pol I synthesizes large rRNA (35S to 45S, depending on the species); Pol II synthesizes mRNAs and some small nuclear (sn) RNAs, such as U1 to U5; and Pol III synthesizes mostly tRNAs and 5S rRNA, as well as U6 snRNA and a few other transcripts (53).

The core promoter orchestrates polymerase recruitment, promoter activity, and response to regulatory input (59, 66). In eukaryotes, TATA promoter elements direct transcription by Pol II of a large subset of (but not all) protein-encoding genes, but often not the far fewer snRNA genes that are transcribed by Pol II. While TATA elements are found in a minute fraction of Pol III genes, they are generally not found in the core promoter regions of Pol I genes (53). Intriguingly, despite the lack of TATA promoter elements, Pols I, II, and III all require TATA-binding protein (TBP) for initiation (17). Archaea use widespread TATA-like promoters and a TBP ortholog to direct transcription by a single Pol of all gene types, those encoding tRNA, rRNA, and mRNAs (reviewed in reference 67). Orthologs of another central initiation factor, TFIIB, cooperate with TBPs in promoter recognition in archaea and eukarya (35, 36). While TBP is shared by the three eukaryotic Pols, TFIIB and related factors exhibit polymerase and promoter specificity, such that TFIIB is used by Pol II, TFIIB-related factor (Brf) is used by Pol III for tRNA and 5S rRNA genes, and a distinct variant, BRFU/TFIIIB50, is used by Pol III for human U6 and related type 3 genes (63, 70). The TFIIB-related proteins bind adjacent to TBP on the promoter, recruit the corresponding polymerase to the transcription start site, and participate in promoter melting, an intermediate step in initiation (27, 30, 51, 58). Unlike archaeal and the eukaryal Pol II and Pol III systems, there is no apparent TFIIB homolog in the Pol I machinery (11, 84). Instead, the factor known as Rrn3p/TIF-IA bridges the core promoter-associated factors and Pol I (2, 44, 54).

Pol III promoters have historically been categorized into three major types. 5S rRNA (type 1) and tRNA (type 2) genes utilize internal TATA-less promoters, whereas U6 snRNA (type 3) promoters contain upstream TATA elements (7, 15, 28, 53, 80). For TATA-containing genes such as U6, TBP-TFIIIB can recognize the upstream DNA directly (46, 78). The tRNA promoter is composed of a proximal A box element located 10 to 20 bp downstream of the start site of transcription and a B box element at various distances farther downstream. Although the regions upstream of eukaryotic tRNA genes are generally AT rich, the sequence in this region is not conserved (33). Rather, the sequence information used to assemble a tRNA transcription complex resides in the internal promoter, which is recognized by TFIIIC. Once bound, TFIIIC recruits the initiation factor Brf and its associated TFIIIB components to the TATA-less upstream DNA (7, 28, 80). TFIIIB is an entity composed of three polypeptides, TBP, Brf, and B" (7, 28). TBP is brought to the upstream region of the tRNA gene by Brf (designated BRF/hTFIIIB90 in the human system) via stable protein-protein interactions that occur in the absence of DNA (22, 31, 75). Therefore, association with Brf provides TBP access to the TATA-less tRNA promoter (31, 40, 41, 77). In this setting, TATA is not required and the TBP in TFIIIB can bind to upstream DNA that contains stretches of only G and C residues (25).

We discovered that TATA motifs reside upstream of nearly all Schizosaccharomyces pombe tRNA and 5S rRNA genes. Here we demonstrate an obligatory role for TATA in homologous 5S rRNA and tRNA expression in S. pombe. We demonstrate differences in the mechanisms of Pol III transcription complex formation in S. pombe and Saccharomyces cerevisiae using in vitro transcription systems. Furthermore, S. pombe Brf associates with TFIIIC and Pol III in vivo, while TBP is conspicuously absent from these complexes, consistent with a TATA-dependent mechanism of TBP recruitment. The cumulative data fit a model of obligatory recruitment of TBP by the TATA element, precluding a stringent need for Brf-mediated recruitment of TBP, and lead to the proposal that this may reflect an ancient Pol III system.

