From the Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202
Received for publication, November 27, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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Yeast Mot1, an essential
ATP-dependent regulator of basal transcription, removes
TATA box-binding protein (TBP) from TATA sites in vitro.
Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in
vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did
not. Complex formation was prevented above 200 mM salt,
consistent with electrostatic interaction. Correspondingly, TBP
variants lacking solvent-exposed positive charge did not bind Mot1,
whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of
TBP), bound TBP normally in vitro, suggesting it forms
nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not
growth-inhibitory. C-terminal deletions were toxic when overexpressed,
and toxicity was ameliorated by TBP co-overproduction. Residues 1-800
of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog,
bound the TBP-like factor, dTRF1. Native Mot1 and derivatives
deleterious to growth localized in the nucleus, whereas nontoxic
derivatives localized to the cytosol, suggesting TBP binding and
nuclear transport of Mot1 are coupled.
In yeast (Saccharomyces cerevisiae), as in other
eukaryotes, many positive and negative regulatory factors act in
concert to ensure that transcription initiation by RNA polymerase II
(pol II)1 is
controlled correctly (1). Promoter accessibility is dictated by
chromatin structure, which is modulated by chromatin-remodeling complexes and histone-modifying enzymes (2). Once a promoter is
available, formation of a preinitiation complex begins with binding of
TFIID, a complex composed of the TATA box-binding protein (TBP) and
several TBP-associated factors (TAFs) (3). TFIID binds to the TATA box
sequence present at nearly all yeast promoters (4, 5). Formation of a
TFIID-DNA complex creates a platform for assembly of the remaining
general transcription factors and pol II (6-8).
Transcriptional activators stimulate transcription initiation via
recruitment of the pol II preinitiation complex to a promoter either
through direct interaction with TBP, TFIIB, and/or components of the
pol II holoenzyme (9, 10) or via intervening "mediator" complexes
(11). Considerable evidence highlights the importance of activator
recruitment of TBP. For example, by using a TBP derivative with altered
DNA binding specificity, it was shown that
activator-dependent engagement of TBP at a promoter is
rate-limiting for transcription in vivo (12). Also, fusion
of TBP to the DNA-binding domains of either LexA or Gal4 allows high
level transcription from LexA- or Gal4-binding sites in the absence of
any other activator (13-15). Finally, as shown by in vivo
cross-linking techniques, promoter occupancy by TBP correlates with
transcriptional activity (16, 17).
Because promoter occupancy by TBP is central to gene activation (18),
TBP is also a prime target of transcriptional inhibitors. A conserved
heterodimeric repressor, NC2/Dr1/DRAP, binds TBP via a histone-like
fold, preventing TBP association with TFIIA and/or TFIIB, thereby
repressing transcription (19-22). S. cerevisiae cells
lacking NC2 Another class of negative transcriptional regulator targeting TBP is
the yeast MOT1 gene product. Like NC2, Mot1 is essential for
viability and evolutionarily conserved. Mot1 homologs exist in
Drosophila (55) and humans (63, 64). A recessive
temperature-sensitive mutation (mot1-1) elevated
transcription of a plasmid-borne reporter gene in the absence of its
normal activator and also increased transcription from several
chromosomal pol II-dependent genes, as well as from a basal
promoter lacking any upstream activation sequence (UAS) (24). An
independently isolated allele (mot1-1033) had a similar
effect on another pol II gene (25). Another recessive allele
(bur3-1, for "bypass UAS requirement," now
mot1-301) was isolated by selecting for mutations permitting
transcription from the SUC2 gene promoter lacking its UAS
(23, 26).
Consistent with a primary role as a negative regulator, Mot1 was found
to be the factor responsible for an
ATP-dependent inhibitory activity
(ADI) in nuclear extracts (27) that removes TATA box-bound TBP in an
ATP-dependent manner (28). Although TBP stimulates RNA
synthesis by all three RNA polymerase types in transcription reactions
in vitro (29), the presence of Mot1 decreases transcription of pol II genes, but not pol I or pol III genes (28). Likewise, Mot1 is
required for repression in vitro of LEU2
expression by the negative regulator, Leu3 (30). Mot1 was also
identified in nuclear fractionation studies as a component (Taf170) of
TBP-containing complexes distinct from TFIID (31).
The C-terminal domain of Mot1 possesses the seven signature motifs of a
superfamily of helicases and nucleic acid-dependent ATPases
(32), many of which modulate the state of assembly of protein-nucleic
acid complexes (33). Mot1 is the prototype of a distinct class that
includes Snf2 (yeast), ERCC6/CSB (human), Brahma
(Drosophila), HepA (Escherichia coli), and others
(24, 34). Despite this homology, no helicase activity has been reported for any member of the Mot1/Snf2 group. Mot1, purified to
apparent homogeneity, does not exhibit even local DNA strand-unwinding activity but does retain full ability to release TBP bound to a TATA
box in an ATP-dependent manner (35). Prior studies (36) suggested that contacts with DNA do not play a critical role in Mot1-TBP interaction. Here we have explored this issue in detail and
present experiments performed both in vivo and in
vitro that define the requirements for, and quantify the affinity
of, the physical association between Mot1 and TBP. We also determined the subcellular localization of Mot1, and the role of TBP interaction in Mot1 compartmentation.
Growth Conditions, Strains, and Recombinant DNA
Method--
Strain W303-1A (MATa ura3-1
his3-11,15 ade2-1 leu2-3,112 trp1-1 can1-100) was used for all
experiments, unless otherwise indicated. For immunofluorescence, an
otherwise isogenic MATa/MAT
Yeast cells without plasmids were grown at 30 °C in rich medium
(YP), and cells with plasmids were grown in minimal medium (SC) lacking
the appropriate nutrient(s) to maintain selection, as described (43),
using glucose (Glc) as the carbon source unless otherwise indicated.
Solid media contained either 2% Glc or 2% galactose and 0.2% sucrose
(Gal/Suc). For expression of genes from the galactose-inducible
GAL1 promoter in liquid media, cells were pre-grown in SC
containing 2% raffinose (Raf) as the carbon source, and then galactose
(Gal) was added to 2% to initiate induction. Plasmids were introduced
into yeast cells using a modification of the lithium acetate
transformation procedure that utilizes single-stranded carrier DNA
(38). Recombinant DNA manipulations were carried out using standard
techniques (44).
Construction of Plasmids for Expression in Yeast--
To
facilitate immunodetection of Mot1, DNA encoding the 16-residue c-Myc
epitope (LEEQKLISEEDLLRKR) recognized by the monoclonal antibody (mAb)
9E10 (45) was fused in-frame to the 3'-end of the MOT1
coding sequence, using a PCR-based method that exploits 3 primers (46)
as follows: an upstream primer (JDM26), a
MOT1/Myc joiner primer (JDM25), and a Myc
epitope-containing primer (MYC) (Table
I). The resulting 674-base pair PCR product was introduced into the
SmaI site of plasmid pRS316 (37) by blunt-end ligation and
verified by DNA sequencing. The resulting product was digested with
BstXI and introduced into BstXI-digested
pRSMOT1, yielding pRSMot1Myc. Addition of the Myc tag did
not detectably alter Mot1 function since pRSMot1 fully complements a
mot1
A series of truncations and internal deletions of MOT1Myc
and MOT1(D1408N)Myc (see Fig. 4)
expressed from the GAL1 promoter and carried on
URA3-containing 2-µm DNA-based plasmids were generated, as
appropriate, from plasmids, pKH7 or pKH14, respectively (28), as
described below. To produce pKH23, pKH33, pKH34, pKH35, pKH43, and
pKH45, plasmid pKH7 was digested with the restriction enzymes SalI-XhoI (pKH23),
CelII-SalI (pKH33),
BstEII-NruI (pKH34),
NruI-CelII (pKH35),
BspEI-NheI (pKH43), or
BspEI-BstEII (pKH45), respectively. The resulting
5'-overhangs were filled in using the Klenow fragment of E. coli DNA polymerase I and dNTPs (except in the case of pKH23), and
the plasmids were religated. Plasmids pKH44 and pKH46 were generated
similarly to pKH43 and pKH45, respectively, except that they were
derived from pKH14. Plasmid pJDSac was constructed by introducing the
2.4-kb SacI-SacI fragment from pRSMot1Myc into YEp352GAL (47). To create pKH1, pRSMot1Myc was digested with NheI and HpaI, treated with mung bean nuclease
(Stratagene), and religated. Plasmid pKH24 was made by swapping the
2.4-kb XhoI-BspEI fragment from pKH1 into pKH7.
