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(Received for publication, April 24, 1996, and in revised form, June 19, 1996)
From the Department of Biological Chemistry, University of California, Irvine, California 92697-1700
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
ABSTRACT 
A new gene, RRN11, has been defined by certain rrn mutants of Saccharomyces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I). We have cloned the gene and found that it encodes a protein of 507 amino acids. We have used a strain with the chromosomal RRN11 deleted and carrying HA1 epitope-tagged RRN11 on a plasmid to isolate a protein complex containing the protein encoded by RRN11. This protein complex complemented rrn6 mutant extracts, which were previously shown to be deficient in the essential pol I transcription factor called Rrn6/7 complex or core factor (CF). The CF complex was previously shown to consist of three proteins, the 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, and the 66-kDa subunit. The results of the above complementation experiments combined with mobility of Rrn11p in SDS-polyacrylamide gel electrophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion that RRN11 encodes the 66-kDa subunit of CF. Glutathione S-transferase-Rrn11p fusion protein was found to bind strongly to 35S-labeled Rrn6p and Rrn7p but only weakly to 35S-labeled TATA-binding protein. Similarly, glutathione S-transferase-Rrn7p fusion protein bound strongly to 35S-labeled Rrn6p and Rrn11p but only weakly to 35S-labeled TATA-binding protein. These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex does not contain TATA-binding protein. RRN11 was shown to be an essential gene, and [3H]uridine pulse experiments demonstrated directly that RRN11 is essential for rDNA transcription by pol I in vivo. Thus all three subunits of CF are essential for rDNA transcription. Because of the resemblance of CF to mammalian essential pol I transcription factor SL1, the amino acid sequences of Rrn11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors (TAFs) in the human SL1, TAFI48, TAFI63, and TAFI110. No significant similarity was detected between two sets of the proteins. Similarity as well as differences between CF and SL1 are discussed.
INTRODUCTION
Transcription of rRNA genes is central to the overall regulation of ribosome synthesis and has been studied extensively. In eukaryotes, the genes for large rRNA (rDNA)1 are tandemly repeated in the nucleolus, and RNA polymerase I (pol I) is devoted to their transcription. For comprehensive understanding of rDNA transcription and its regulation, it is essential to identify and characterize molecular components of the transcriptional machinery that participate in rDNA transcription. Since there is some evidence that growth rate regulation of rDNA transcription takes place at the step of transcription initiation (for reviews, see Refs. 1, 2, 3, 4), knowledge of the molecular components of transcription initiation factors as well as of pol I may be particularly important for understanding of regulation of rDNA transcription.
Structures of the rRNA gene promoters appear to be similar among various eukaryotes including the yeast Saccharomyces cerevisiae. The rRNA gene promoter, which covers about 150 base pairs upstream from the transcription start site and extends slightly into the transcribed region, consists of two domains, a core promoter element covering the transcription initiation site and an upstream element. In a variety of experimental systems, it has been shown that the core element is necessary and sufficient for specific initiation, while the upstream element greatly stimulates initiation. These two cis-elements probably operate in concert to achieve efficient transcription from the promoter (for reviews, see Refs. 1, 2, 3, 4).
Regarding trans-acting protein factors, extensive studies using metazoan in vitro transcription systems have identified at least two factors, UBF and SL1 (5), also known by other names, e.g. TIF-IB (6). UBF interacts with both the upstream element and the core element and helps the binding of SL1, which enables recruitment of pol I. The genes for UBF, which is a single polypeptide, have been cloned and characterized from a variety of organisms (4, 7). In contrast to the cellular abundance of UBF, SL1 is present in much smaller amounts (5), but its purification from human (8) and mouse (9) has been achieved, revealing the presence of TBP and three other polypeptides (TAFs). The genes for the three human TAFs have recently been cloned and sequenced, and active human SL1 has been reconstituted from these four recombinant proteins (10, 11). Despite the impressive progress coming from these in vitro studies, however, many questions remain. For example, UBF is a DNA-binding protein with low sequence specificity (12, 13) and binds not only to the upstream element but also to the core element as well as other regions of the template. Thus, the relationship between the stimulatory activity of the upstream element as a cis-element and that of UBF as a trans-factor has remained unclear. The question of how SL1, singly or together with UBF, makes pol I recruitment possible has also remained unexplored.
