ABSTRACT
The transcription termination factor TTF-I binds specifically to an 18 bp DNA element in the murine ribosomal gene
spacer and mediates termination of RNA polymerase I transcription. In this
study, we have compared DNA binding and termination activity of recombinant
full-length TTF-I (TTF-I
p130
) with two deletion mutants lacking 184 and 322 N-terminal amino acids, respectively. All three proteins exhibit similar
termination activity, but the DNA binding of TTF-I
p130
is at least one order of magnitude lower than that of the deletion mutants,
indicating that the N-terminus represses the interaction of TTF-I with DNA. The inhibitory effect of the N-terminus can be transferred to a heterologous DNA binding
domain and is separable from other activities of TTF-I. We show by several methods that TTF-I
p130
, the N-terminal domain alone, and fusions of the N-terminus with the DNA binding domain of Oct2.2 form stable oligomers
in solution. Thus, in contrast to previous studies suggesting that activation
of TTF-I occurs through proteolysis, we demonstrate that full-length TTF-I
mediates
termination of rDNA transcription
in vivo
and
in vitro
and that the oligomerization state of TTF-I may influence its DNA binding activity.
Termination of mouse ribosomal gene transcription requires binding of the
nucleolar transcription termination factor TTF-I to a repeated DNA element, termed `Sal box', located downstream of the
rRNA coding region (
1
,
2
). An additional terminator element is found 170 bp upstream of the rDNA
transcription start site (
3
,
4
). Even though there is marked sequence divergence between terminator elements
from different organisms, such as mammals, frog and yeast, the mechanism of RNA
polymerase I (Pol I) transcription termination is probably similar or even
identical in these diverse species. All characterized Pol I terminators function in only one orientation and bind a protein which
presumably contacts the elongating RNA polymerase I (for review, see ref.
5
). In mammalian cells, this protein is known as Transcription Termination Factor
(TTF-I). Cloning and deletional analysis of the cDNAs for human and murine TTF-I has revealed distinct regions of the protein which may represent
different functional domains (
6
,
7
). The DNA binding domain which resides within the C-terminal half of the protein is highly conserved between human and mouse (
7
). Significantly, this part of TTF-I shows striking homology both to the DNA binding domain of the proto-oncoprotein c-Myb (
8
) and the yeast transcription factor Reb1p (
9
). The functional significance of this sequence homology has been demonstrated
by site-directed mutagenesis. Mutation of the conserved tryptophan residues known
to be important for specific DNA binding by c-Myb (
8
) prevented TTF-I binding to the `Sal box' motif (
6
). Moreover, Reb1p bound to its target site within the enhancer of yeast rDNA
stops Pol I transcription and, therefore, represents the yeast equivalent to
mammalian TTF-I (
10
).
The N-terminal half of TTF-I exhibits a much less pronounced sequence conservation between
human and mouse. The central region, between residues 430 and 445, seems to be
important for termination
per se
, since deletion of this part impairs termination without affecting DNA binding
(
6
). The N-terminal domain (amino acids 1-322) can be deleted without affecting the function of TTF-I in termination assays (
6
). In a previous effort to purify TTF-I from mouse cells we have isolated a heterogeneous group of polypeptides
(p65, p80, p90, p100) which form distinct DNA-protein complexes on `Sal box' DNA and mediate transcription termination
(
11
). This heterogeneity of TTF-I has been attributed to proteolysis of the full-length protein, TTF-I
p130
. In these previous studies full-length TTF-I
was not detected because the ability to interact with the `Sal box' was strongly
impaired in TTF-I
p130
compared to proteolytic derivatives (
11
). This observation, together with the finding that N-terminal deletion mutants of recombinant TTF-I efficiently interacted with the `Sal box' target sequence,
suggested that the N-terminus represses specific DNA binding of TTF-I.