Plasmids pKH36, pKH37, pKH41, pKH42, and pKH47 were generated by
swapping the 3-kb D1408N mutation-containing NdeI fragment
from pKH14 into pKH24, pKH34, pKH33, pKH35, and pJDSac, respectively.
pKH25 was constructed by using PCR to introduce a XhoI site
just 3' to codon 1261 and then deleting the
XhoI-XhoI fragment corresponding to the sequences
coding for amino acids 1262-1867, but retaining the c-Myc epitope tag.
A 472-base pair fragment of MOT1 was amplified using primers
KHP5 and KHP6, digested with XhoI and AflII, and
ligated to XhoI-AflII-digested pKH7, to generate
pKH25. All deletions of MOT1 were sequenced to confirm that
the junctions were in-frame and at the positions expected.
Templates for Coupled in Vitro Transcription and
Translation--
MOT1and MOT1(D1408N) were
transcribed from the T3 promoter using plasmids pKH21 and pKH22,
respectively. pKH21 was constructed by swapping the 2.4-kb
SacI fragment from pKH7 into pKH2 (28). pKH22 was made in an
identical manner, except that the SacI fragment was derived
from pKH14. As a negative control, a construct expressing RAD3 from the T3 promoter (48) was transcribed.
SPT15 (yeast TBP), with an in-frame N-terminal
(His)6 tag, was transcribed from the T7 promoter in plasmid
pKH19, which was constructed (39) by inserting a 1.5-kb
NdeI-BamHI fragment containing the
SPT15 coding sequence from pM1 (gift of Robert Tjian,
University of California, Berkeley) into
NdeI-BamHI-digested pET15b (Novagen). Three TBP
mutants, spt15(K133L K138L),
spt15(K133L K145L), and spt15(K138T Y139A), were expressed from plasmids
pKH39, pKH40, and pKH48, respectively, which were constructed by
transferring the Bsu36I-BamHI fragment containing
the appropriate portion of the SPT15 open reading frame
(ORF) from pT7-IID(K133L,K138L), pT7-IID(K133L,K145L) (41), and
TBP(N2-1) (49), respectively, into pKH19. pKH38 is identical to pKH39
and pKH40, except that it expresses wild-type SPT15 and was
generated by swapping the Bsu36I-BamHI fragment
from pT7-IID (41) into pKH19. The NdeI-BamHI fragment from pT7-IID(R105H) (gift of Dr. Steve Buratowski, Harvard Medical School) was transferred into NdeI-BamHI-
digested pKH19 to yield plasmid pKH49, which expresses
spt15(R105H) from the T3 promoter. pKH38 (instead
of pKH19) was used to prepare wild-type TBP for those experiments in
which normal TBP was compared with TBP mutants because pKH38 was
constructed in parallel with the plasmids expressing the TBP mutants.
To express MOT1,
mot1(
C-terminal deletions (see Fig. 4) were constructed by linearizing
plasmid pKH2 with restriction enzymes at sites within the ORF, allowing
for run-off transcription. In the case of enzymes leaving 3'-overhangs,
the 3'-exonuclease activity of T4 DNA polymerase was used to digest the
overhanging ends to flush ends, so as to prevent spurious initiation by
T3 RNA polymerase. The deletions and restriction enzymes used are as
follows:
Most N-terminal deletions were made by PCR using T3 promoter-containing
primers. The resulting PCR products were then transcribed directly. The
mot1( In Vitro Transcription and Translation--
Proteins to be used
in coimmunoprecipitation experiments were produced by coupled in
vitro transcription and translation in the presence of
[35S]Met (PerkinElmer Life Sciences) using the Promega
TNTTM Coupled Reticulocyte Lysate System, according to the
manufacturer's directions. To increase yield, 0.2 mg/ml yeast tRNA
(Sigma) was added to each translation mixture. Prior to use,
PCR-derived or linearized DNA templates were extracted with
phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with
ethanol. After translation, proteins were partially purified by
precipitation with 50% ammonium sulfate (50), which also allowed for
quantification of translation yield as described in detail elsewhere
(51). Briefly, the concentration of translated protein can be estimated
based on the percent incorporation of [35S]Met, the
number of Met residues known to be present in the sequence of the
protein, the molecular weight of the protein, and the concentration of
unlabeled methionine in the extract (~5-10 µM). The
ammonium sulfate precipitates were resuspended in Buffer A (20 mM Tris-HCl (pH 7.5), 75 mM potassium acetate,
10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.1% Tween 20, and 12.5%
glycerol) at a volume equal to the starting volume.
Immunoprecipitation--
Immunoprecipitations were performed
following the procedures described (51). Radiolabeled proteins (~0.5
pmol each), prepared by coupled in vitro transcription and
translation as described above, were mixed in a final volume of 200 µl of Buffer A, incubated for 30 min at 4 °C, and then clarified
by centrifugation at maximum speed in a microcentrifuge at 4 °C for
15 min. In some experiments (see Fig. 1), unlabeled yeast TBP (yTBP),
which was expressed in and purified from E. coli according
to the method of Ref. 52, was used. After clarification, the resulting
supernatant solution was withdrawn and mixed with 15 µl of a 50:50
slurry of protein A-agarose beads (Oncogene Sciences) in Buffer A and 2 µl of polyclonal anti-yTBP antibodies (gift of Dr. Grace Gill,
Harvard Medical School) in a 1.5-ml microcentrifuge tube. After
incubation at 4 °C for 1 h on a roller drum, the agarose beads
were collected by centrifugation for 5 s in a microcentrifuge. The
supernatant solution was removed by aspiration using a 25-gauge needle,
and the pellets were washed 3 times with 1 ml of ice-cold Buffer A. The
bead-bound immune complexes were resuspended in SDS sample buffer,
boiled, and resolved on a 10% SDS-polyacrylamide gel, along with a
lane containing 14C-labeled molecular weight markers
(Amersham Pharmacia Biotech). After electrophoresis, gels were fixed
for 30 min in 10% methanol and 10% acetic acid, dried under vacuum,
and analyzed by autoradiography using x-ray film (Kodak Biomax MR) or
quantified using a PhosphorImagerTM (Molecular Dynamics).
Immunoblotting--
Cultures of strain W303-1A harboring
plasmids containing the various MOT1Myc derivatives were
grown to an approximate density of A600 nm = 0.7 in SCRaf medium lacking uracil, induced by addition of Gal, and
grown for an additional 3 h. Cells were harvested by
centrifugation and washed once with ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM
phenylmethylsulfonyl fluoride, 20 µg/ml pepstatin A, 1 mM
DTT). All subsequent steps were carried out at 4 °C. An amount of
cells (equivalent to 10 A600 nm units) was
resuspended in 200 µl of lysis buffer and lysed by vortex mixing with
glass beads as described in detail (53). Unbroken cells and large cell
debris were removed by low speed centrifugation (2000 rpm for 5 min in
a Sorvall SS-34). For immunodetection of Mot1, a sample of the lysate
equivalent to 30 µg of total protein, as determined by the
dye-binding method of Bradford (54), was resolved by SDS-PAGE on an 8%
gel, transferred to a nitrocellulose filter, and incubated with
anti-c-Myc mAb 9E10 at a 1:10,000 dilution for several hours.
Filter-bound immune complexes were detected by incubating the filters
with a 1:5000 dilution of horseradish peroxidase-conjugated sheep
anti-mouse antibodies (Amersham Pharmacia Biotech) and visualized using
a chemiluminescence detection system (ECLTM, Amersham
Pharmacia Biotech) as recommended by the manufacturer.
Indirect Immunofluorescence--
Diploid strain W303D
carrying plasmids expressing Mot1 or Mot1 variants was grown in SCRaf
medium to A600 nm = 0.5, induced with Gal, and
incubated for an additional 3 h. Cells were fixed, converted to
spheroplasts, and incubated with primary and secondary antibodies
essentially as described (53). Primary antibody (anti-c-Myc mAb 9E10)
was used at a 1:300 dilution. Secondary antibody (fluorescein
isothiocyanate-conjugated goat anti-mouse F(ab')2 fragment;
Jackson ImmunoResearch) was used at a 1:100 dilution.