We have been studying transcription of rDNA by pol I in the yeast S. cerevisiae using both genetic and biochemical approaches. By using a fusion gene (pGAL7-35 S rDNA), which consists of the 35 S rRNA coding region fused to the galactose-inducible GAL7 promoter (pGAL7), we isolated mutants (rrn mutants) that showed no or only very poor growth on glucose media but could grow on galactose media because of transcription of the 35 S rDNA by pol II (14). Mutants were classified into at least 12 complementation groups defining genes whose function is specifically involved in rDNA transcription by pol I (14, 15, 16).2 Five of these genes, RRN1, RRN2, RRN4, RRN12, and RRN13, were shown to encode pol I-specific subunits of the polymerase, the A190, A135, A12, A43, and A49 subunits, respectively (14, 15, 17).2 By analyzing RRN6 and RRN7, we have previously demonstrated that these genes encode the 102- and 60-kDa subunits, respectively, of a complex (Rrn6/7 complex) that also contains another protein (p66) with an apparent molecular mass of about 66 kDa (18). By in vitro transcription experiments using crude extracts from rrn6 or rrn7 mutant cells, it was demonstrated that the Rrn6/7 complex is essential for forming a transcription-competent preinitiation complex, but its stable binding to the promoter depends on the initial binding of some other factor(s) (18). More recently, we have demonstrated that the stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF, which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. It was also found that UAF alone interacts with the upstream element and forms a stable complex, helping to recruit other factor(s) including the Rrn6/7 complex to the promoter and committing the template to transcription (16). Like the upstream element of the promoter, UAF is greatly stimulatory but not absolutely required for rDNA transcription. In contrast, the Rrn6/7 complex is essential; rDNA transcription requires the presence of the Rrn6/7 complex regardless of whether template contains the upstream element or not (16, 18)3; that is, this complex must function in conjunction with the core element of template. Thus, we now call this complex core factor or CF. As discussed previously (18) and also in this paper, CF interacts, although weakly, with TBP and appears to be similar to SL1 in metazoan systems.
By studying a newly defined RRN gene RRN11 we have now demonstrated that this gene encodes the previously uncharacterized subunit p66 of the Rrn6/7 complex or CF. In this paper, we first describe cloning and characterization of RRN11 and then examine interactions of the three CF subunits, Rrn6p, Rrn7p, and Rrn11p among themselves and with TBP in vitro. Independently, RRN11 was also cloned and characterized by Reeder and co-workers.4
MATERIALS AND METHODS
Yeast strains and plasmids used
are listed in Table I and Fig. 1. The two plasmids
pNOY345 and pNOY346 (see Fig. 1A) were isolated from the
ATCC77164 yeast genomic library (CEN6 ARS4 TRP1) prepared
from the strain YPH15 (provided by C. Connelly
and P. Hieter). pNOY349 (CEN6 ARS4 TRP1 RRN11) was
constructed by cloning the 2.6-kb ApaI-NheI
fragment from pNOY346 into pRS314 (19) between ApaI and
SpeI (see Fig. 1B). pNOY350 (CEN6 ARS4 URA3
RRN11; Table I) is a derivative of pRS316 (19) carrying the 2.6-kb
KpnI-SacI fragment from pNOY349 cloned between
KpnI and SacI sites. pNOY351 (CEN6 ARS4
TRP1 rrn11
::LEU2) was constructed from pNOY349 by
replacing the 1.8-kb SpeI-Eco47III
RRN11 fragment with the 2.2-kb
XbaI-HpaI LEU2 fragment isolated from
YEp351 (20) (see Fig. 1B). pNOY354 (Table I) was constructed
as follows: a NotI site was first inserted immediately
upstream of the RRN11 stop codon of plasmid pNOY349 by
site-directed mutagenesis as described by Kunkel et al.