Recent reports from several groups have challenged the dogma that DNA binding
domains function autonomously. Several transcription factors, including TATA-binding protein (TBP), p53, NF-[kappa]B and members of the
ets
family, bind to DNA with higher affinity when truncated than as full-length protein (
12
-
15
). Moreover, it has been shown that domains distinct from the DNA binding region
of some proteins actually negatively influence the DNA binding activity of
these factors (
15
,
16
). In this report we have examined the function of the N-terminal domain of TTF-I. We find that the DNA binding activity of recombinant full-length TTF-I is at least one order of magnitude lower than that of
N-terminally truncated versions. Nevertheless, in partially purified
transcription systems, both TTF-I
p130
and N-terminal deletion mutants exhibit similar termination activities which
suggests that cellular proteins may interact with the N-terminus of TTF-I and relieve its repressive effect. Consistent with the idea that
this part of TTF-I may be involved in mediating protein-protein interactions, we demonstrate that the N-terminal domain forms stable oligomers in solution and acts
autonomously in repressing DNA binding activity when fused to a heterologous
DNA binding domain.
Constructs containing histidine-tagged TTF-I
p130
, TTF[Delta]N185 and TTF[Delta]N323 in pRSET (Invitrogen) were described by Evers
et al
. (
6
). For expression in baculovirus infected Sf9 cells,
Nde
I-
Hin
dIII fragments containing the histidine-tag and TTF-I sequences from the pRSET constructs were cloned into pBacPAK9
(Clontech). TTF1-320, TTF1-184/Oct and TTF185-320/Oct were cloned into pRSET. Cloning strategies are
available on request. Oct2 POU was expressed from pMT-pKA-Oct2-POU (
17
).
Proteins were expressed by infecting 2.5 * 10
8
Sf9 cells with recombinant baculovirus. After 48 h, the cells were harvested, rinsed in PBS and
resuspended in 3 vol lysis buffer (50 mM HEPES-KOH, pH 7.8; 300 mM KCl; 5 mM MgCl
2
; 1 mM PMSF; 1 [mu]g/ml leupeptine). Cells were lysed by sonification followed by addition of
0.5% NP-40 and centrifugation. Imidazole (1 mM) was added to the supernatant and incubated with NTA-agarose beads (Quiagen) for 30 min at 4oC. The beads were washed with 20 column volumes of buffer 1 (50
mM HEPES-KOH, pH 7.8; 300 mM KCl; 5 mM MgCl
2
; 0.5% NP-40; 1 mM imidazole; 1 mM PMSF; 1 [mu]g/ml leupeptine), 20 vol of buffer 2 (same as buffer 1 with 1 M KCl)
and 20 vol of buffer 3 (same as buffer 1 with 10 mM imidazole). Proteins were
eluted 20 mM HEPES-KOH, pH 7.8; 100 mM KCl; 5 mM MgCl
2
; 200 mM imidazole; 1 mM PMSF; 1 [mu]g/ml leupeptine and dialysed against buffer AM-100 (20 mM Tris-HCl, pH 7.9; 5 mM MgCl
2
; 0.1 mM EDTA; 20% glycerol; 2 mM DTE; 100 mM KCl).
Electrophoretic mobility shift assays were performed as described (
11
,
18
). For proteolytic treatment of TTF-I, DNA-protein complexes were incubated for 15 min at room temperature
before 100 ng of V8 protease were added and incubation was continued for
another 15 min before electrophoresis on 8% native polyacrylamide gels. Binding
of
in vitro
translated Oct2 POU or TTF-I/Oct fusion proteins to the octamer probe was described by Annweiler
et al
. (
19
).
To determine DNA binding by indirect immunoprecipitation of protein-bound DNA,
32
P-labeled `Sal box' or octamer oligonucleotides were incubated with purified
recombinant TTF-I or with
in vitro
translated proteins under standard binding conditions. After 30 min incubation
at room temperature, the reactions were diluted 2-fold with buffer AM-100
and incubated for 30 min at room temperature with [alpha]-N(2-17), [alpha]-TTF[Delta]N323 or [alpha]-Oct antibodies bound to
protein A-Sepharose beads. The immunoprecipitates were washed 3 times with buffer
AM-100, dissolved in loading buffer (10 mM Tris-HCl, pH 8.0; 50 mM EDTA; 1% SDS; 30% glycerol) and analyzed on a
12% native polyacrylamide gel.