Bacterial Expression and Use of GST Fusions for Protein Binding
Assays--
Certain proteins used for binding assays were expressed in
and purified from E. coli strain BL21(DE3) (Novagen).
Plasmid pGEX-4T (Amersham Pharmacia Biotech) was used to express GST
alone. Plasmid pGST-89B (gift of Naomi Zak, Hadassah Medical School,
The Hebrew University, Jerusalem, Israel) was used to prepare a fusion
of GST to the first 825 residues of Drosophila 89B protein
(55). Plasmids pHA-dTRF1 (56, 57) and pHA-dTBP were the gifts of Dr.
Michael Holmes, University of California, Berkeley. To purify GST and
GST-89B, cells were grown overnight at 37 °C in LB-Amp and used to
inoculate 100 ml of pre-warmed LB-Amp to an
A600 nm = 0.1. Cells were grown to an
A600 nm = 0.5 and then induced by addition of
isopropyl-1-thio-
For protein binding assays, equimolar amounts of purified GST and
GST-89B were mixed with equal volumes of the extracts containing either
HA-dTRF1 or HA-dTBP in binding buffer (TEG-100 containing 0.2% Triton
X-100 and protease inhibitors as above). All steps were performed at
4 °C. The mixtures were clarified by centrifugation for 5 min at
16,000 × g and then incubated for 2 h on a rotary mixer with glutathione-Sepharose beads that had been pre-washed in
TEG-100 containing 0.25 mg/ml bovine serum albumin and 0.2% Triton
X-100 (TEG-100-BT). The beads were collected by centrifugation for 5 min at 500 × g, washed 3 times in 1 ml each of
TEG-100-BT, and resuspended in 2× SDS-PAGE sample buffer. Bound
proteins were eluted by boiling for 5 min and analyzed by SDS-PAGE and
immunoblotting, as described above, using mouse anti-HA mAb 12CA5 (gift
of Prof. Robert Tjian, University of California, Berkeley) at 1:10,000 dilution.
Direct and High Affinity Interaction of Mot1 and yTBP--
To
determine whether Mot1 and yTBP associate in the absence of DNA,
radiolabeled Mot1 (and various derivatives) was produced by coupled
in vitro transcription and translation and mixed in solution
with a 2-fold excess of purified bacterially expressed yTBP. The
mixture then was precipitated with bead-bound anti-yTBP antibodies, and
the amount of coimmunoprecipitated Mot1 was examined (Fig.
1). As one control, Mot1 alone (in the
absence of yTBP) was treated in the same way to measure nonspecific
binding to the beads. The amount of anti-yTBP antibodies used was
sufficient to capture 20-30% of the yTBP added, as estimated from
experiments in which the unlabeled protein was replaced with
radiolabeled yTBP prepared by in vitro translation (see for
example, Fig. 2). Among the advantages of
this method for assaying protein-protein interaction are that the vast
excess of unlabeled proteins in the reticulocyte lysate reduces
nonspecific adsorption and helps ensure specificity, that radiolabeling
allows quantitation of the results and, thereby, estimation of the
affinity constant (Kd), and that various derivatives
of the proteins of interest can be made readily in vitro
(50, 51).
The calculated molecular mass of Mot1 is 210 kDa; however, the
full-length protein prepared by in vitro translation (28) or
the native protein purified from yeast (35, 36) migrates on SDS-PAGE
with an apparent size of ~180 kDa, compared with standard molecular
weight markers. Hence, as expected, radiolabeled Mot1 and an
ATPase-defective point mutant, Mot1(D1408N) (28, 39), appeared as a
prominent 180-kDa doublet corresponding to full-length and near
full-length protein (with minor amounts of lower
Mr species representing both internal initiation
and premature termination products) (Fig. 1, lanes 1 and
2). In the absence of yTBP, only a trace of Mot1 was
nonspecifically associated with the beads; in contrast, in the presence
of yTBP, bead-bound Mot1 increased at least 10-fold (Fig. 1, lane
4 versus lane 5), suggesting that Mot1 and yTBP
physically interact.
Some DNA-binding proteins appear to associate with each other when, in
fact, the interaction is mediated through their concomitant binding to
DNA; however, the presence of the intercalating dye, ethidium bromide,
effectively blocks such indirect interactions (58). Addition of
ethidium bromide in large excess (100 µg/ml) to these
immunoprecipitation reactions did not detectably reduce the amount of
bead-bound Mot1 in either the absence or presence of yTBP (data not
shown). Conversely, addition of an excess of a TATA-containing DNA
fragment did not increase the amount of bead-bound Mot1 in either the
absence or presence of yTBP (data not shown). Finally, as a negative
control, radiolabeled Rad3, a demonstrated 90-kDa helicase (in the same
superfamily, but only distantly related to Mot1) and a known component
of yTFIIH (48), showed only nonspecific adsorption to the beads that
was unaffected by yTBP (Fig. 1, lanes 8 and 9).
By using the same method, rabbit polyclonal antibodies directed against
the C-terminal domain of Mot1 (28, 39) were able to coimmunoprecipitate
yTBP in either the absence or presence of ethidium bromide (data not
shown). These results clearly demonstrated first, formation of
Mot1-yTBP complexes in dilute solution, and second, that this
interaction does not require mutual binding of these proteins to DNA.
An approximate Kd for the Mot1-yTBP-binding
reaction2 could be calculated
by determining the concentrations of Mot1 and yTBP present in the
reaction from their specific radioactivity, by estimating the fraction
of the total input yTBP immunoprecipitated, and by quantitating the
fraction of the total input Mot1 coimmunoprecipitated (see Ref. 51 for
additional details of this method). The Kd value,
estimated from many such experiments conducted at different input
levels of the reactants, always fell in the range 20-50 nM. This value may underestimate Mot1-yTBP binding affinity
because some antibodies in the polyclonal anti-yTBP antiserum could
interfere with complex formation and because Mot1 concentration in each reaction was most likely overestimated (given that a significant portion of the radioactive species was not full-length and was unable
to interact with TBP; see Fig. 1). Nonetheless, the estimated Kd is in reasonably good agreement with a value
(Kd = 100 nM) determined from a totally
independent method, the concentration dependence of the stimulation of
the ATPase activity of purified Mot1 by purified yTBP (35).
Radiolabeled Mot1(D1408N) (28, 39) displayed the same binding behavior
as wild-type Mot1 (Fig. 1, lane 6 versus
lane 7), indicating that catalytic activity of the
C-terminal ATPase domain is not required for yTBP binding. Consistent
with this result, addition of ATP or a nonhydrolyzable ATP analog
(AMP-P-NH-P) neither enhanced nor reduced binding of either normal Mot1
or Mot1(D1408N) to yTBP in this assay (data not shown). Mutation of the
equivalent residue in another member of this ATPase superfamily
(eIF-4A) prevents ATP hydrolysis but not ATP binding (59).
Mapping the Mot1-binding Region of yTBP--
In the absence of
ATP, purified Mot1 forms a ternary complex with yTBP bound to
TATA-containing DNA; in the presence of ATP, Mot1 acts catalytically to
dissociate yTBP-DNA complexes (35, 36). The Mot1-containing ADI
fraction of yeast nuclear extracts was unable to dissociate yTBP-DNA
complexes prepared using yTBP variants with point mutations in the
so-called basic region (27), which corresponds to a solvent-exposed
Radiolabeled forms of wild-type yTBP and all four mutants were
produced in equivalent amounts (Fig. 2B, Input)
and mixed in 2-fold excess with radiolabeled Mot1. As observed before,
only a trace of Mot1 was nonspecifically adsorbed to the anti-yTBP beads, whereas in the presence of wild-type yTBP, the amount of bead-bound Mot1 increased more than 10-fold (Fig. 2B). The
mutant, Spt15(R105H), with an alteration within the concave DNA-binding face of yTBP, bound Mot1 as well as normal yTBP. In contrast, all three
mutants with alterations in Helix 2 were unable to bind Mot1 (Fig.
2B), as reproducibly observed in three independent trials
(Fig. 2C). Thus, contacts in Helix 2 of yTBP mediate its interaction with Mot1.
Mot1-yTBP Interaction Is Largely Electrostatic--
The fact that
Mot1 binding was abolished by mutations that eliminated positive charge
on yTBP suggested that the interaction has an electrostatic component.