(21). The 0.5-kb NotI-SphI fragment, which
carries the nucleotide sequence encoding three tandem copies of the HA1
epitope peptide (HA1)3 (for the exact sequence of the
peptide, see Ref. 16) and part of the ADC1 terminator and
was isolated from plasmid pNOY330 (16) was then inserted into the
corresponding NotI-SphI sites, yielding pNOY354.
Similarly, pNOY303 (Table I) was constructed in the following way: A
NotI site was first created immediately downstream of the
RRN7 methionine codon of pNOY3165, which is a pBluescript II
KS(
) derivative carrying an RRN7 fragment between
XhoI and XbaI sites, yielding pNOY3246. The
0.1-kb NotI fragment carrying (HA1)3 from the
GTEP1 plasmid (22) was then inserted into the NotI site of
this plasmid. Next, the KpnI-EcoRI fragment of
the resultant plasmid was cloned between the corresponding
KpnI and EcoRI sites of pNOY252, which carries
HA1-RRN7 and is similar to pNOY209 but is a derivative of
pRS314 rather than pRS316 (18), replacing HA1-RRN7 by
(HA1)3-RRN7 and yielding pNOY303.
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::LEU2). Vector
DNA regions are shown by thick lines. The chromosomal insert
is shown by thin lines and with cross-hatched
arrows that represent open reading frames and indicate the
direction of transcription. The locations of the Ty1 elements that are
present in the DNA sequences in the data bank
(GenBankTM/EMBL Data Bank accession numbers SC9827, SC8054,
and SC8337) but are absent in the DNA fragments cloned in this work are
indicated in A by open triangles. Structures of
pNOY351 carrying the rrn11::LEU2 and of its
parent (pNOY349) are shown in B. Restriction enzyme sites
shown are: A, ApaI; Bg, BglII; Ec,
Eco47III; H, HpaI; N, NheI; P,
PstI; S, SacI; Sp, SpeI; X, XbaI.
Haploid segregants obtained in tetrad analysis of NOY728 shown in
C were grown in yeast-extract-peptone-glucose (24) for 5 days at 30 °C.
pNOY3244 (Table I) was constructed by cloning the 1.7-kb
NotI-XbaI fragment carrying RRN7 from
plasmid pNOY3246 (see above) into the NotI-XbaI
sites of pNOY3247, fusing the RRN7 protein coding region to
the glutathione S-transferase (GST) coding region. pNOY3247
is a derivative of pGEX-3X (Pharmacia Biotech Inc.) and was constructed
by inserting the NotI linker 5
-AGCGGCCGCT-3 into the
SmaI site and the XbaI linker 5-CTCTAGAG-3 into
the EcoRI site (which had been blunted with the Klenow
enzyme) of pGEX-3X. Similarly, pNOY3241 (Table I) was constructed by
cloning the 1.5-kb NotI-SpeI fragment from
plasmid pNOY355 carrying the RRN11 into pNOY3247 that was
cut by NotI and XbaI. pNOY355 was constructed
from pNOY349 (see Fig. 1) by creating a NotI site
immediately downstream of the methionine initiation codon of the
RRN11 gene using site-directed mutagenesis (21).
Plasmids used to make 35S-labeled TBP, Rrn6p and Rrn11p
in vitro, pNOY3171, pNOY3173, and pNOY3245, respectively
(Table I), were all constructed as derivatives of pNOY3168. Plasmid
pNOY3168 is a derivative of pBS() (Stratagene) and was constructed by
inserting a 1.7-kb EcoRI-StuI fragment from
pCITETM-1 vector (Novagen) between EcoRI and
SmaI of pBS(), placing this fragment carrying the CITE
sequence between the T7 promoter and several cloning sites of pBS().