Widening our search revealed TATA elements upstream of the S. pombe genes for U1 to U5 snRNAs and 37S rRNA genes, which do not use TATA elements in several other species. Mutation of the TATA element in the Pol I core promoter abolishes rRNA expression in vivo. The data indicate that all three Pols require TATA promoter elements for efficient expression in fission yeast. These results suggest that S. pombe may represent an ancient eukaryotic transcription system that was intermediate between that in archaea and the more diversified eukaryotic model systems that are described in textbooks.

	MATERIALS AND METHODS

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Construction and expression of TATA-less tRNA, 5S rRNA, and 37S rRNA genes and F-Ret1. The S. cerevisiae tRNA^ser gene (on chromosome IX) was amplified from genomic DNA, cloned into the pJK vector, and named SctRNA^serUCA. TATA was then introduced at 30; this changed the upstream sequence TATCTACAA to TATATATAA. Plasmid pFL20/18SPst5.8Si4, containing full-length S. pombe ribosomal DNA (rDNA) and 500 bp of upstream sequence producing a sequence-tagged 5.8S* rRNA (23), was left unaltered or changed at the TATAAA sequence to GGATCC by site-directed mutagenesis to create pFL20/18SPst5.8Si4-Mu2-6 (and -Mu2-7). The plasmids were used to transform S. pombe strain yAS50, and transformants were selected and grown on Edinburgh minimal medium lacking uracil. The 5S rDNA gene with flanking sequences (43) was amplified by PCR from S. pombe DNA and cloned into pGEM-T (Promega, Madison, Wis.) to create p5S-T, which was mutagenized at four nucleotide positions to create plasmid p5Smu. The 5Smu-containing fragment was subcloned into the NcoI/NdeI sites of pRep90X (21) to create pRep90X-5Smu. The TATA element of p5Smu was mutagenized and cloned into the NcoI/NdeI sites of pRep90X to create pRep90X-5Smu-Bam. These were used to transform yAS50, and transformants were selected on EMM lacking leucine. Ret1⁺ was amplified from genomic DNA. The product was cloned into the SalI and SmaI sites of pREP3X, resulting in pREP3X-Ret1, which was digested with PstI and BamHI and cloned into pBluescript-ura4, resulting in pBluescript-Ret1a. Ret1 5'-flanking sequence was obtained by PCR of S. pombe genomic DNA. The product was cloned into the KpnI and SalI sites of pBluescript-Ret1a, resulting in pBluescript-Ret1b. A 4.2-kb KpnI/BamHI fragment of pBluescript-Ret1b was transformed into yHL6382 (21) to create yYH3272 (h⁺ his3-D1 leu1-32 ura4-D18 ade6-M216 ret1::[F-ret1, ura4⁺]). The genomic structure of F-ret1 in yYH3272 was confirmed by PCR (not shown). All constructs were verified by sequencing.

Immunoaffinity purification of Pol III and TFIIIC complexes. Extracts were prepared according to the method of Hamada et al. (16) with modifications. Cells were broken with a French press two times, and the lysate was prepared by adding 3.5 M (NH₄)₂SO₄ to a final concentration of 0.4 M. The resulting precipitate was dissolved in BC100 (20 mM HEPES, pH 7.9, 20% glycerol, 0.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 100 mM KCl) and dialyzed into BC100. For immunoaffinity purification, the extract was adjusted to 0.05% NP-40. Two milligrams of extract protein was incubated with 40 µl of M2 agarose beads (Sigma) at 4°C for 4 h. The beads were washed five times with 0.5 ml of BC100) 0.05% NP-40) and eluted two times with 40 µl of BC100 containing 200 µg of FLAG peptide (Sigma)/ml.

Antisera and immunoblotting. The following antigens were used for antiserum production: N-terminal peptides of Ret1p, spB", and Rpc39p, and a C-terminal peptide of spBrf. Anti-TBP, anti-Sfc1p, ant-Sfc3p, anti-Sfc6p, anti-Sfc4p, and anti-spLa (Sla1p) were described previously (21). Samples were separated by 4 to 20% polyacrylamide gel electrophoresis, transferred to nitrocellulose, probed with appropriate antibodies, and processed using an ECL kit (Amersham).