Competing salt ions weaken electrostatic interactions but strengthen
hydrophobic interactions (60). Consequently, radiolabeled Mot1 and
radiolabeled yTBP were mixed in the presence of no additional salt
(above that present in the buffers used to prepare these proteins) or
in the presence of increasing concentrations of potassium acetate, and
their interaction was assessed by the coimmunoprecipitation method. As
ionic strength increased, the amount of Mot1 bound to yTBP decreased;
in contrast, even the highest level of salt tested had no effect
whatsoever on the efficiency of immunoprecipitation of yTBP by the
anti-yTBP antibodies (Fig. 3A). Strongest interaction of
Mot1 with yTBP was observed in the absence of added salt; however,
under these conditions, there was slightly more nonspecific interaction
of Mot1 with the antibody-coated beads than at the lowest salt
concentration tested (75 mM). Consequently, subsequent
immunoprecipitations were routinely performed in 75 mM
potassium acetate. The striking monotonic relationship between increasing salt concentration and decreasing Mot1 recovery (Fig. 3B) suggests that Mot1-yTBP binding is mediated mainly by
electrostatic contacts.
Genetic Mapping of the yTBP-binding Region of Mot1--
The
C-terminal domain of Mot1 (residues 1255-1867), when expressed and
purified from E. coli, possesses robust ATPase activity that
is abolished by the D1408N mutation (28, 39). Full-length Mot1(D1408N)
is unable to complement either the mot1-1ts or
mot1
Full-length MOT1 and MOT1(D1408N), and
a series of deletions in each protein, each marked with a C-terminal
c-Myc epitope tag, were tested for their phenotype when overexpressed
in a MOT1+ strain (W303-1A). Equivalent
expression was expected because all constructs were generated from the
same parental plasmid and, thus, shared the same marker, the same
origin of replication, the same promoter (the Gal-inducible and
Glc-repressible GAL1 promoter), the same 5'-untranslated and
initial N-terminal sequence (except for Mot1(
To examine the effect of overexpression, cells carrying each construct
were serially diluted and spotted on either Glc- or Gal-containing agar
medium. On the repressing Glc medium, all derivatives grew well and
indistinguishably (data not shown). On Gal medium, overexpression of
Mot1 had no deleterious effect on growth (Fig.
5A, left panel), compared with
cells carrying empty vector, whereas overexpression of Mot1(D1408N)
markedly inhibited growth (Fig. 5A, right panel), as
observed previously (28, 39). Similarly, two C-terminal truncations
that removed the catalytic domain of Mot1 were growth inhibitory when
overexpressed (Fig. 5A, left panel, bottom); one,
As found previously, yTBP overexpression in a wild-type cell, or in a
cell overproducing Mot1, has no phenotype (28, 39). If Mot1 deletions
retaining the N-terminal 800 residues are deleterious to growth when
overexpressed because they bind yTBP (but are otherwise nonfunctional),
then co-overexpression of yTBP should ameliorate their
growth-inhibitory effect. Indeed, co-overexpression of SPT15 (yTBP) from a constitutive promoter (ADH1), but not the
empty vector, alleviated toxicity of overexpressed
MOT1(D1408N) (28) and each of the four
growth-inhibitory derivatives ( Mutations in the Mot1-binding Region of yTBP Relieve Mot1(D1408N)
Toxicity--
Helix 2 mutations in yTBP crippled its interaction with
Mot1 in vitro (Fig. 2). Just as mutations in Mot1 that
presumably eliminate its interaction with yTBP mitigated the toxic
effect of Mot1(D1408N) (Fig. 5A), mutations in yTBP that
prevent its interaction with Mot1 should also reduce the
growth-inhibitory effect of Mot1(D1408N). To test this prediction,
yeast strains were constructed in which the chromosomal
SPT15 locus was deleted and replaced by wild-type yTBP
(Spt15), Spt15(K133L,K138L), or Spt15(K133L,K145L), each expressed from
the endogenous SPT15 promoter on a low copy number
(CEN-based) plasmid. Strains carrying either spt15(K133L,K138L) or
spt15(K133L,K145L) as the sole source of yTBP
were temperature-sensitive for growth, as previously reported (41). All
three strains were cotransformed with a multicopy plasmid
overexpressing either wild-type MOT1 (Fig. 5C,
left) or MOT1(D1408N) (Fig. 5C,
right) from the GAL1 promoter. On Glc medium at
25 °C, all of these strains grew equivalently (data not shown). On
Gal medium at 25 °C, overexpression of MOT1 had no
deleterious effect on the growth, regardless of the nature of yTBP
present (Fig. 5C, left). In contrast, toxicity of
MOT1(D1408N) observed in cells expressing
wild-type yTBP was reduced in cells expressing Spt15(K133L,K138L) and
reduced quite dramatically in cells expressing Spt15(K133L,K145L).
These data provide independent and compelling evidence that
Mot1(D1408N) is toxic in vivo because of its ability to
sequester yTBP and that Helix 2 is a critical determinant for Mot1
recognition of yTBP. Thus, the fact that deletions within the
N-terminal 800 residues of Mot1(D1408N) alleviated its
growth-inhibitory effect suggests that this region of Mot1 is
responsible for yTBP binding. On the other hand, since yTBP is located
in the nucleus (61), it was possible that derivatives of Mot1(D1408N)
that are no longer growth-inhibitory are innocuous because they are unable to enter the nucleus.
Localization of Mot1 by Indirect Immunofluorescence--
We
examined the subcellular localization of epitope-tagged Mot1 and its
deletion derivatives by indirect immunofluorescence after brief Gal
induction in diploid cells (their larger size makes visualization of
subcellular compartments easier) to confirm that Mot1 is a nuclear
protein, consistent with its function as a transcriptional regulator.
In the absence of any Myc-tagged protein, there was little (if any)
background staining. Cells expressing MOT1-Myc displayed
bright fluorescence that was completely congruent with the cell
nucleus, revealed by co-staining the cells with the DNA-specific dye,
4',6-diamidino-2-phenylinole (Fig. 6).
Although not as bright, an identical pattern was observed when cells
expressing MOT1-Myc from its endogenous promoter on a low
copy number (CEN-based) plasmid were examined (data not shown). Thus, as expected, Mot1 is a nuclear protein.
Localization of the deletion derivatives fell into three distinct
classes; representative examples of each class are shown in Fig. 6.
First, full-length Mot1(D1408N) and three toxic Mot1 deletions
(
Residues 195-209 of Mot1 (-KK(X8)RRKKK-) match
the consensus bipartite NLS first recognized in frog nucleoplasmin
(62). Three deletion derivatives found in the cytosol ( Biochemical Mapping of the yTBP-binding Region of
Mot1--
Deletion mutants of Mot1 that retain its N-terminal 800 residues (
To try to narrow down the yTBP-binding region of Mot1 further (39),
additional deletions were made and tested for coimmunoprecipitation with yTBP (data not shown). No loss of yTBP binding was observed for
C-terminal truncations removing all residues up to 1087. A fragment
corresponding to residues 1-801 bound yTBP efficiently. However, all
attempts to generate smaller N-terminal deletions capable of binding
yTBP failed. Removal of as few as 100 N-terminal residues totally
abolished yTBP binding activity. Thus, the yTBP-binding segment of Mot1
appears large either because contact residues are noncontiguous or
because the entire region is needed to form a properly folded domain.
Conservation of the TBP-binding Region of Mot1--
Two orthologs
of Mot1 have been identified, Drosophila melanogaster 89B
protein (55) and human TAF172 (63, 64). The three proteins possess
~35% identity (~50% similarity) over their first 800 residues and
share ~55% identity (~70% similarity) in their C-terminal
catalytic (ATPase) domains. The hTAF172 exhibits biochemical properties
(63, 64) rather similar to those of Mot1 (35, 36), including
ATP-dependent dissociation of TBP-TATA DNA complexes and
TBP-stimulated ATPase activity. 89B protein has not been characterized
biochemically but is localized to the nucleus and appears to be
associated with distinct loci on polytene chromosomes (55). However,
when expressed in yeast, neither 89B (65) nor hTAF170 (64) can
complement a mot1 mutation.