Transcription in S-100 extracts and partially purified reconstituted systems were performed
as previously described (
1
,
11
,
20
). Assays (25 [mu]l) contained 20-30 ng of linear template DNA (pMrT
2
/
Eco
RI), varying amounts of recombinant TTF-I, and either 6 [mu]l of S-100 extract or 10 [mu]l of a mixture of partially purified RNA polymerase I, TIF-IA, TIF-IC, TIF-IB and UBF (
20
). After preincubating the proteins with template DNA, the reactions were started by the addition of nucleotides and incubated for 60 min at 30oC. Transcription in the tailed template system was performed as described (
21
).
TTF-I
p130
and TTF[Delta]N185 were expressed and purified from baculovirus infected Sf9 cells,
mixed with buffer AM-100 (to a total volume of 100 [mu]l) and layered on top of a 3 ml 10-30% glycerol gradient containing buffer AM-100, 3% sucrose and 0.5 mM DTE. Samples were centrifuged at 4oC for 16 h at 45 000 r.p.m. in a SW60 rotor. Fractions (180 [mu]l) were collected, 8 [mu]l were treated with V8 protease and tested
for DNA binding activity in the electrophoretic mobility shift assay. For analysis of TTF1-320, 50 [mu]l of an
in vitro
translation reaction were used. The fractions were precipitated with
trichloroacetic acid, analyzed by SDS-PAGE, and the amount of recovered protein was quantified with a
PhosphorImager. For gel filtration, 25-50 [mu]l translation reactions were centrifuged and passed over a Superdex200 FPLC (Pharmacia) column in buffer AM-100 without glycerol. The fractions were precipitated and analyzed by SDS-PAGE followed by autoradiography.
The [alpha]-TTF[Delta]N323 antibodies have been described before (
6
). The antibody [alpha]-N(2-17) was raised against a synthetic peptide containing the amino acids 2-17 from TTF-I. For immunoprecipitations, cytoplasmic extract and recombinant TTF-I
p130
were preincubated for 30 min under transcription conditions and then incubated
for 30 min at room temperature with either [alpha]-TTF[Delta]N323 or [alpha]-N(2-17) antibodies that were coupled to
protein A-Sepharose.
For metabolic labeling, 1.6 * 10
5
NIH 3T3 cells were incubated for 16 h with 0.7 mCi/ml [
32
P]orthophosphate (
22
). The cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.5% sodium deoxycholate; 0.5% NP-40; 0.5% SDS; 10 mM EGTA; 20 mM KF; 1 mM sodium orthovanadate; 10
mM potassium phosphate) supplemented with 1 mM PMSF, 50 [mu]g/ml pepstatin A, 1 [mu]g/ml aprotinin and 1 [mu]g/ml leupeptin. After preclearing, lysates were incubated for 3 h at
4oC with the bead-bound antibodies, the immunoprecipitates were collected, washed three
times with RIPA buffer, and, after electrophoresis, labeled TTF-I was detected by autoradiography.
To gain insight into the function of the N-terminus of TTF-I, we expressed full-length TTF-I (TTF-I
p130
) and two N-terminal deletion mutants, TTF[Delta]N185 and TTF[Delta]N323, in baculovirus infected insect cells, purified the
proteins (Fig.
1
A) and compared their properties. In Figure
1
B, the relative DNA binding activity of equivalent amounts of purified TTF-I
p130
, TTF[Delta]N185 and TTF[Delta]N323 was compared using an electrophoretic mobility shift assay.
Clearly, no DNA-protein complexes were formed with TTF-I
p130
(lanes 1 and 2). Deletion of 184 N-terminal amino acids (TTF[Delta]N185), on the other hand, allowed the formation of discrete high
affinity complexes (lanes 5 and 6). A more extensive N-terminal deletion (TTF[Delta]N323) did not increase DNA binding further (lanes 9 and 10). This
observation suggests that the 184 N-terminal amino acids of TTF-I exert a negative effect on DNA binding. If this assumption is
correct, limited proteolysis should reveal the DNA binding activity of TTF-I
p130
. Consistent with earlier observations (
11
), digestion of TTF-I
p130
with V8 protease resulted in the formation of a distinct DNA-protein complex (lanes 3 and 4) whose mobility and intensity was similar
to the protease-resistant core of the deletion mutants (lanes 7, 8, 11 and 12). Thus,
either limited proteolysis or deletion of the N-terminus unmasks the DNA binding activity of TTF-I
p130
.
