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Molecular and Cellular Biology, October 2001, p. 6870-6881, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.6870-6881.2001
Widespread Use of TATA Elements in the Core
Promoters for RNA Polymerases III, II, and I in Fission Yeast
Mitsuhiro
Hamada,1,
Ying
Huang,1
Todd M.
Lowe,2 and
Richard J.
Maraia1,*
Laboratory of Molecular Growth Regulation,
National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland,
20892-27531 and Department of Genetics,
Stanford University School of Medicine, Stanford, California
94305-51202
Received 22 June 2001/Accepted 9 July 2001
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ABSTRACT |
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.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
Construction and expression of TATA-less tRNA, 5S rRNA, and
37S rRNA genes and F-Ret1.
The S. cerevisiae tRNAser gene (on chromosome IX)
was amplified from genomic DNA, cloned into the pJK vector, and named
SctRNAserUCA. 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).
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RESULTS |
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).
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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.
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Figure
1A shows the upstream sequence (the first base of the mature
tRNA is designated M1 under the
A. thaliana Logo) and
the
first half of the tRNA sequence (ending after position 37),
followed by
a gap representing introns and sequences extending
through the variable
stem-loop (not shown), followed by the B
box region and continuing to
the end of the tRNA sequence (CCA
is not included). The positions of
the A box and B box and some
features of tRNA structure are indicated.
The largest amount of
information content coincides with the A and B
box promoter elements.
In the upstream regions, the information content
is higher for
S. pombe than for the other species. The most
concentrated upstream
information appears as a TATAAA motif at a
position typical of
TATA promoter elements (recall that Pol III
initiation occurs
a variable distance [
5 to 10 bp] upstream of the
first base of
the mature tRNA). This degree of information was not
found in
the upstream regions of the
S. cerevisiae,
H. sapiens, and
D. melanogaster tRNA sequences (not shown,
but see below). TATA-like
information also appeared upstream of
A. thaliana tRNA genes,
although the information content was
not as high nor as well defined
as for
S. pombe (Fig.
1A),
consistent with the documented presence
of the 4-nucleotide sequence
TATA (
13).
tRNAscan-SE used the first base of the mature tRNA sequences to
isolate the upstream sequences. However, Pol III initiates
at various
distances (
5 to 15 bp) upstream of the mature tRNA
sequence.
Prealignment of the sequences by an alignment program
led to a
significant increase in the information content of the
TATA region in
the
S. pombe and, to a lesser degree,
A. thaliana sequences (Fig.
1B) but not those of the other species (not
shown).
To our surprise, prealignment also produced a prominent A that appeared
in the
A. thaliana and
S. pombe sequences about
six
positions upstream of the mature tRNA (Fig.
1B). An increase in
the
information content of this position in association with the
increased
information content of the TATA motif that appeared
in the aligned
sequences (more so for
S. pombe than
A. thaliana),
in conjunction with the 30-bp spacing of the prominent
A and TATA,
argued that the A represents a consensus transcription
initiation
site (+1). While prealignment led to a moderate increase in
the
TATA content of the
A. thaliana sequences, a more
substantial
increase was observed around the predicted +1 site
(underlined),
which appeared in the form of ANC
AA
(Fig.
1B). ANC
AA may be analogous
to the initiator element
described for certain Pol II (Inr) and
Pol I (rInr) core promoters
(
56,
66).
Inspection of individual
S. pombe sequences revealed that
while 35% matched at seven or more TATATATA positions, the great
majority matched at five or more positions (not shown). Thus,
most, if
not all,
S. pombe tRNA genes appear to have at least
a
component of an upstream TATA element around the
30 region
(not
shown). Our ability to examine tRNA transcription in fission
yeast led
us to focus on
S. pombe for the remainder of this
study.
G10, which occupies the third position of the A box and is invariant in
all
S. cerevisiae tRNA genes and highly conserved
in those
of other species (
14,
15), is much less conserved
in
S. pombe (Fig.
1A). Analysis of the subset of the
S. pombe non-G10 genes revealed that they were similar to
the full set
of sequences in several features, including an upstream
TATA,
A-to-B box distance, percentage of genes with introns, +1
information,
and B box consensus (not shown). We conclude that upstream
TATAs
are typical of the great majority of
S. pombe tRNA
genes. As will
be described in more detail below, template commitment
and other
in vitro transcription assays using
S. pombe
extract indicate
that although tRNA genes with G10 appear to compete
better than
non-G10 genes for a limiting TF (probably TFIIIC [see
below]),
these nonetheless require upstream TATA elements regardless
of
whether G occupies the number 10 position, i.e., even when the
A and
B boxes exhibit perfect matches to the
S. cerevisiae consensus.
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 tRNAserUGA
(16). Two suppressor genes were described,
tRNAserUGA-W, which encodes a wild-type
full-strength suppressor, andtRNAserUGA-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.