If 89B protein is a Mot1 ortholog, then it should interact with
Drosophila TBP (dTBP). However, it has been demonstrated
recently that metazoan genomes contain additional TBP-like molecules
(56, 57, 66-68). In Drosophila, one such TBP-related
factor, TRF1, has intriguing parallels with 89B protein as follows:
both are essential for viability; both are expressed primarily in the
central nervous system and germ cells; and both colocalize at discrete loci on polytene chromosomes (55, 56, 69). Therefore, we investigated
interaction between the N-terminal domain (residues 1-825) of 89B
protein, expressed as a GST fusion in bacteria (Fig. 8A), and both dTBP and dTRF1,
each also expressed in bacterial cells. We reproducibly observed that
beads coated with GST-89B(1-825) specifically and efficiently adsorbed
dTRF1 from extracts of bacterial cells, whereas GST alone, although
present in much greater amounts (Fig. 8A), did not.
Likewise, beads coated with GST-89B(1-825) also bound dTBP (Fig.
8B). Even though dTBP always exhibited a higher level of
nonspecific interaction with GST alone than did dTRF1, increased
binding of dTBP to GST-89B was reproducibly observed in each of several
independent trials. Thus, the N-terminal segment of 89B protein binds
to a TBP-related factor and to dTBP itself. Likewise, it has been shown
that hTAF170 can also interact physically with hTBP (63, 64). Hence,
the function of the N terminus of Mot1 in yTBP binding has been
evolutionarily conserved. Our findings suggest that 89B protein plays a
role in dissociating dTRF1-DNA and dTBP-DNA complexes.
By using a coimmunoprecipitation method, we found that Mot1
and yTBP form a high affinity complex (Kd ~50
nM) in the absence of DNA, ATP, or any other yeast protein.
Our results explain why Mot1 was found in TBP-containing complexes
isolated from yeast extracts (30, 31, 71). This Kd
value is in good agreement with an estimate we obtained by an
independent kinetic method (35). It has been demonstrated both in
vitro (27) and in vivo (28) that Mot1 action is
antagonized by the TBP-binding general transcription factor, TFIIA. In
the absence of DNA, yeast TFIIA interacts with yTBP rather weakly, with
an estimated Kd in the 1-2 µM range
(72).3 Hence, if Mot1 and
TFIIA were present in solution at equivalent concentrations, Mot1-yTBP
complexes would be greatly favored over TFIIA-yTBP complexes. However,
as we have shown here and elsewhere (35), the presence of DNA does not
significantly enhance the affinity of Mot1 for yTBP, whereas the
Kd for interaction of TFIIA with TATA element-bound
TBP is estimated to be in the low nanomolar range (72,
73).4 Thus, when the target
is yTBP bound to a TATA box, TFIIA-yTBP-DNA complexes would be greatly
favored over Mot1-yTBP-DNA complexes.
Mot1(D1408N), which lacks detectable ATPase activity (28, 39),
bound yTBP with the same affinity as wild-type Mot1. The fact that
Mot1-yTBP complexes formed in solution in the absence or presence of
ATP (or a nonhydrolyzable analog) and that Mot1(D1408N) did not display
a higher affinity for yTBP than normal Mot1 suggests (albeit
indirectly) that ATP binding and hydrolysis drive neither formation nor
dissociation of Mot1-yTBP binary complexes. Yet, ATP hydrolysis is
required for dissociation of Mot1-yTBP-DNA ternary complexes (27, 28).
Therefore, energy derived from ATP binding and hydrolysis must be
channeled into conformational changes in yTBP that weaken its
interaction with DNA (but not its association with Mot1), in agreement
with other observations we have made (35). In vivo, however,
formation of Mot1-yTBP complexes is clearly reversible because even
high level overexpression of Mot1 is not detectably deleterious to
cells. What process or factor is responsible for competing with Mot1
for binding to yTBP?
TFIIA appears to be the best candidate for a physiologically
relevant antagonist of Mot1 action for several reasons. First, we have
shown that co-overexpression of TOA1 and TOA2,
the genes encoding the subunits of yTFIIA, partially suppresses the
dominant-negative phenotype of overexpressed Mot1(D1408N) (28). Second,
addition of purified TFIIA inhibits ADI (Mot1)-dependent
dissociation of yTBP-DNA complexes in vitro (27). Third,
TFIIA bound to TBP contacts the DNA upstream of the TATA box (73-75),
which is the same segment of the DNA that Mot1 contacts when it
associates with TATA box-bound yTBP (28). Fourth, we have shown here
that alteration of basic residues in Helix 2, including Lys133, Lys138, and Lys145, nearly abolish yTBP association with Mot1, as judged by
direct protein binding in vitro and by the resistance
conferred by these mutations to the dominant-negative effects of
overexpressed Mot1(D1408N) in vivo. Two of these mutants,
Spt15(K133L,K138L) and Spt15(K133L,K145L), remain bound to a TATA box
even in the presence of Mot1 and ATP (27). Likewise, the same yTBP
mutants are impaired in their ability to associate with TFIIA (41, 49, 76), suggesting that TFIIA contacts Helix 2. Indeed, recent NMR studies
performed with TFIIA-yTBP complexes containing full-length Toa1 (77) or
model Toa1-derived peptides (78) have revealed that basic residues in
Helix 2 of yTBP (including LysK133, Lys138, and Lys145)
contribute significant contacts with TFIIA that were not observed by
x-ray diffraction because the crystallographic studies were performed
on complexes containing versions of Toa1 with large deletions (74, 75).
Finally, a single charge-reversal mutation in yTBP, Spt15(K145E), is
sufficient to prevent yTBP binding to both Mot1 and TFIIA (79). Thus,
cumulative evidence suggests that binding of Mot1 and TFIIA to
TATA-bound yTBP is mutually exclusive.
By using deletions and independent strategies to assess
interaction with yTBP both in vitro and in vivo,
we delimited the yTBP-binding region of Mot1 to residues 1-800. In
this respect, our results are in substantial agreement with the
findings of Auble et al. (36), although we found no evidence
that the C-terminal portion of Mot1 contributes to recognition and
binding of yTBP. Our results support the conclusion that overexpression
of ATPase-defective Mot1(D1408N) is toxic to cells because it forms a
nonproductive complex with yTBP. Deletions that removed the ATPase
domain still bound to yTBP with near-normal affinity and displayed the
same growth-inhibitory phenotype. However, C-terminally truncated Mot1 derivatives that bound yTBP in vitro, like
Mot1( The results we obtained in vivo and in
vitro demonstrated that the N-terminal 800 residues of Mot1 are
necessary and sufficient for yTBP binding. Moreover, we found that
evolutionarily conserved, positively charged residues on the surface of
yTBP are necessary for Mot1 binding. Our observation that the
N-terminal domain of 89B protein bound dTRF1 and dTBP suggested that
clusters of evolutionarily conserved, surface-exposed, and negatively
charged residues within the first 800 amino acids of Mot1, 89B protein,
and hTAF170 might be candidates for residues involved in TBP binding.
Two sequence elements in Mot1
(Asp462-Asp463-Asp464 and
Asp503-Asp504-Asp505) fit these
criteria and are also conserved in apparent Mot1 orthologs in
Arabidopsis thaliana and Schizosaccharomyces
pombe (GenBankTM accession numbers CAB71002.1 and
CAB37625.1, respectively). Hence, two corresponding Mot1 derivatives,
Mot1(D462A,D463A,D464A) (mot1-462) and
Mot1(D503A,D504A,D505A) (MOT1-503), were constructed by
site-directed mutagenesis (65, 70). Both mutant proteins were produced
at the same level as normal Mot1, and Mot1-503 fully complemented a
mot1 Perhaps multiple redundant elements within the N-terminal
domain of Mot1 contribute to its high affinity binding of yTBP. Consistent with this suggestion, analysis of the human homolog of Mot1
(TAF(II)170) suggests that multiple segments of the N-terminal domain
participate in TBP binding.5
In this same regard, recent in silico analysis suggests that Mot1 contains eleven HEAT repeats (82) (see Fig.
4A). HEAT repeats and related helical repeat elements are
clearly involved in protein-protein interactions over extended
protein-protein interfaces (83). Seven of the 11 putative HEAT repeats
in Mot1 fall within the first 800 residues. The mot1-462 and
MOT1-503 alleles altered residues in different candidate
HEAT repeats (448-479 and 492-523, respectively). Perhaps one or more
of the remaining five HEAT repeats within the N-terminal region of Mot1
provides the contacts critical for yTBP recognition.