Plasmid pNOY3171 carries a 1.1-kb fragment DNA inserted between the
BalI and BamHI sites of pNOY3168, placing the
entire TBP coding region as an open reading frame starting from the AUG
codon of the CITE. The 1.1-kb DNA was derived from pAB24 (23) and its
5-end was created by polymerase chain reaction so that the required
proper fusion took place in the final construct. Plasmid pNOY3173 was
similarly constructed by inserting a 3.1-kb fragment carrying
RRN6 between the BalI and PstI sites
of pNOY3168, placing the entire RRN6 coding region as an
open reading frame starting from the CITE AUG codon. The fusion to the
CITE AUG codon was achieved by the use of polymerase chain reaction. It
should be noted that the second amino acid of Rrn6p encoded by the
final construct (pNOY3173) is now glycine (GGT) rather than serine
(AGT) as a result of the fusion to the CITE sequence. Plasmid pNOY3245,
which carries the RRN11 coding sequence fused to the CITE
AUG codon, was also constructed. A 1.6-kb DNA fragment, which carries
RRN11 (from the codon encoding the second amino acid) and
the distal nontranslated DNA sequence present in pNOY349 (Fig.
1B), was inserted between BalI and
SphI of pNOY3168. The fusion was achieved by
oligonucleotide-directed mutagenesis, which resulted in the insertion
of a glycine codon (GGC) between the first ATG (for methionine) and the
second TTT (for phenylalanine) codons of RRN11 in the
the final construct (pNOY3245). Plasmid pNOY221 used to make
35S-labeled HA1-Rrn7p was constructed from pNOY209 (18) by
deleting the AflII-KpnI fragment.
All genetic techniques are standard procedure (24). NOY728 (Table I)
was constructed by transformation of the
EagI-SmaI fragment (carrying
rrn11::LEU2), which was obtained from plasmid
pNOY351 (Fig. 1B), into NOY397, selecting Leu+.
NOY729, NOY730, and NOY731 were constructed from NOY728 using pertinent
plasmids as indicated in Table I. NOY732 was obtained after
transformation of yeast NOY553 (same as NOY552 but Mat
) (18) with
pNOY303 and eliminating the resident pNOY209 plasmid using
5-fluoroorotic acid.
A complex containing (HA1)3-Rrn11p was isolated from extracts of strain NOY731 expressing (HA1)3-Rrn11p using anti-HA1 antibodies attached to protein G-Sepharose as described previously (18). Detail of in vitro transcription reactions was also described previously (18). Transcription extracts PC-300 (eluting from the phosphocellulose column at 300 m KCl) and D-300 (eluting from DEAE column at 300 m KCl) fractions (18) were prepared from rrn6 deletion mutant strain NOY567. The DNA template used was a circular supercoiled DNA (pSIRT) containing a yeast rDNA minigene which generates a specific transcript of approximately 765 nucleotides that is apparently unprocessed and correctly terminated (18). Reactions were carried out for 40 min at room temperature, and radioactive RNA synthesized was analyzed by 5% urea-PAGE followed by autoradiography.
Protein Binding AssaysGST-Rrn7p and GST-Rrn11p fusion
proteins were prepared by induction of Escherichia coli
strains carrying pNOY3244 and pNOY3241, respectively, with
isopropyl-
--thiogalactoside, followed by affinity
purification of fusion proteins using glutathione-agarose beads
(Sigma) as described previously (25).