Next, we investigated whether the termination activity of the different TTF-I derivatives corresponds to their DNA binding activity. Three
transcription systems with successively higher levels of purity were tested.
Two promoter-driven systems utilized the template pMrT
2
which contains the mouse rDNA promoter fused to a 3' terminal spacer fragment, including the termination site T
2
(
1
). Terminated transcripts are 174 nt; readthrough transcripts are 236 nt in
length. In cytoplasmic extracts (S-100), which contain very low levels of cellular TTF-I (
23
), the majority of RNA products are readthrough transcripts (Fig.
2
A, lane 1). Addition of equivalent amounts of either TTF-I
p130
(lanes 2-4) or mutant TTF-I (lanes 5-10) produced the same amounts of terminated transcripts
relative to readthroughs. Therefore, TTF-I
p130
and the two deletion mutants had comparable termination activities.
Previously, it was suggested that limited proteolysis is required to unmask the
DNA binding domain of full-length TTF-I and that proteolyzed forms of TTF-I mediate transcription termination (
11
). Since in the crude transcription system TTF-I
p130
had the same termination activity as TTF[Delta]N185 or TTF[Delta]N323, we tested whether proteolysis of TTF-I occurred in cell extracts. For this, TTF-I was incubated with cytoplasmic extract proteins and
then immunoprecipitated with antisera that recognize different regions of TTF-I. The supernatants of the precipitation reactions were analyzed for
termination activity. The serum [alpha]-N(2-17) was raised against a peptide containing amino acids 2-17
from TTF-I and, therefore, precipitates TTF-I
p130
, but not the N-terminal deletion mutants. The polyclonal antibody [alpha]-TTF[Delta]N323 (raised against amino acids 323-833), on the other hand, precipitates all forms
of TTF-I. As expected, preimmune sera did not precipitate exogenous TTF-I
p130
and, therefore, terminated transcripts were produced (Fig.
3
A, lanes 1 and 3). However, both TTF-I antibodies depleted transcription termination activity (lanes 2 and 4)
indicating that termination in cell extracts is mediated by TTF-I
p130
and not by a proteolyzed form that lacks the N-terminus.
In order to determine the size of native TTF-I proteins, mixtures of protein size markers and purified TTF-I
p130
and TTF[Delta]N185, respectively, were analyzed by glycerol gradient sedimentation.
Individual fractions were analyzed for DNA binding after limited protease
treatment. TTF[Delta]N185 sedimented as a single symmetrical peak with an apparent molecular mass of 100 kDa as expected for a monomer
(Fig.
5
A). In contrast, a similar analysis of TTF-I
p130
revealed a broad non-symmetrical peak that trailed from the bottom of the tube to the size of
monomeric protein (Fig.
5
B). The same size distribution was observed with
in vitro
translated TTF-I
p130
and TTF[Delta]N185 (data not shown). This result suggests that the presence of the N-terminal 184 amino acids facilitates the formation of non-distinct oligomeric structures. To further address this
issue, we performed the sedimentation analysis with
35
S-labeled TTF1-320, a polypeptide encompassing the N-terminal half of TTF-I (Fig.
5
C). Significantly, most of the protein was found at the bottom of the tube and
only a small portion sedimented at approximately 140 and 450 kDa: these
molecular masses are much larger than expected for monomeric TTF1-320, and therefore support the view that the N-terminal domain of TTF-I has the potential to oligomerize with itself and to confer
oligomerization on TTF-I
p130
. Since comparable results were obtained for both baculovirus expressed and
in vitro
translated TTF-I
p130
, it is not very likely that this oligomerization may be dependent on other
factors present in the
in vitro
translation lysate.
The sedimentation of TTF1-320
as large complexes suggests that this domain can oligomerize when separated from
the rest of TTF-I, and that oligomerization may occlude the DNA binding domain of TTF-I
p130
. If this is the case, then the N-terminal domain should repress DNA binding of a heterologous protein. To
test this possibility, we examined the DNA binding properties of chimeric
proteins in which different sections from the N-terminal region of TTF-I, namely amino acids 1-320, 1-184 and 185-320, were fused to the DNA binding domain of
Oct2.2 (Oct2 POU). In electrophoretic mobility shift assays, Oct2 POU formed a
specific DNA-protein complex with the octamer probe (Fig.