	RESULTS

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TATA elements upstream of S. pombe tRNA genes. For this study, tRNAscan-SE (42) was used to identify the tRNA gene locations, and then the upstream regions were extracted from S. pombe, S. cerevisiae, Homo sapiens, Drosophila melanogaster, and Arabidopsis thaliana genomic databases. The sequence sets were then used to generate sequence Logos (Fig. 1A) (62). In the Logo display, the bases are stacked on top of each other for each position, the height of each letter being proportional to its frequency, and the letters are sorted with the most common one on top. The height of each stack is adjusted according to the information content (i.e., conservation) and plotted, in bits, on the base 2 logarithmic scale on the y axis (62).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Genomewide analysis reveals TATA motifs upstream of S. pombe tRNA genes. (A) Sequence Logos of the genomic 5' flanking regions of tRNA sequences at 174 S. pombe, 275 S. cerevisiae, 425 D. melanogaster, 625 H. sapiens, and 600 A. thaliana loci. The positions of the A and B box promoter elements are indicated above the Logos. Note the relatively low level of sequence information content in the S. pombe A box, at the no. 10 position indicated by the asterisk, compared to that of S. cerevisiae. Although the full sets of sequences were used for the upstream regions and the first half of the tRNA for all species, for H. sapiens, A. thaliana, and D. melanogaster, the B box and downstream regions were limited to 200, 215, and 250 sequences, respectively, for practical reasons. Approximately 20% of the H. sapiens sequences were identified by tRNAscan-SE as "possible pseudogenes," probably reflective of tRNA-like repetitive elements in the human genome (42). Approximately 1% each of the A. thaliana, S. pombe, and D. melanogaster sequences and none of the S. cerevisiae sequences were identified as possible pseudogenes. Common features of tRNA structure are indicated below the Logos. M1 indicates the first base of mature tRNA. A gap representing introns and sequences extending through the variable stem-loop (not shown) is designated i/v. ac, anticodon. (B) Sequence Logo of the S. pombe and A. thaliana sequences after prealignment of the upstream regions by the Clustal program. The numbering under the upstream region is relative to the putative consensus +1 site.

TATA promotes tRNA expression in S. pombe and in a homologous in vitro transcription system. We recently characterized an in vivo suppression assay that is sensitive to the expression level of tRNA^ser_UGA (16). Two suppressor genes were described, tRNA^ser_UGA-W, which encodes a wild-type full-strength suppressor, andtRNA^ser_UGA-M, which is comparably active for Pol III transcription but is less active for suppression (16). The naturally occurring TATATAAA sequence was altered in both of these genes, and their suppressor activities in S. pombe were determined (Fig. 2A). While both genes containing the wild-type TATA were active (16), the TATA-less genes were inactive.

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Upstream TATA is a positive determinant of tRNA expression in vivo and in a homologous in vitro S. pombe system. (A) The TATATA motifs of two opal suppressor tRNA genes, tRNAserUGA-W (W) and tRNAserUGA-M (M), were left unchanged (+) or replaced with GGATCC () as indicated along the horizontal axis and examined for suppressor activity in vivo as previously described (16, 24). (B) In vitro transcription in an S. pombe-derived extract was performed using three tRNA genes, containing (+) or lacking () upstream TATA elements, and empty plasmid control (c) as indicated below the lanes. Lanes 1 and 2, S. cerevisiae-derived tRNAserUCA gene; lanes 3 and 4, S. pombe-derived tRNAserUGA-M gene; lane 5, control plasmid containing no tRNA gene; lane 6, S. pombe-derived tRNAserUGA-M-3T gene (produces longer transcript [16]). The S. cerevisiae and S. pombe genes differ in size due to a 15-nt intron in the latter, and their A and B box elements are identical except at one position, G10 in the former and T10 in the latter. Transcript bands are indicated by arrows. -RM indicates a recovery marker added to the reactions, and -IC indicates an extract-derived internal control.