[35S]Methionine-labeled TBP, Rrn6p, Rrn7p, and Rrn11p were synthesized in vitro using rabbit reticulocyte lysate systems (Promega), and pNOY3171, pNOY3173, pNOY221, and pNOY3245, respectively, were used as template (Table I; see above). These 35S-labeled proteins were incubated with GST-Rrn11p (or GST-Rrn7p or control GST), which were attached to glutathione beads and prewashed with and suspended in buffer A (Tris-HCl, pH 7.5, 10 m; MgCl2, 5 m; CaCl2, 5 m; NaCl, 200 m; Nonidet P-40, 0.1%) containing 0.2% bovine serum albumin in a final volume of 200 µl. After 1 h at room temperature, the beads were recovered and washed extensively with buffer A (without bovine serum albumin) and proteins bound were analyzed by SDS-PAGE.
RESULTS
Like many other rrn mutants (14), three mutants were independently isolated from NOY418, which carries the GAL7-35 S rDNA fusion on a plasmid (pNOY103) as mutants that grow on galactose but not on glucose. By standard genetic crosses with other rrn mutants and with each other, we have shown that these three mutations represent a new complementation group, which now defines the RRN11 gene.
We used one of the mutants (NOY727, isolation number 1016) carrying
rrn11-1 to clone the RRN11 gene. The mutant
cells were transformed with a yeast genomic library, and transformants
with the growth characteristics of the wild type (RRN11)
were isolated on glucose plates, the plasmids were recovered, and
partial sequencing of isolated yeast genomic inserts showed that the
region covering the RRN11 locus had already been determined
as part of the yeast genome sequencing project. By constructing the
plasmid pNOY349 (Fig. 1B), which complements
the mutation, we were able to conclude that RRN11
corresponds to an open reading frame called YM9827.09c and is located
between PRP39 and CAT2 on chromosome XIII. The
amino acid sequence of Rrn11p deduced from the nucleotide sequence is
shown in Fig. 2. The protein (Rrn11p) encoded by
RRN11 is 507 amino acids in size with a calculated molecular
weight of 59,200 and a calculated isoelectric point of 5.9. No
significant homology to any sequence in the current data banks was
observed. We note that the DNA sequence in the data bank indicates the
presence of two Ty1 elements that are absent in the DNA fragments we
recovered from the genomic library (Fig. 1A). This
difference reflects a DNA polymorphism caused by transposition of Ty1
element and is consistent with the known preference of Ty1 integration
near tRNA genes (26).
We carried out standard gene disruption experiments. A diploid strain (NOY728) with one wild-type copy of RRN11 and one copy completely deleted and replaced by LEU2 (Fig. 1B) was constructed and sporulated, and tetrads were dissected. Tetrad analysis showed the expected two viable and two nonviable segregation pattern at all temperatures studied, which ranged from 20 to 37 °C, and cosegregation of spore viability and leucine auxotrophy, demonstrating that RRN11 is an essential gene (Fig. 1C).
We constructed a haploid strain with the chromosomal RRN11
deleted and carrying the pGAL7-35 S rDNA on the pNOY103
plasmid. This strain, (NOY730) was used to examine effects of the
deletion of RRN11 on rDNA transcription in vivo.
The strain was grown in galactose medium, shifted to glucose medium to
repress rRNA synthesis from the GAL7 promoter, and then
incorporation of [3H]uridine into large rRNAs was
examined and compared with the control wild-type (RRN11)
strain. As shown in Fig. 3, the synthesis of large rRNAs
(18 S, 25 S, and 5.8 S rRNAs and other precursor rRNAs) was not
detected in the mutant in glucose medium, while the synthesis of 5 S
RNA and tRNAs continued as in galactose medium (Fig. 3, lane
4 compared to lane 3). Quantitation showed that the
synthesis of large rRNAs relative to 5 S RNA plus tRNAs in the mutant
is less than 1% of that in the wild type. The results confirm the
conclusion that RRN11 is an essential gene and is
specifically required for transcription of rDNA.
::LEU2 mutant and
the wild-type control strains before and after shift from galactose to
glucose media. A wild-type control strain NOY418 (wt, lanes
1 and 2) and rrn11::LEU2 mutant
strain NOY730 (rrn11, lanes 3 and
4) were grown at 30 °C, and [3H]uridine
pulse-labeling was carried out as described previously (16) before
(GAL, lanes 1 and 3) and after (GLU, lanes
2 and 4) shift from galactose to glucose media. RNA
samples containing approximately equal radioactivity were subjected to
electrophoresis. An autoradiogram of the dried gel is shown.