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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.
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The effects of TATA on the efficiency of in vitro transcription in
S. pombe-derived extract are shown in Fig.
2B. In addition
to the
S. pombe tRNA
serUGA-M
gene, we also examined an
S. cerevisiae
tRNA
ser gene that contains perfect matches to the
consensus A and B box
promoter elements and, as is typical of
S. cerevisiae tRNA genes,
contains no upstream TATA element.
Specifically, the A and B box
elements in the two yeasts'
tRNA
ser genes used here are identical except for
one position in the
A box, T10 in the
S. pombe
tRNA
ser gene and G10 in the
S. cerevisiae tRNA
ser gene. These genes were
used for in vitro transcription (Fig.
2B). Nucleotides in the wild-type
TATA-less
S. cerevisiae tRNA
ser gene
were replaced to create a TATA element. The TATA-less
S. cerevisiae gene was inactive, while the substitutions that created
the TATA element activated it (Fig.
2B, lanes 1 and 2). This
demonstrated
that a yeast tRNA gene with perfect matches to the
consensus A
and B box elements requires an intact upstream TATA for
efficient
transcription in the
S. pombe system. The
S. pombe tRNA
serUGA-M gene whose TATA
was mutated was inactive, while the gene containing
wild-type TATA was
active (Fig.
2B, lanes 3 and 4). A negative
control, showing the
products of a reaction containing a control
plasmid that does not
contain a tRNA gene, is shown in lane 5,
and another version of the
TATA-containing tRNA
serUGA-M gene that
differs only in the sequence at the Pol III terminator
and therefore
produces a distinctively longer transcript (
16)
is shown
in lane
6.
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.
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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.
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To investigate if the upstream TATA is important for expression, we
examined 5S rRNA production from a pRep90X-derived plasmid
containing
an
S. pombe 5S rRNA gene (
43). We introduced
four
substitutions in the 5S sequence that correspond to nonconserved
residues (derived mostly from
S. cerevisiae 5S). These
substitutions
allow specific detection by oligonucleotide hybridization
that
distinguishes expression from the test gene, designated 5S*, from
that of endogenous 5S rRNA and are also compatible with the predicted
5S rRNA secondary structure in this region (not shown). The wild-type
upstream TATATAA was then changed to GGATCCA or left unaltered,
and the
resulting plasmids, which differed only at the TATA sequence,
were used
to transform
S. pombe to a selectable
leu1+ phenotype. Both plasmids yielded
transformation efficiencies
comparable to that of the empty control
plasmid, and the transformants
exhibited indistinguishable growth
phenotypes (not shown). Total
RNAs from duplicate sets of transformants
were examined by Northern
blotting (Fig.
3B). Hybridization with an
antisense oligonucleotide
probe revealed that 5S rRNA was expressed
from the 5S* gene containing
the wild-type TATA (Fig.
3B, lanes 2 and
5) but not from the TATA-less
5S* gene (lane 1 and 4) or from the
control plasmid lacking an
insert (lanes 3 and 6). To estimate the
relative activity of the
TATA-containing 5S* gene and to examine for
differences in loading,
the blot was stripped and rehybridized with an
oligonucleotide
specific for the wild-type 5S rRNA sequence under the
same conditions
as for the 5S* probe (Fig.
3C). Quantitation led to the
estimate
that 20 to 25% of the total 5S rRNA in the
TATA-containing lanes
was produced by the plasmid-borne 5S* rRNA
gene containing the
wild-type TATA element, while the TATA-less gene
produced background
levels of 5S* rRNA (not shown). These results
indicated that the
conserved TATA found upstream of
S. pombe
5S rRNA genes is required
for 5S rRNA expression in vivo. Sequence
analysis revealed that
these constructs differed only in the upstream
TATA element, as
expected (not shown). Therefore, the large difference
in the amount
of 5S* rRNA production from the TATA-containing and
TATA-less
5S* rRNA genes suggests that TATA-dependent transcription
represents
an obligatory pathway of Pol III recruitment in
S. pombe cells.
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.