We found that Mot1 is located in the nucleus. Deletions that
removed the putative NLS (62, 84) were excluded from the nucleus
(summarized in Table II). Unexpectedly,
however, two deletions (
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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(BUR6/NCB1 gene product) grow very
poorly; cells lacking NC2
(NCB2 gene product) are
inviable (21, 23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
diploid
(W303D) was used. Strain KHY18 was constructed in the following manner.
Strain YPH501 (37) was transformed (38) with an
XhoI-NotI fragment of plasmid pKH4 containing the
mot1-
2::LEU2 allele in which the
LEU2 gene replaces codons 79-1822 of the
MOT1-coding sequence (39). After confirming correct
transplacement of the MOT1 locus on one homolog of
chromosome XVI by Southern blotting, the resulting
LEU2+ diploid (KHY1) was transformed with
pRSMOT1, a derivative of the URA3-marked
CEN-based vector, pRS316 (37), containing a 7.1-kb genomic
BglII fragment expressing MOT1 from its
endogenous promoter (24). After sporulation and tetrad dissection, one of the resulting URA3+
LEU2+ MATa spore clones
was designated KHY18 and was used to study two site-directed mutants
(mot1-462 and mot1-503) generated in this study
(see below). Strain WPY1 was generated by transformation of strain
W303D with a BamHI-SnaBI fragment of plasmid
pKA23 containing the spt15
::LEU2
mutation, in which a portion of the SPT15 coding sequence
has been replaced with the LEU2 gene (40). After selection of LEU2+ transformants and verification for
proper integration at the SPT15 locus using the polymerase
chain reaction (PCR) with appropriate primers, WPY1 was transformed
with a TRP1-marked CEN-based plasmid expressing
wild-type SPT15 from its endogenous promoter (pUN45IID) or
the same plasmid expressing either of two TBP mutants
(K133L,K138L or K133L,K145L) (41). After sporulation, the cells
were treated with diethyl ether to enrich for spores (42), and
LEU2+ TRP1+ haploids
carrying the spt15
::LEU2 mutation,
maintained by low copy plasmids expressing TBP or TBP mutants, were
selected on medium lacking Leu and Trp.
mutation (data not shown). A Myc-tagged version of
the dominant-negative ATPase-defective MOT1(D1408N) allele (28) was prepared
in the same fashion, yielding pRSMot1(D1408N)Myc.
Synthetic oligonucleotide primers used for PCR
25-243),
mot1(
494-801),
mot1(
802-1259),
mot1(
1262-1867), and
mot1(
1089-1867) by in
vitro transcription, PCR was used to amplify sequences containing
the MOT1 deletion mutants from plasmids pKH7, pKH43, pKH34,
pKH35, pKH25, and pKH23, respectively. In each case, a 5'-primer
(KHP22) that corresponds to a segment of the T3 promoter fused to the
MOT1 translation start site was used in combination with a
primer (T7) that anneals 3' to the MOT1 ORF.
1823-1867, BstXI;
1676-1867,
EcoNI;
1386-1867, NdeI;
1280-1867,
HpaI;
1258-1867, CelII;
1203-1867,
BspBI;
1167-1867, PvuI;
1120-1867,
AflII;
1087-1867, SalI;
842-1867,
MunI; and
800-1867, NruI.
1-897) fragment was made with
primers KHP12 and KHP14, using linearized pKH2 as the template. The
mot1(
1-1253), mot1(
1-655), and
mot1(
1-513) fragments were made in
a similar manner except that primers, KHP13, KHP15, and KHP16,
respectively, were used in place of KHP12. Primers KHP13 and KHP14 were
used to make mot1(
1-1253). The
mot1(
1-100) fragment was made using primers T7 and KHP23. The
mot1(
1-1100) fragment was
transcribed from plasmid pKH27 (39), which was made by digesting pKH2
with XhoI and SalI and religating the vector. The
PCR product generated using primers KHP9 and JDM6 with pKH2 as the
template was digested with XhoI and SalI and
introduced into pKH2 digested with the same enzymes to create plasmid
pKH29, which expresses mot1(
1-1026) from the T3 RNA polymerase promoter.
-D-galactoside to a final concentration of 0.1 mM. The cultures were shifted to 30 °C and grown
for a further 3 h to an A600 nm = ~2.
Cells were harvested by centrifugation, washed in TEG buffer (25 mM Tris-HCl (pH 7.6), 0.5 mM EDTA, 10%
glycerol) containing 300 mM NaCl (TEG-300), and recollected. All subsequent steps were performed at 4 °C. Cell pellets were resuspended in 3 volumes of ice-cold TEG-300 plus 1 mM DTT, 1 mM 4-(2-aminoethyl)benzenesulfonyl
fluoride, and 1 µg/ml leupeptin. Cells were lysed by three 10-s
bursts of sonication on ice and then Triton X-100 (10% stock) was
added to a final concentration of 1%. The detergent-containing extract
was clarified by centrifugation for 10 min in a microcentrifuge at top
speed (16,000 × g) to remove cell debris, and the
supernatant fraction was transferred to a fresh tube. The GST proteins
were purified by batchwise binding to glutathione-Sepharose beads
(Amersham Pharmacia Biotech), as recommended by the supplier. After
elution, purified proteins were dialyzed exhaustively against TEG-100, and protein concentration was determined by a modification (Protein Assay Kit, Bio-Rad) of the dye-binding method of Bradford (54), using
bovine serum albumin as the standard. Cells expressing HA-dTRF1 and
HA-dTBP were grown, induced, and lysed as described above. To remove
nucleic acids from the clarified extract, polyethyleneimine (10%
stock) was added slowly with mixing to a final concentration of 1%.
The extract was stirred on a rotary mixer for 15 min and then the
resulting precipitate was sedimented by centrifugation for 20 min at
16,000 × g. The supernatant fraction was transferred to a fresh tube, diluted to a final protein concentration of 5 mg/ml in
TEG-100, and frozen in aliquots at
80 °C.
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Mot1 and Mot1(D1408N) coimmunoprecipitate
with yTBP. [35S]Met-labeled Mot1, Mot1(D1408N), and
Rad3 (as a control) were produced by in vitro transcription
and translation and partially purified by ammonium sulfate
precipitation. For each coimmunoprecipitation reaction, 0.5 pmol of the
indicated protein was incubated in either the absence (lanes 4, 6, and 8) or the presence (lanes 5, 7, and
9) of a 2-fold molar excess of purified unlabeled yTBP at
4 °C for 30 min and then with 2 µl of rabbit polyclonal -yTBP
antibodies for 1 h at 4 °C. The resulting immune complexes were
recovered by binding to protein A-agarose beads. After brief
centrifugation, the supernatant solution was removed, and the beads
were washed once with buffer. The bead-bound immune complexes were
solubilized by boiling in SDS-PAGE sample buffer and analyzed by
SDS-PAGE and autoradiography, along with portions (2%) of the initial
amount of material that was added to each reaction (Input, lanes
1-3).
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Fig. 2.
Basic residues in Helix 2 of yTBP are
required for the binding of Mot1. A, schematic
representation of the -carbon backbone of yTBP (gray);
side chains of the basic residues mutated in the yTBP derivatives used
in this study (black). Coordinates from Ref. 52 were
obtained from the Protein Data Bank (Brookhaven National Laboratory)
and visualized using MacImdadTM (Molecular Applications
Group). B, [35S]Met-labeled Mot1 was incubated
alone (lane 7) or with [35S]Met-labeled yTBP
(lane 8) or with each of the four yTBP mutants indicated
(lanes 9-12), and then immunoprecipitated with
-yTBP
antibodies, as described in the legend to Fig. 1. The resulting immune
complexes were analyzed by SDS-PAGE and autoradiography, along with a
portion (2%) of the initial amount of Mot1 that was added to each
reaction (Input, lane 1) and portions (20%) of the initial
amount of yTBP and each yTBP variant that was added to each reaction
(Input, lanes 2-6). C, Mot1 coimmunoprecipitated
by yTBP and each yTBP mutant was quantified using a
PhosphorImagerTM (Molecular Dynamics), normalized to the
amount of yTBP immunoprecipitated and expressed as a percentage of the
amount of Mot1 coimmunoprecipitated by wild-type yTBP (defined as
100%). Values represent the average of three separate experiments, and
the error bars represent the S.D.