RRN11 Encodes the p66 Subunit of the Rrn6/7 Complex (CF)
Based on two observations, we considered the possibility
that RRN11 encodes the p66 subunit of the Rrn6/7 complex
(18) (CF), the essential transcription factor containing Rrn6p, Rrn7p,
and uncharacterized p66. First, the RRN11 gene is essential
like RRN6 and RRN7 both for viability and for
rDNA transcription in vivo and is different from
RRN5, RRN9, and RRN10, which are not
essential for viability and are not absolutely required for rDNA
transcription, although greatly stimulatory both in vivo and
in vitro. Second, the calculated molecular mass of Rrn11p is
59.2 kDa and is not very different from the apparent molecular mass (66 kDa) of p66. In order to test this possibility, we constructed a
haploid strain (NOY731) with the chromosomal RRN11 deleted
and carrying a triple-HA1-tagged RRN11 gene
(HA1)3-RRN11 on a centromeric plasmid. This
strain grew at the same growth rate as the isogenic wild-type strain,
indicating that the epitope-tagged protein functions like the native
protein. Extracts were prepared from this haploid strain, and protein
complexes containing Rrn11p were isolated by an immunoaffinity
purification method using monoclonal antibodies against the HA1
epitope. If Rrn11p is identical to the p66 subunit of CF, the
affinity-purified preparation obtained in this way should contain CF
and should be able to complement extracts prepared from an
rrn6 mutant (or an rrn7 mutant) which are missing
the intact CF. The results shown in Fig. 4 demonstrate
that this is indeed the case. The preparation complemented the
rrn6 extracts (lanes 5 and 6 compared
to a positive control, lane 2, and the negative control,
lane 1). The mock-purified control preparation obtained from
control wild-type (RRN11) cells without HA1 epitope-tagging
had no complementation activities (lanes 3 and
4). We conclude that Rrn11p is complexed with Rrn6p and
Rrn7p and is thus most likely identical to p66.
In previous work (18) the purified Rrn6/7 complex (CF) was analyzed by
SDS-PAGE and the apparent molecular masses of the three components,
Rrn6p, p66, and Rrn7p, were found to be 115, 66, and 56 kDa,
respectively. Since the molecular mass of Rrn11p calculated from the
DNA sequence (59.2 kDa) is near the apparent molecular mass of both
Rrn7p (56 kDa) and p66, we examined the mobility of Rrn11p in SDS-PAGE
relative to Rrn7p. We analyzed the same affinity-purified preparation
containing triple-HA1-tagged Rrn11p ((HA1)3-Rrn11p) by
SDS-PAGE followed by Western blot using anti-HA1 antibodies. For
comparison, we also analyzed a sample containing
(HA1)3-Rrn7p prepared from strain NOY732. The mobility of
(HA1)3-Rrn11p relative to that of (HA1)3-Rrn7p
(and relative to molecular weight markers) was consistent with what we
expect from Rrn11p being identical to the protein observed as p66 in
the previous work (data not shown). In addition, as described below, we
synthesized 35S-labeled Rrn6p, Rrn7p, and Rrn11p in
vitro using reticulocyte translation systems. The mobilities of
these radioactive proteins in SDS-PAGE were also consistent with the
results obtained in the previous work for the three components of CF,
Rrn6p, Rrn7p, and p66 (Fig. 5). Thus, Rrn11p must be
identical to the p66 of CF observed in the previous work.