An essential TATA element upstream of 5S rRNA genes in S. pombe. 5S rRNA genes are dispersed in S. pombe, flanked by sequences that vary among copies (43). The upstream flanking regions plus the first G of the 5S rRNA sequences of multiple 5S rDNA loci are shown as a sequence Logo in Fig. 3A. Although variability is apparent at many positions, a TATA motif is prominent and typical for the majority of 5S genes.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Essential TATA motifs upstream of S. pombe 5S rRNA genes. (A) Sequence Logo of the upstream regions of multiple dispersed S. pombe 5S rDNA loci. The first base (G) of mature 5S rRNA was included as the last base on the right. (B) Northern blot analysis. A neutral sequence tag allows detection of the tagged transcript, designated 5S* rRNA, which is distinguishable from endogenous 5S rRNA. In vivo expression of plasmid-borne TATA-containing (+) and TATA-less () 5S* rRNA genes was monitored with a 5S*-specific probe. C, control. (C) The blot in panel B was stripped and rehybridized to detect endogenous 5S rRNA.

The Brf subunit of TFIIIB copurifies with fission yeast TFIIIC and Pol III with no associated TBP. Extracts of a previously described S. pombe strain carrying FLAG-tagged Sfc3p, the S. pombe homolog of the B box-binding subunit of TFIIIC (21); a new strain carrying FLAG-tagged spRet1p, the S. pombe homolog of the second-largest subunit of Pol III; and a control strain, yAS50, were subjected to immunoaffinity purification using monoclonal anti-FLAG antibody (M2) cross-linked to agarose (Fig. 4). The input, flowthrough, and affinity-eluted materials were subjected to immunoblotting to detect the proteins indicated to the right of each panel. The spBrf homolog was found associated with Sfc1p, Sfc4p, Sfc6p, and the FLAG-tagged Sfc3p (Fig. 4C to F and H, lanes 6), while two other TFIIIB homologs, spTBP and spB", were not associated (Fig. 4G and I, lanes 6). The relative amounts of Brf in the negative control (lanes 3) and the Sfc3p-TFIIIC complex (lanes 6) are comparable to the ratio seen for Sfc6p, a genuine TFIIIC component (lanes 3 and 6). Quantitative analysis using known amounts of recombinant proteins indicated that a substantial fraction of spTFIIIC is specifically associated with Brf (not shown). The Pol III subunit homologs, spRet1p and spRpc39p were not associated with the S. pombe TFIIIC complex (Fig. 4A and B, lanes 6), nor was the Pol III nascent transcript-binding protein, spLa (Fig. 4J, lane 6), further indicating specificity of the Brf association. The same profile was also reproducible when a strain carrying epitope-tagged Sfc6p was used for purification (not shown). These results indicate that in S. pombe, Brf is specifically associated with a substantial fraction of TFIIIC, and most significantly, this occurs in the absence of TBP.

View larger version (69K): [in this window] [in a new window]   FIG. 4.   Fission yeast Brf and B" associate with Pol III TF complexes in the absence of stably associated TBP. Extracts prepared from a wild-type strain, the FLAG-Sfc3 strain, and a FLAG-spRet1p strain were incubated with anti-FLAG immunoglobulin G (M2)-agarose. After incubation, the supernatants were collected as the flowthrough, the agarose was washed five times with buffer containing 250 mM NaCl, and the bound material was eluted. The input (I), flowthrough (F), and eluate (E) of the M2-agarose from the three extracts were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting using antisera to the proteins as indicated on the right.

Requirement for an upstream TATA element to program a functional Pol III preinitiation complex. Since tRNA expression in S. pombe involves the widespread use of upstream TATA elements, we wanted to examine another hallmark feature of class III genes, their ability to program stable transcription complexes (15). While the interaction of TFIIIC with certain tRNA (and VA1) genes generates a complex that is stable on subsequent challenge with orthologous promoter DNA, this is not true for other tRNA genes, in large part due to variances in the A and/or B box elements (1, 12, 29, 37). However, inclusion of TFIIIB in the TFIIIC-DNA complex does program stable complex formation (33, 37). According to the assembly pathway elucidated using S. cerevisiae components, TFIIIC binds to the internal promoter and then recruits TFIIIB to the upstream DNA through contacts made principally to Brf (6, 57, 60). Thus, on TATA-less promoters, TFIIIC recruits and induces Brf and associated TBP to bind upstream DNA (6, 29, 47, 57, 60; reviewed in reference 7). This assembly pathway leads to a preinitiation complex that is stable upon challenge by homologous promoters and is consistent with the assemblages elucidated for human, Drosophila, and Xenopus (3, 30, 37, 59, 76). Using currently available activities, we can examine complex assembly and stability with a template commitment assay (3, 37, 61). In this assay, the first template is allowed to assemble with TFs and a second template is then added to challenge the stability of the first transcription complex. Because several templates were compared in S. cerevisiae and S. pombe extracts (Fig. 5), multiple observations regarding cis-acting elements and trans-acting factors are noteworthy.