-helix (Helix 2) in the C-terminal lobe of yTBP (52). Mutations in
this same region of yTBP decrease its ability to form complexes with
TFIIA (41), and TFIIA competes with Mot1 for binding to yTBP-DNA
complexes (28). To determine whether mutations in Helix 2 affect
Mot1-yTBP interaction in solution and in the absence of DNA, three yTBP variants carrying mutations in this segment (Fig. 2A),
Spt15(K133L,K138L), Spt15(K133L,K145L) (41), and Spt15(K138T,Y139A)
(49), and a mutant, Spt15(R105H), that alters a residue on the surface
of yTBP that contacts TATA DNA (52), were tested for their ability to
bind Mot1 by the coimmunoprecipitation method.
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Fig. 3.
Mot1-yTBP interaction is disrupted at high
ionic strength. A, [35S]Met-labeled Mot1
was incubated alone or with [35S]Met-labeled yTBP in the
absence of any added salt or in the presence of the indicated
concentrations of potassium acetate, subjected to immunoprecipitation,
and analyzed, along with portions (Mot1, 1%; yTBP, 20%) of the
initial amount of each protein added to the reaction. B,
Mot1 coimmunoprecipitated by yTBP was quantified, as described in the
legend to Fig. 2 (at each salt concentration, the amount of Mot1
nonspecifically associated with the antibody-coated beads in the
absence of yTBP was subtracted from the amount of Mot1
coimmunoprecipitated by yTBP).
mutations and, when overexpressed, acts to inhibit
growth in a dominant-negative fashion, which can be overcome by
co-overexpression of yTBP (SPT15) (28, 39). Although unable
to hydrolyze ATP, Mot1(D1408N) still binds yTBP with apparently normal
affinity (Fig. 1, lanes 6 and 7). Hence, an
explanation for its dominant-negative phenotype is that
Mot1(D1408N)-yTBP complexes are nonfunctional, and this sequestration
reduces the available yTBP pool below the level necessary for
viability. Presumably, yTBP overexpression overcomes this problem by
restoring a sufficient level of free yTBP. By this model, the toxic
effect of Mot1(D1408N) should also be relieved by mutations that ablate
its ability to bind yTBP. We used this approach to delineate the
regions of Mot1 required for its association with yTBP in
vivo.
1-1100), which is
initiated from an internal Met codon), and the same C-terminal tag.
Indeed, on Gal-containing medium, all of the internal deletions and
C-terminal truncations constructed, whether in Mot1 (Fig.
4A) or Mot1(D1408N) (data not shown), were expressed at similar levels and at their expected molecular masses, as judged by SDS-PAGE and immunoblotting with anti-Myc mAb 9E10 (Fig. 4B).
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Fig. 4.
Structure and expression of Mot1
deletions. A, schematic depiction of wild-type Mot1
(open box) and the various deletion and truncation
derivatives used in this study. Top, positions of consensus
HEAT repeat motifs (82) (cross-hatched boxes), putative
bipartite NLS (vertical black stripe), relative positions of
the triple-alanine substitution mutations in, respectively, the
mot1-462 and mot1-503 alleles
(asterisks), and the seven signature motifs found in the
superfamily of nucleic acid-dependent ATPases and helicases
(gray boxes) are indicated. Results presented here
demonstrate that residues 1-801 are required for both yTBP binding and
for nuclear localization. We have shown previously that the C-terminal
domain is a catalytically active ATPase (28). The function of the
region between residues 801 and 1254 is unknown. Bottom,
relative positions of the conserved ATPase domain (solid
box) and the putative bipartite NLS (black stripe) are
indicated. The extent of the sequence removed is indicated by the
numbers to the left of each construct and by the missing
area (connected by thin lines for internal deletions). All
constructs were tagged with a C-terminal c-Myc epitope (not shown).
B, wild-type yeast cells (strain W303-1A) were transformed
with an empty multicopy vector (YEp352GAL) or the same vector
expressing normal Mot1 or the Mot1 variants shown in A from
the galactose-inducible GAL1 promoter, grown to
mid-exponential phase in Glc medium, shifted to Gal medium for 3 h, harvested, and lysed with glass beads. Samples of the resulting
extracts (30 µg of total protein) were resolved by SDS-PAGE and
analyzed by immunoblotting with anti-c-Myc mAb 9E10. The migration
positions of molecular weight markers are shown
(left).
1262-1867, was nearly as toxic as Mot1(D1408N). Likewise, in the
context of either Mot1 or Mot1(D1408N), derivatives (
1090-1259 and
802-1259) that retained the first 800 N-terminal residues of Mot1
displayed a detectably deleterious effect on growth (Fig. 5A,
left and right panels), whereas all of the remaining
constructs, in which portions or all of these residues were removed
(
494-801,
244-1280,
25-491,
25-243, and
1-1100),
had no effect on growth. Given that all inhibitory derivatives had the
same phenotype in the context of either Mot1 or Mot1(D1408N), these
deletions presumably abolish Mot1 activity (and presence of the D1408N
mutation thus becomes irrelevant for their dominant-negative effect).
Indeed, each deletion (Fig. 4A) was incapable of restoring
viability to mot1-1ts or mot1
cells
(39).
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Fig. 5.
In vivo assay for Mot1-yTBP
interaction. A, strain W303-1A was transformed with an
empty URA3-marked multicopy vector (YEp352GAL) or the same
vector expressing from the GAL1 promoter either wild-type
MOT1 and the various deletion derivatives of it indicated
(left panel) or MOT1(D1408N) and the
various deletion derivatives of it indicated (right panel).
The resulting transformants were grown to saturation in SCGlc-Ura
medium (to maintain selection for the plasmid). Cultures were diluted
to A600 nm = 1 with water, followed by four
5-fold serial dilutions, which were then spotted on SC-Ura plates
containing either 2% Glu (not shown) or 2% Gal to 0.2% Suc and
incubated at 30 °C for 3 days. B, strain W303-1A was
transformed with an empty vector (YEp352GAL) or with the same vector
expressing either MOT1, MOT1(D1408N),
or the indicated deletions and cotransformed with either an empty
LEU2-marked vector (pAD4M) ( ) or the same vector
expressing yTBP (SPT15 gene product) from the
ADH1 promoter (+). The resulting transformants were then
grown, diluted, plated on SC-Ura-Leu plates containing either 2% Glc
(not shown) or 2% Gal to 0.2% Suc, and incubated as in A. C, strain WPY1
(spt15
::LEU2) expressing either
wild-type SPT15 (yTBP) or either of two yTBP mutants
(K133L,K138L or K133L,K145L) from the endogenous SPT15
promoter on a TRP1-marked CEN-based plasmid
(pUN45IID) were transformed with a URA3-marked multicopy
vector (YEp352GAL) expressing either wild-type MOT1 or
MOT1(D1408N) from the GAL1 promoter,
as indicated. The resulting transformants, propagated on SCGlc-Trp-Ura,
were diluted (as in A) and then plated on either the same
medium (not shown) or on SCGal/Suc-Trp-Ura medium, and incubated as in
A.
1090-1259,
802-1259,
1262-1867, and
1089-1867) (Fig. 5B).
View larger version (66K):
[in a new window]
Fig. 6.
Mot1 is a nuclear protein. A wild-type
diploid strain (W303D) was transformed on Glc medium with an empty
vector (YEp352GAL) or the same vector expressing c-Myc epitope-tagged
versions of either MOT1 or the deletions indicated, shifted
to Gal medium for 3 h, fixed, and analyzed by indirect
immunofluorescence, as described under "Experimental Procedures."
DNA was visualized by 4',6-diamidino-2-phenylinole (DAPI)
staining (left panels); Mot1 was visualized by incubating
mouse anti-c-Myc mAb 9E10, as the primary antibody, followed by
fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin, as
the secondary antibody (right panels).
1090-1259,
1262-1867, and
1089-1867) were localized exclusively to the nucleus, like Mot1 itself (Fig. 6). Second, the
fourth toxic deletion,
802-1259, was localized primarily in the
nucleus, but cytosolic staining was also readily visible (Fig. 6).
Third, all five nontoxic deletions (
25-491,
25-243,
1-1100,
494-801, and
244-1280) were primarily, if not exclusively, in
the cytosol (Fig. 6).