In Vitro Interactions among CF Subunits and TBP
Because we have now cloned all the genes for the components of CF, one can design experiments to examine interactions of these protein components with each other and with other proteins involved in rDNA transcription such as TBP, protein components of UAF, or subunits of pol I. As a first step in this direction, we prepared GST-Rrn11p and GST-Rrn7p fusion proteins and studied their interactions with 35S-labeled Rrn6p, Rrn7p, Rrn11p, and TBP. The labeled proteins were synthesized in vitro using reticulocyte translation systems. As shown in Fig. 5A, Rrn6p and Rrn7p bound with a high efficiency to GST-Rrn11p fusion protein attached to glutathione-agarose beads in the presence of 200 m NaCl. Similarly, Rrn6p and Rrn11p also bound with a high efficiency to GST-Rrn7p (Fig. 5B). Under the same conditions, 35S-labeled TBP bound more weakly to GST-Rrn11p and GST-Rrn7p but not to GST (Fig. 5, A and B). The amounts of 35S-labeled proteins bound to GST fusion proteins in these experiments were quantified. The following values (as percent of input) were obtained as averages from two experiments (one shown in Fig. 5): A, binding to GST-Rrn11p: TBP, 10.3 ± 3.1%; Rrn7p, 33.8 ± 6.8%; Rrn6p, 33.2 ± 7.5%. B, binding to GST-Rrn7p: TBP, 7.9 ± 1.4%; Rrn11p, 42.3 ± 8.3%; Rrn6p, 29.9 ± 1.5%. It should be noted that the amounts of GST fusion proteins used were approximately the same and that 35S-labeled Rrn6p, Rrn7p, and Rrn11p were added in approximately equal molar amounts, while the amount of 35S-labeled TBP added was about 5 times higher relative to these three probes. We also note that we have not succeeded in expressing a GST-Rrn6p fusion protein in E. coli and hence have not studied binding of 35S-labeled TBP to GST-Rrn6p. Although the interaction of TBP with the three individual CF subunits as well as with the assembled CF complex needs further study, we conclude that interactions among three individual CF subunits are all strong, suggesting that these three subunits also interact with each other within the assembled CF complex.
DISCUSSION
As stated in the introduction, two transcription initiation factors have been characterized in the yeast pol I system. One is UAF, which interacts with the upstream element of the promoter and is greatly stimulatory but is not essential. (UAF contains three proteins encoded by RRN5, RRN9 and RRN10, respectively, and probably two additional proteins.) The second is CF, which is essential for rDNA transcription and is composed of three proteins, two proteins encoded by RRN6 and RRN7, respectively, and p66. Identification of RRN11 as the gene encoding the previously recognized p66 by the present study as well as by Reeder and co-workers4 completes cloning and sequencing of all the three components of CF.
We have shown that the three components of CF interact strongly with each other in vitro. Under the same conditions, Rrn11p and Rrn7p interact with TBP more weakly; the interaction of the third CF subunit, Rrn6p, with TBP has not been studied in the same way. These observations are consistent with the fact that one can purify the complex consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex (CF) does not contain TBP (18). In addition, we have found that a temperature-sensitive rrn7 mutation can be suppressed by the presence of a multicopy plasmid carrying RRN6,6 suggesting that the direct interaction between Rrn6p and Rrn7p observed in vitro also takes place in vivo. Regarding the weak interactions observed between the TBP and Rrn7p (or Rrn11p), although they appear to be specific in vitro, their significance in vivo has to be examined by other means. Using a yeast two-hybrid system, we have observed an interaction between Rrn6p and TBP within yeast cells, but we have not demonstrated a similar interaction between TBP and Rrn7p (or Rrn11p) with this method.3 It should be noted that apparently specific physical interactions observed in vitro do not necessarily prove functional significance of such interactions in vivo. For example, it has been demonstrated that the ability of TBP to interact with transcriptional activators in vitro is not directly relevant to its ability to support activated transcription in vivo (27). Purified CF, consisting of Rrn6p, Rrn7p, and Rrn11p, binds to GST-TBP only very weakly in vitro. The interaction was observed in the presence of 200 m potassium glutamate but not under conditions similar to those used in the present study, that is, in the presence of 200 m KCl.3 Thus, it is possible that some direct interactions between TBP and Rrn11p (or Rrn7p) observed in this study, although they are apparently specific, might not take place in the mixture of TBP and assembled CF complex even under the same in vitro conditions, perhaps because of masking of the TBP-interacting sites in uncomplexed Rrn11p (or Rrn7p). This and related questions are under current study.