6
A, lanes 1 and 2). TTF1-320/Oct, on the other hand, did not bind (lanes 3 and 4). TTF1-184/Oct
produced two complexes whose mobilities closely correspond to that of Oct2 POU
alone, and therefore presumably represent proteolytic cleavage products of TTF1-184/Oct (lanes 5 and 6), as confirmed below. In contrast, TTF185-320/Oct efficiently bound to the octamer sequence producing a
complex that migrated much more slowly than that of Oct2 POU (lanes 7 and 8).
Since no binding was observed with TTF1-320/Oct and the complexes formed with TTF1-184/Oct were due to proteolytic cleavage,
we conclude that the N-terminal 184 amino acids inhibit DNA binding of Oct2 POU.
The sedimentation behaviour of TTF-I
p130
and
TTF[Delta]N185 indicated that the N-terminus mediates oligomerization. To determine whether fusion of
the N-terminus of TTF-I to the Oct2 POU domain would produce oligomers of the chimeric
proteins, Oct2 POU and TTF-I/Oct derivatives were subjected to gel filtration on a Superdex200 FPLC
column. The elution profiles revealed drastic differences in the sizes of the
individual proteins (Fig.
7
). Consistent with a monomeric structure, Oct2 POU eluted at a volume corresponding to 25 kDa and TTF185-320/Oct at ~55 kDa. In contrast, TTF1-184/Oct reproducibly eluted at a volume corresponding to 550
kDa. Also after centrifugation in a linear glycerol gradient, TTF1-184/Oct was found at the bottom of the tube and in the size range of ~600 kDa, indicating that it formed large complexes. TTF185-320/Oct, on the other hand, exhibited a sedimentation behaviour
as expected for monomeric proteins (data not shown). These experiments
demonstrate that the N-terminus of TTF-I harbours a homophilic protein-protein interaction domain and imply that oligomerization
mediated by this domain results in inhibition of DNA binding.
Masking or repression of the DNA binding domain of transcription factors may be
a way of regulating their activity. For instance, NF-[kappa]B1&2 are both synthesized as precursor proteins and require
proteolytic processing for maturation (for review, see refs
25
and
26
). In the cases of TBP, p53, members of the
ets
family of transcriptional regulators and POZ domain proteins, the DNA binding
affinity has been shown to be regulated by internal inhibitory regions (
12
,
13
,
16
,
27
). Inhibition can be released through partial proteolysis or by the formation of
complexes with protein partners (
28
). Similarly, limited protease treatment of cellular TTF-I (
11
) or deletion of the N-terminus of recombinant TTF-I is required to reveal specific DNA binding to the `Sal box' target
sequence. These results suggested that the N-terminal domain of TTF-I represses its own DNA binding function. To elucidate the molecular
mechanism by which the N-terminus affects the function of TTF-I, we compared the properties of recombinant full-length TTF-I with two N-terminal deletion mutants. We found that, despite the dramatic reduction in
DNA binding activity of TTF-I
p130
when compared to N-terminal deletion mutants or protease treated TTF-I, intact TTF-I efficiently mediated transcription termination in partially
purified transcription systems. However, the reduced DNA binding activity of TTF-I
p130
is more closely
reflected in a tailed template assay in which transcription occurs in the
presence of highly purified RNA polymerase I without any auxiliary factors. In
this assay, TTF[Delta]N185 and TTF[Delta]N323 mediated efficient termination and TTF-I
p130
exhibited a strongly reduced activity. This is an interesting observation which
implies that other activities in the cell may regulate the DNA binding activity
of TTF-I, possibly by covalent modifications or direct interaction. Consistent
with this view, we found that the vast majority, if not all, of cellular TTF-I is the unproteolyzed protein. Moreover, using the yeast two hybrid
system we have recently isolated a cellular partner that interacts with TTF-I
p130
(data not shown). Such an interaction with other proteins could produce
complexes with different functional properties or may mediate changes in either
the oligomerization state or the structure of TTF-I to allow DNA binding.
Models of intramolecular inhibition propose that inhibitory regions can either
sterically or allosterically affect the function of the DNA binding domain.