View larger version (91K): [in this window] [in a new window]   FIG. 5.   An upstream TATA is required to program a functional Pol III preinitiation complex on an A box- and B box-containing tRNA gene in S. pombe. Transcription complexes were monitored using template exclusion assays performed in parallel in S. cerevisiae (top) and S. pombe (bottom) in vitro transcription systems. The tRNA genes used for Fig. 2B were tested for the ability to form stable transcription complexes. After incubation of the first template (the order of addition is indicated above the lanes) with transcription buffer (previously determined to be optimal at 20 min), nucleoside triphosphates and [-32P]GTP were added along with the second template (indicated above the lanes), and transcription was allowed to proceed. The reaction was stopped, and RNA was extracted and separated on 6% polyacrylamide-8 M urea. The templates were as follows: A, sc-tRNASer TATA; B, sc-tRNASer +TATA; C, sp-tRNASer TATA; D, sp-tRNASer +TATA; E, pJK148 (empty vector); F, sp-tRNASer-3T +TATA. The arrows point to the different transcripts synthesized.

A TATA element in the S. pombe Pol I core promoter. TATAAAA was also found in the region upstream of the transcription initiation sites of the genes that encode large rRNAs. In this promoter, TATAAAA overlaps the 30 region of the previously determined Pol I start site (Fig. 6A) (8). We examined 5.8S rRNA expression from a plasmid that harbors the TATA-containing wild-type 37S rRNA gene of S. pombe. The 5.8S rRNA produced from this plasmid is marked with a 4-nucleotide sequence tag that allows its transcript (designated 5.8S*) to be distinguished from endogenous 5.8S rRNA by slower mobility on polyacrylamide gels (23). The upstream TATAAA was changed to GGATCC (Fig. 6A) or left unaltered, and the resulting plasmids were used to transform ura4-D18 S. pombe cells to the ura4⁺ phenotype. The two plasmids yielded comparable transformation efficiencies, and the transformants exhibited indistinguishable growth phenotypes (not shown). Total RNA was prepared from the transformants and examined by polyacrylamide gel electrophoresis and ethidium bromide staining (Fig. 6B). As described, the tagged 5.8S* rRNA was visible as a clear band with slower mobility than 5.8S rRNA (23), produced from the wild-type TATA-containing plasmid (lane 3). Cells containing plasmids bearing the TATA-less promoter (lanes 1 and 2) and the control plasmid lacking the rRNA gene (lane 4) did not express the 5.8S* rRNA. As a control, the TATA-less promoter was converted back to a TATA-containing promoter by site-directed mutagenesis, and this rescued 5.8S* rRNA (not shown). Hybridization with an antisense probe confirmed that the tagged 5.8S* rRNA was expressed from the gene containing the wild-type TATA (Fig. 6C, lane 3) but not from the TATA-less genes or the control plasmid (lanes 1, 2, and 4). An oligonucleotide complementary to wild-type 5.8S and 5.8S* rRNAs revealed comparable loading in all lanes (Fig. 6D); again as expected, 5.8S* was expressed only from the TATA-containing plasmid (Fig. 6D, lane 3). These results indicated that the TATAAA sequence that is naturally found upstream of large rRNA genes in S. pombe is important for rRNA expression in vivo.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Pol I-transcribed large-rRNA genes use an upstream TATA motif in the core promoter region. (A) Sequence of promoter region (40 through 20 relative to +1), with TATAAA motif underlined, of S. pombe rDNA (top line) (8) and TATA mutant (bottom line); dots indicate identical residues. (B) Ethidium bromide-stained polyacrylamide gel. Total RNA was isolated from S. pombe strains that had been transformed with plasmids containing 37S rRNA genes in which the 5.8S rRNA sequence contained a unique sequence tag (5.8S*; see the text). Lanes 1 and 2, two independent isolates in which the TATA element was mutated by site-directed substitution; lane 3, wild-type TATA; lane 4, control plasmid containing no rRNA gene. (C) Northern blot analysis using oligonucleotide probe conditions specific for the tagged 5.8* rRNA; the lanes are the same as in panel B. (D) The Northern blot in panel B was stripped and hybridized using an oligonucleotide probe that recognizes both wild-type (endogenous) 5.8S rRNA and 5.8* rRNA; the lanes are the same as in panel B.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