As mentioned above, the Rrn6/7/11 complex, CF, appears to interact with TBP specifically, although weakly, and thus resembles SL1 of metazoan systems, which consists of TBP and three TBP-associated proteins (TAFs). Because of the resemblance of CF to mammalian SL1, the sequences of Rrn6p and Rrn7p were previously compared with those of the three TAFs in the human SL1, TAFI48, TAFI63, and TAFI110. No sequence similarity was detected between two sets of the proteins (10, 18). With the sequence of Rrn11p now available, we compared it with the sequences of the three TAFs in the human SL1. No significant similarity was detected. In view of the well recognized evolutionary divergence of components in the pol I transcription system, the absence of primary sequence similarity between human SL1 and yeast CF components may not be surprising. Nevertheless, it is noteworthy that some unique sequence features are not shared. For example, TAFI63 in the human SL1 contains two putative zinc fingers, and it was suggested that they might be involved in binding of TAFI63 to promoter DNA as observed in ultraviolet cross-linking experiments (10). No such zinc finger motif is present in Rrn11p (or Rrn6p or Rrn7p). Similarly, as noted previously (18), the leucine zipper-like motif found in Rrn6p, which might be involved in an interaction with some other protein, is not present in human TAFI proteins. Thus, it is still premature to conclude that CF and SL1 are really functionally homologous. It should also be noted that although CF appears to interact with TBP specifically, UAF also shows a specific interaction with TBP, and this interaction is in fact stronger than that between CF and TBP in vitro.3 Elucidation of the exact functional roles of TBP in both the yeast and the metazoan pol I transcription systems is an important subject of future study and may also help to settle the question of whether CF and SL1 are functional homologues.
As stated above, previous studies have demonstrated that UAF is greatly stimulatory but is not essential for rDNA transcription, whereas CF is essential for rDNA transcription (16, 18). This conclusion was obtained first from in vitro experiments and then confirmed by in vivo analyses using a set of yeast mutants which carry a deletion in one of the three genes, RRN5, RRN9, and RRN10, which encode subunits of UAF, or a deletion in RRN6 or RRN7, which encodes a subunit of CF. Strains carrying rrn5, rrn9, or rrn10 deletion are viable, and in vivo [3H]uridine pulse-labeling experiments also indicated the presence of weak but detectable rDNA transcription by pol I in these deletion strains (16). In contrast, strains carrying rrn6 or rrn7 deletion are nonviable (16, 18), and no residual rDNA transcription by pol I was detected in [3H]uridine incorporation experiments (16). Experiments described in this paper have demonstrated that a strain with a complete RRN11 deletion is nonviable, and no pol I-dependent rDNA transcription was detected in similar [3H]uridine pulse-labeling experiments in vivo. Thus, all three subunits of CF, Rrn6p, Rrn7p, and Rrn11p, appear to be essential for the function of CF, the essential transcription factor of the yeast pol I. Now that the genes for all three subunits are available, we should be able to study in more detail functional roles of this essential transcription factor in rDNA transcription in relation to other molecular components participating in this important process.
FOOTNOTES
To whom correspondence should be addressed. Tel: 714-824-4564;
Fax: 714-824-3201.
Acknowledgments
We thank K. Sutton, L. Vu, and D. A. Keys for their participation and help in some aspects of the present work, J. Keener for critical reading of the manuscript, and M. Oakes and D. Semanko for help in preparation of the manuscript. We also thank Dr. R. H. Reeder and colleagues for communicating their findings prior to publication.
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