Both mechanisms predict that conformational changes in the protein may
accompany DNA binding. For the transcription factor Ets-1 it has been shown that the full-length protein exhibits a reduced DNA binding activity compared to N-terminal truncations (
15
). A model based on circular dichroism analysis suggests that the N-terminal inhibitory domain makes intramolecular contacts with both the C-terminal inhibitory region and the ETS domain in the absence of DNA.
The interplay of two inhibitory regions is needed in Ets-1 to display a reduced DNA binding activity. DNA binding has been shown to
be accompanied by a conformational change which might be stabilized by an
interacting protein. By analogy to TTF-I, deletion of one inhibitory region in Ets-1 relieves inhibition.
Alternatives to this intramolecular mechanism are models in which intermolecular
interactions are inhibitory to DNA binding. Our experiments using chimeric
proteins containing the Oct2 POU domain fused to the N-terminus from TTF-I are more consistent with this latter mechanism for repression.
Testing the chimeras for binding to the octamer oligonucleotide demonstrated
that the 184 N-terminal amino acids of TTF-I inhibit DNA binding of the heterologous Oct2 POU domain, whereas
the region of TTF-I from amino acid 185 to 320 had no effect. Size determination studies of
TTF-I
p130
and the N-terminal domain (amino acids 1-320), as well as TTF-I/Oct fusion proteins, further suggest that inhibition of DNA
binding may be due to the formation of stable oligomers of proteins containing
184 N-terminal amino acids of TTF-I. This finding is reminiscent of proteins containing the POZ
domain. The POZ domain is a protein-protein interaction motif present in a large family of proteins involved
in DNA- or actin-binding, many of which have been shown to form large aggregates (
29
). For instance, the POZ domain of the zinc finger proteins ZID and Ttk promotes
homophilic protein-protein interactions which result in protein
oligomerization. ZID and Ttk form very large complexes in sedimentation
analyses and the presence of the POZ domain results in the formation of large,
uniform protein complexes that were visible in electron microscopy. The POZ-mediated protein oligomerization, in turn, inhibits the interaction of the
associated zinc finger regions with DNA (
16
). The intriguing correlation between repression of DNA binding and protein
oligomerization suggests that similar molecular mechanisms may be involved in
inhibition of DNA binding by the N-terminus of TTF-I and the POZ domain, respectively. Actually, we observe sequence
similarity between the N-terminus of TTF-I and part of the consensus motif for the POZ domain (
6
,
16
).
The ability of TTF-I
p130
to oligomerize highlights additional possibilities for the function of the N-terminal domain. The proximity of enhancer and terminator sequences in the
rDNA spacer of yeast cells has inspired models proposing a functional link
between transcription termination and initiation (
30
). In these models, enhancer and promoter elements of different transcription
units are physically associated, forming a complex from which coding and spacer
regions are looped out. In this scenario, Pol I terminated at the 3'-end of the gene could pass directly to the promoter of either the
same or the downstream gene without being released into the free pool. This
hypothesis is supported by the observation that active rDNA transcription units
have been visualized as loops separated by intergenic spacers (
31
). In micrographs of
Bombyx mori
and
Drosophila
tissue culture cell nuclear chromatin spreads, each ribosomal gene appears as a
small loop with the intergenic spacers flanking the gene in contact. This
configuration could facilitate high transcriptional activity by recycling RNA
polymerase and associated factors from the 3' tail to the 5' head of an active gene. The property of the N-terminal domain to promote TTF-I oligomerization, together with the existence of a
conserved TTF-I binding site upstream of the Pol I initiation site and a series of
terminator elements at the 3'-end of each transcription unit, suggests that the functional link
of the initiation and termination reaction might be mediated by multimerization
of TTF-I bound upstream and downstream of the promoter. Therefore, in addition to
its role in modulating DNA binding, the N-terminal domain of TTF-I might play a crucial structural role in organizing the rDNA
transcription units and spacer regions.
We thank Thomas Wirth for the initial Oct2 POU construct and for [alpha]-Oct antibodies, and Peter Seither for purified mouse RNA polymerase
I. This work was supported, in part, by the Deutsche Forschungsgemeinschaft
(SFB 229) and the Fonds der Chemischen Industrie.
REFERENCES
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