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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.
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Analysis of the spRet1p complex revealed the presence of the associated
Pol III subunit, spRpc39p, as expected (Fig.
4A and
B, lanes 9). In
this case, spBrf and spB" homologs were reproducibly
found associated
with the complex, while spTBP, spLa, and four
spTFIIIC subunits were
not associated (Fig.
4C to J, lanes 9).
The specificity of the Brf
association with the TFIIIC and Pol
III complexes was reproducible in
multiple experiments and was
observed with affinity-purified antibody
raised against a terminal
peptide of spBrf (Fig.
4), as well as with
two antisera raised
against recombinant spBrf (not shown). The same
profile of associated
and nonassociated proteins was observed when a
strain carrying
the spRPC53p subunit of Pol III was epitope tagged and
used for
purification (not shown; as will be described elsewhere,
FLAG-tagged
Pol III is active for promoter-dependent transcription, as
well
as efficient termination and recycling on preassembled
transcription
complexes isolated on immobilized template
DNA).
It was noted that TFIIIB components could not be detected when the
epitope-tagged TFIIIC220 human counterpart of Sfc3p was
used to isolate
TFIIIC complexes from HeLa cells (
74), suggesting
that the
Brf-TFIIIC association may be a unique characteristic
of
S. pombe. TBP together with Brf was readily found associated
with
epitope-tagged Pol III isolated from
S. cerevisiae
(
7).
spTBP could not be detected in our Brf-containing
spTFIIIC complex
or the Brf-containing Pol III complex, even though TBP
is found
tightly associated with Brf (in the absence of DNA) in other
systems
(
20,
22,
31,
45,
55,
75). Thus, compared to the
TFIIIB
subunit associations in other systems, the
S. pombe
TFIIIB subunits
appear to be arranged differently, although in a manner
that is
appropriate for the unique promoter architecture of
S. pombe tRNA
genes (see below). Furthermore, we have been unable to
demonstrate
association between TBP and Brf in
S. pombe
extracts by using
direct immunoprecipitation or epitope-tagging
approaches (Y. Huang,
M. Weindel, and R. Maraia, unpublished data). The
apparent weak
association of TBP and Brf would appear to reflect a
functional
feature of the
S. pombe Pol III system:
principally TATA-mediated,
rather than principally Brf-mediated,
recruitment of TBP to the
core promoter regions of tRNA
genes.
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.
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|
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.
|
|
Three tRNA gene templates, in TATA-containing and TATA-less versions,
were examined simultaneously in
S. cerevisiae and
S. pombe extracts (Fig.
5, top and bottom, respectively). The
S. cerevisiae tRNA
ser gene contains A
and B elements that match the consensus, while
the A and B box elements
of the
S. pombe tRNA
ser genes differ
from those of the
S. cerevisiae gene at one position
only, T
at position 10. Both the TATA-containing and TATA-less
versions of the
S. cerevisiae tRNA
ser gene (Fig.
5,
templates A and B) were efficiently transcribed
and excluded
transcription of the challenging tRNA gene (template
F) in
S. cerevisiae extract (top, lanes 1 and 2). In contrast
to this,
while either of the
S. cerevisiae tRNA genes could exclude
the second template in
S. pombe extract, only the
TATA-containing
gene (template B) was active for transcription while
the TATA-less
gene (template A) was inactive (bottom, lane 1 and 2).
Most significantly,
this indicates that although the tRNA gene is able
to interact
stably with limiting
S. pombe TFs, this alone is
not sufficient
to program a functional transcription complex in the
absence of
a TATA
element.
Both versions of the
S. pombe
tRNA
serUGA gene excluded transcription
from the second template in
S. cerevisiae extract (Fig.
5,
top, lanes 3 and 4), although the TATA-containing gene (template
D) was
transcribed more efficiently than the TATA-less gene (template
C).
Prior data suggest that the relatively low-level transcription
of the
S. pombe gene in
S. cerevisiae extract is almost
certainly
due to the single nonconsensus nucleotide, T10
(
48) (Fig.
5,
top, compare lanes 1 and
3).