A major conclusion of this study is that transcription in S. pombe involves widespread use of TATA promoter elements. This is significant not so much because of the number of promoters involved but more so because TATA use is widespread in the Pol I and III systems. It has generally been found that, except for rare exceptions (see below), eukaryotic tRNA gene promoters are TATA-less. The results presented here should extend the known diversity of gene structure and promoter function in eukaryotes (59, 66). Although, as detailed below, scattered reports indicate TATA elements upstream of some tRNA and 5S rRNA genes, this is the first report that indicates TATA function for the great majority of tRNA, 5S rRNA, and large-rRNA genes. Moreover, for these genes, TATA-mediated transcription is an obligatory pathway of expression, indicating that functional use of TATA elements is the rule for tRNA transcription in S. pombe rather than the exception (see below). The inability to detect expression from TATA-less tRNA and 5S rRNA genes in S. pombe provides strong evidence to suggest that the mechanism to bring TFIIIB activity to the core promoter in the absence of a TATA element that has been elucidated for other model organisms is not available to and/or not used in S. pombe. This further suggests a fundamental difference between fission yeast and other model systems in the mechanisms that bring the central TF, TBP, to the core promoter. The presence of TATA elements upstream of a substantial fraction of A. thaliana tRNA genes suggests that plants and perhaps other branches of the evolutionary tree share this feature with fission yeast. Thus, this study provides a basis to suggest that, contrary to what has been generally accepted, a significant portion of eukaryotes may use TATA-dependent mechanisms for the great majority of their Pol III transcription.

S. pombe Pol III promoters: TATA elements are the rule. As derived from Fig. 1, the consensus sequences 8TRG/NYNNARNNG18 and 53RTTCRANYY62 represent the A and B boxes of S. pombe tRNA genes. For the S. pombe 7SL and U6 RNA genes, sequence matches to the A box begin 13 and 17 bp, respectively, downstream of their first nucleotide. These genes also match the B box consensus sequence beginning at positions 60 and 69 of 7SL and U6, respectively. Substitution of conserved B box residues in the S. pombe U6 gene rendered this template inactive in the S. pombe in vitro transcription system (M. Hamada and R. Maraia, unpublished data). Thus, in S. pombe, tRNA, U6, and 7SL genes exhibit similar promoter architectures, in which TATA appears as one of multiple promoter elements (Fig. 7A).

View larger version (41K): [in this window] [in a new window]   FIG. 7.   Conserved TATA motifs in the promoters of small RNA genes in S. pombe. (A) Pol III-transcribed genes. The upstream TATA, internal control region (ICR) (for 5S only), A box and B box (for tRNAs and U6 and 7SL RNAs), and terminators (T) are shown. The bent arrows indicate the +1 positions (as previously determined). Consensus sequences for the A and B box elements that fit all tRNA genes, as well as U6 and 7SL genes, are shown below the schematics; boldface letters are invariant or very highly conserved. The U6 B box resides in an intron (shaded; see the text) (B) Upstream regions of Pol II-transcribed snRNA genes are presented both in Logo (above) and as individual aligned sequences, ending at +1.