In contrast to the stability observed in the
S. cerevisiae
system with the
S. pombe
tRNA
serUGA gene, this gene did not exclude
transcription from the second
gene (template F) in the
S. pombe system (Fig.
5, lanes 3 and
4). Because the promoters of the
S. cerevisiae and
S. pombe genes
differ at only
one position in the internal promoter (G10 versus
T10, respectively),
which is a significant determinant of transcription
(
48),
and because prior studies indicate that interaction of
TFIIIC with the
A box can be a determinant of template exclusion
(
1,
37,
61,
65), it is reasonable to deduce from the
data in Fig.
5 that
S. pombe TFIIIC stably interacts with the
consensus promoter
(templates A and B, lanes 1 and 2) but not
with the nonconsensus
promoter (templates C and D, lanes 3 and
4) and that this is the basis
of template exclusion in the former
but not the latter. Moreover, Fig.
5 provides additional information
that is relevant to a major
conclusion of this study: even in
the case where a stable complex is
formed in the
S. pombe system
(Fig.
5, bottom, lanes 1 and
2), presumably reflecting a stable
interaction of TFIIIC with the
consensus promoter (
37), this
is not sufficient to program
a functional preinitiation complex
in the absence of a TATA element. By
contrast, stable TFIIIC binding
is sufficient to recruit functional
TFIIIB in the
S. cerevisiae system (
29,
30,
33,
61,
65) (Fig.
5, top). The cumulative
data in Fig.
5 argue strongly
that a TATA element is required
for the functional programming of an
S. pombe transcription complex
even when all of the
necessary activities are
present.
The results support the model in which the TFIIIC-mediated placement of
TFIIIB, which leads to a functional transcription
complex in
S. cerevisiae, does not appear to occur efficiently,
if at all, in
the
S. pombe system (Fig.
5, bottom, lanes 1 and
2). A
second point that is suggested by the data is that the
trans-acting
factors that recognize TATA to promote
transcription need not
be recruited into a highly stable complex in the
S. pombe system,
since even when a functional complex is
assembled, it is not stable
on challenge by the orthologous promoter
(Fig.
5, bottom, lane
4). This unexpected observation suggests that
S. pombe TFIIIB
interacts less stably with tRNA
transcription complexes than does
S. cerevisiae TFIIIB (see
Discussion). The demonstrable differences
in the two transcription
systems were confirmed and strengthened
by results obtained with
control templates and were also observed
when the order of addition of
the genes was switched (lanes 5
to 10). The results provide convincing
biochemical evidence that
the
S. pombe and
S. cerevisiae transcription systems interact
differently with tRNA
genes but in a manner that is consistent
with the associations of
TFIIIB subunits shown in Fig.
4.
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.
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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 |
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).
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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.
|
|
The data in Fig.
5 suggest that although tRNA genes with A box residues
other than G at the number 10 position represent a
significant fraction
of genes, these may interact with TFIIIC
in a less stable manner than
those with G at this position. Attempts
to identify other features of
these genes that distinguished them
from the main set of tRNA genes was
not successful. It is also
interesting in this regard that the A box
sequences of the
S. pombe U6 and 7SL genes contain a G at
the corresponding position
of their A box sequences. In considering the
potential implications
of these findings, it may be important to recall
that both the
G10 and the non-G10 tRNA genes required an upstream TATA
for expression
in our assays. While the results suggest a potential for
effects
of the A box residue corresponding to G10 related to
transcription
complex formation and/or stability, the physiological
significance
of this, if any, remains an open
question.
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).
The requirement for an upstream TATA may explain, at least in part,
observations that indicate differential expression of
tRNA genes in the
two yeasts such that while
S. pombe tRNA genes
are
efficiently expressed in
S. cerevisiae, the TATA-less tRNA
genes from
S. cerevisiae were not expressed in
S. pombe (
34,
79).
Intriguingly, the data in Fig.
1 reveal an inverse correlation between
the prevalence of guanosine at position 10 and the
presence of an
upstream TATA motif, most readily observable for
S. cerevisiae,
S. pombe, and
A. thaliana. Since
G10 is invariant
or highly conserved in species that rely on TATA-less
transcription,
the data suggest that G10 may be a significant
determinant of
TATA-less transcription. This is consistent with the
data in Fig.
5 (top). For templates bearing T10 but no TATA,
transcription
activity is low relative to the same template with TATA
(compare
lanes 3 and 4), while for templates containing G10, the
presence
or absence of TATA makes little if any difference (compare
lanes
1 and
2).