Species specificity of Pol III-dependent tRNA transcription. It is important to note that the data indicate that the role of TATA is not to provide simple additive transcriptional activity to the internal promoters of tRNA and 5S rRNA genes in S. pombe; rather, the homologous transcription machinery is highly dependent on and functionally adapted to the TATA element, even when the internal promoter is strong (Fig. 2, bottom, lanes 1 and 2).

TATA and tRNA transcription: exceptional cases in conventional model organisms. Although in certain cases TATA elements direct Pol III transcription of tRNAs, these appear to be unusual instances. The transcription start site for the gene encoding Xenopus laevis selenocysteine tRNA^[ser]sec is dictated by an upstream promoter that includes a TATA element (5, 39). However, this gene utilizes a unique pathway of expression, as evidenced by the Pol III initiation site, which occurs at a highly unusual position for a tRNA, coinciding with the 5' end of the mature tRNA (38). The sequence of tRNA^[ser]sec does not contain an A box promoter element, and start site selection is instead mediated by the TATA element (5, 52). An upstream TATA has also been shown to control expression of a tRNA gene in the insect Bombix mori, in which two genes contribute to silk gland-specific tRNA^Ala synthesis in a tissue-regulated manner. In this case, an upstream TATA directs expression of the more active of the two tRNA^Ala genes in a TBP-dependent manner (50). This tRNA^Ala gene was also analyzed in D. melanogaster cells, where its transcription was stimulated by increased TBP levels (71). Figure 1A demonstrates that TATA elements are not found upstream of most tRNA genes in the insect D. melanogaster, for which it was recently reported that TBP-related factor, TRF, rather than TBP itself, directs tRNA transcription (68). The cumulative data are consistent with a model in which TATA and TBP may be used for special cases of tRNA expression in insects and other organisms. In summary, prior to the present report, TATA elements had been characterized as promoters of tRNA transcription in exceptional cases.

A functional TATA element in a Pol I core promoter. Our data demonstrate that substitution of the TATA element that overlaps the 30 region of the S. pombe Pol I promoter abolishes rRNA expression in vivo. Although our results do not address the mechanism by which TATA promotes expression by Pol I in S. pombe, they nonetheless indicate an important role of TATA in rRNA expression in fission yeasts. The location of the TATAAA sequence, positioned 35 bp upstream of the previously determined start site of Pol I transcription, suggests that TATA-TBP functions as part of the core promoter in S. pombe.

TATA elements are also more widespread in the Pol II-transcribed genes in S. pombe than in other species. Although TATA promoter elements are a recognized hallmark of protein-encoding genes, not all Pol II promoters contain TATA elements, as some mRNA-encoding genes use other core promoter elements for transcription (4, 66). Another class of genes, those that produce snRNAs (e.g., U1 to U5), are transcribed by Pol II from TATA-less promoters in yeast, human, and Drosophila (18, 73, 82). In contrast to this, TATA motifs have been noted in the promoter regions of some S. pombe snRNA genes that are transcribed by Pol II (reference 83 and references therein). Alignment of the upstream regions of the U1 to U5 snRNA genes of S. pombe revealed a prominent TATA motif in addition to an upstream TTAC sequence (Fig. 7B).

Evolutionary implications. It seems reasonable to consider S. pombe a tentative link to an ancient ancestor that used ubiquitous TATA elements to promote transcription of a wide range of genes, if not of all genes. The ubiquitous TATA-like elements of an archaeonlike organism would have remained after divergence of the three nuclear Pols. Thus, while S. pombe may have retained TATA elements as essential components of the Pol I, II, and III systems, many genes of other organisms would have lost their TATAs. This scenario is consistent with a residual requirement for TBP and/or TBP-related factors by all three Pols, even though most of their target genes do not use TATA elements in many species. It is also consistent with the use of TBP-related factors in metazoans (19, 68, 69, 72). Although plant tRNA genes are also flanked by upstream TATAs (13) (Fig. 1), we are unaware of a simple line of inheritance between plants and S. pombe that could account for this common feature. 5S rRNA genes are also dispersed in Neurospora crassa, and transcription was also reported to require a TATA at 29 (64). TATA elements upstream of a substantial fraction of plant and other species' 5S and tRNA genes suggests that fission yeasts are not unique in the structure of their Pol III promoters.