TATA elements have been noted upstream of tRNA genes in
A. thaliana, and a TATA element has been reported to effect
reinitiation
of tRNA transcription in tobacco (
13,
81). A
CAA motif was
noted to be a transcription initiation site for plant
tRNA (
81).
The analysis in Fig.
1B suggests that this
motif may be extended
to ANC
AANA/T (the underlining
reflects a +1 site), reminiscent
of initiator elements (Inr)
found in some Pol I and Pol II genes
(
56,
66). However,
several differences distinguish the patterns
of sequence information
content in
S. pombe and
A. thaliana tRNA
genes
(Fig.
1B): (i) the information content is higher at TATA
for
S. pombe, (ii) the information content is higher at position
10 (greater propensity for G10) in
A. thaliana, and (iii) the
information content is higher around +1 for
A. thaliana. The
A. thaliana pattern is consistent with data that show
partial or
little effect of TATA mutations but larger effects of CAA
mutations
on plant tRNA expression (
9,
81). Thus, the
cumulative evidence
indicates that although fission yeasts and plants
exhibit TATA
elements upstream of their tRNA genes,
S. pombe
is more obligatorily
dependent on the TATA promoter element for
transcription.
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
tRNAAla synthesis in a tissue-regulated manner.
In this case, an upstream TATA directs expression of the more active of
the two tRNAAla genes in a TBP-dependent manner
(50). This tRNAAla 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.
The transcription system characterized here suggests that Pol III
initiation in
S. pombe may be comparable to type 3 gene
transcription and other systems that use TATA elements to recruit
TBP.
Although for tRNA transcription, the yeast and mammalian
TFIIIB
subunits appear to be completely orthologous, the situation
is more
complex for U6 snRNA expression in metazoa (e.g., type
3 genes), as a
distinct variant of BRF known as BRFU/TFIIIB50
characterizes the
TFIIIB activity that mediates type 3 gene transcription
(
63,
70). The BRFU/TFIIIB50 variant that acts at the U6
TATA-containing
promoter differs from Brf in its lack of a C-terminal
domain that
in Brf has been shown to interact with TBP (
10,
26,
32),
and consistent with this, it may not stably bind TBP, since
association
of TFIIIB with TBP was notably weak (
70). This
suggests principal
recruitment of TBP by the TATA element, which may
obfuscate a
requirement for stable interaction between TBP and BRFU in
the
absence of DNA (
70). In this regard, the
S. pombe Pol III initiation
system may be comparable to Pol II, in
which TBP and TFIIB do
not form a stable association in the absence of
DNA. Instead,
association of TBP with the TATA element appears to
create a binding
site for TFIIB, in part due to the specific bending
effect of
TBP on TATA DNA (
49). While our results indicate
a significant
role for TATA in tRNA transcription in
S. pombe, they also leave
open the possibility that Brf-mediated
recruitment of TBP occurs
at some level in vivo and that some tRNA
genes are less dependent
on the TATA element for TBP recruitment than
others. However,
the cumulative data suggest that TATA-mediated
recruitment of
TBP plays a major role in Pol III transcription in
S. pombe, while
Brf may be brought to the upstream region in
part by TFIIIC and
in part by recognition of the TATA-TBP complex. In
any case,
S. pombe has revealed a novel Pol III system that
should be useful
for studying eukaryotic transcription initiation and
the function
of the core
promoter.
| |
ACKNOWLEDGMENTS |
We are grateful to W. Makalowski for alignments and for
suggesting Logo. We thank R. Intine for pFL20/18SPst5.8Si4 and
technical advice, E. P. Geiduschek, N. Hernandez, R. Roeder, D. Setzer, and D. Reinberg for discussions, and I. Willis for
encouragement and expert advice.
Mitsuhiro Hamada and Ying Huang contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 6 Center Dr.,
Rm. 416, Bethesda, MD 20892-2753. Phone: (301) 402-3567. Fax: (301)
480-6863. E-mail: maraiar{at}mail.nih.gov.
Present address: Department of Biochemistry, Saitama Medical
School, Moroyama, Iruma-gun, Saitama, 350-0495, Japan.
| |
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Molecular and Cellular Biology, October 2001, p. 6870-6881, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.6870-6881.2001
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Abstract
Introduction