25-491,
25-243, and
1-1100) remove this sequence (Fig. 4A,
NLS represented by black bar). However, two of the deletions
that localized to the cytosol,
494-801 and
244-1280, retain the
putative NLS (and the 494-801 region has no obvious secondary NLS). In
any case, because Mot1 mutants with deletions within the first 800 amino acids were found in the cytoplasm (Fig. 6), their lack of
toxicity upon overexpression could merely be due to their failure to
enter the nucleus. Hence, it was not possible to conclude with
confidence that such deletions alleviate the toxicity of Mot1(D1408N)
in vivo because they prevent yTBP binding. Therefore, as an
alternative method to delineate the yTBP-binding region of Mot1,
interaction of the Mot1 deletion mutants with yTBP was examined
in vitro using the coimmunoprecipitation assay.
802-1259,
1262-1867, and
1089-1867) all displayed
efficient coimmunoprecipitation with yTBP (Fig.
7B), equivalent to (or better than) that of wild-type Mot1 (Fig. 7A). Reassuringly, two
deletions that lack portions of the first 800 residues of Mot1
(
25-243 and
494-801) failed to show detectable interaction with
yTBP (Fig. 7A). Thus, despite the uncertainty raised by
localization of the nontoxic Mot1 variants in the cytosol, the in
vitro binding experiments were completely consistent with the
genetic approach we used to map the yTBP-binding domain of Mot1
in vivo. The fact that two derivatives (
494-802 and
244-1280), which are nontoxic in vivo and fail to bind
yTBP in vitro, contain the presumptive NLS yet fail to enter
the nucleus suggests that interaction of Mot1 with yTBP may be
important for unmasking of the NLS. Hence, residues 1-801 of Mot1 are
necessary for its association with yTBP, and interaction of Mot1 with
yTBP may be necessary for localization of Mot1 to the nucleus.
View larger version (59K):
[in a new window]
Fig. 7.
In vitro binding assay delineates
the N-terminal region of Mot1 as the yTBP-binding domain.
[35S]Met-labeled full-length Mot1 (A) and the
deletions indicated (A and B) were produced by
in vitro transcription/translation, and their interaction
was assessed by coimmunoprecipitation in the presence and absence of
radiolabeled yTBP that was prepared in the same fashion, as described
in the legend to Fig. 1. The resulting bead-bound immune complexes were
solubilized and analyzed, along with samples (2%) of the amount of
these proteins initially added to these reactions, as described in the
legend to Fig. 1.
View larger version (33K):
[in a new window]
Fig. 8.
The N terminus of Drosophila
89B protein interacts with dTBP and the TBP-related factor,
dTRF1. A, S. japonicum glutathione
S-transferase (GST) and a GST-89B(1-825) fusion protein
were purified from E. coli, as described under
"Experimental Procedures." Equal volumes of the final purified
fractions were mixed with SDS-PAGE sample buffer, boiled briefly,
resolved by electrophoresis on a 10% gel, and visualized by staining
with Coomassie Blue dye. B, equimolar amounts of the
purified proteins shown in A were mixed with equal amounts
of extracts of E. coli cells that had expressed at a high
level HA epitope-tagged versions of either dTRF1 (28 kDa) or dTBP (45 kDa), as indicated. The resulting mixtures were then incubated with
glutathione-agarose beads, as described under "Experimental
Procedures." After washing, bead-bound proteins were eluted by
boiling in SDS-PAGE sample buffer, resolved by SDS-PAGE, and analyzed
by immunoblotting with anti-HA mAb 12CA5, along with samples of the
dTRF1- and dTBP-containing extracts (equivalent to 2% of the amount
added to each pull-down reaction) (input). Some extraneous
E. coli proteins weakly cross-react with the anti-HA
antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1262-1867), were less toxic than Mot1(D1408N) when
overexpressed in vivo. Co-overexpression of yTBP with
full-length Mot1(D1408N) does not restore a completely wild-type growth
rate, whereas co-overexpression of yTBP with Mot1(
1262-1867) does,
implying perhaps that the C-terminal domain of Mot1(D1408N) may
interact with other factors. Other Mot1-interacting proteins could
contribute to the observed promoter specificity of Mot1 action (24, 80,
81).
mutation at each of three different temperatures tested (26, 30, and 37 °C). In contrast, cells expressing Mot1-462 as the sole source of Mot1 exhibited an obvious slow-growth phenotype at all three temperatures. However, overexpression of yTBP did not
rescue the slow-growth phenotype of these cells. Moreover, using the
coimmunoprecipitation assay, Mot1-462 did not display any detectable
decrease in yTBP binding in vitro, compared with normal
Mot1. Thus, neither of the segments of Mot1 that we selected for
site-directed mutagenesis make contacts critical for yTBP binding.
494-801 and
244-1280) that did not
remove the NLS, but did destroy yTBP binding, were also excluded from
the nucleus. These deletions may remove sequences in Mot1 responsible
for regulating its nuclear localization by phosphorylation or some
other post-translational modification. However, a more parsimonious
interpretation of the behavior of these mutants is that nuclear
localization of Mot1 requires its binding to yTBP. A requirement for
yTBP binding before nuclear import could provide a simple homeostatic
mechanism for maintaining the proper Mot1-yTBP ratio in the nucleus.
Perhaps yTBP binding induces a conformational change that exposes the NLS in Mot1; alternatively, nuclear translocation of Mot1 may occur via
"piggy-backing" on yTBP. In this regard, the importin, Kap114,
plays a major role in nuclear entry of yTBP (61, 85); whether it is
free yTBP or yTBP sequestered in larger complexes that enters the
nucleus via this route is not known. It will be interesting to test
whether kap114 mutations have any effect on the subcellular
distribution of Mot1. However, because spt15
and
mot1
mutants are inviable, whereas kap114
mutants are viable, both yTBP and Mot1 must have at least one other
Kap114-independent route for entering the nucleus.
Summary of Mot1 mutant phenotypes
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Steve Buratowski, Grace Gill, Mike Holmes, Kevin Struhl, Robert Tjian, and Naomi Zak for the gifts of reagents and advice. We also thank Karen Arndt, Frank Pugh, Steve Hahn, and Marc Timmers for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a predoctoral fellowship from the Howard Hughes Medical Institute (to J. I. A.), by a University fellowship from the Graduate Division of the University of California, Berkeley, and a predoctoral fellowship from the National Science Foundation (to K. E. H.), by a University of California President's Undergraduate research fellowship (to W. A. P.), by Postdoctoral Fellowship PF-3308 from the American Cancer Society, California Division (to J. L. D.), and by Research Grant GM21841 from the National Institutes of Health and facilities provided by the Berkeley campus Cancer Research Laboratory (to J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Current address: Dept. of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139.
¶ Current address: Cell Genesys, Inc., 342 Lakeside Dr., Foster City, CA 94404.
To whom correspondence should be addressed: Dept. of
Molecular and Cell Biology, Division of Biochemistry and Molecular
Biology, Rm. 401, Barker Hall, University of California, Berkeley, CA
94720-3202. Tel.: 510-642-2558; Fax: 510-643-6791; E-mail:
jeremy@socrates.berkeley.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010665200
2
For the reaction Mot1 + yTBP Mot1·yTBP complex, Kd = ([Mot1]eq × [yTBP]eq)/[Mot1·yTBP]eq, where
[Mot1]eq = [Mot1]0
[Mot1·yTBP]eq and [yTBP]eq = [yTBP]0
[Mot1·yTBP]eq. A typical immunoprecipitation reaction might contain ~0.5 pmol of Mot1 and 1 pmol of yTBP in 200 µl (2.5 and 5 nM, respectively).
Therefore, in this example, if the amount of radioactivity recovered in
the immunoprecipitated Mot1·yTBP complex was 2% of the input Mot1, then the concentration of the Mot1·yTBP complex present was 0.25 nM (the actual value, 0.05 nM, was multiplied
by 5 to take into account the fact that the amount of anti-yTBP
antibodies added were sufficient to pull down reproducibly only 20% of
the input yTBP or yTBP-containing complexes). Hence, in this case,
Kd = ([2.5
0.25][5
0.25]/[0.25]) = 43 nM.
3 B. F. Pugh, personal communication.
4 S. Hahn, personal communication.
5 L. A. Pereira, J. A. van der Knaap, V. van den Boom, F. J. van de Heuvel, and H. T. M. Timmers, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pol II, eukaryotic RNA polymerase II; AMP-P-NH-P, 5-adenylyl-imidobisphosphate; BSA, bovine serum albumin; dTBP, D. melanogaster TATA box-binding protein; dTRF1, D. melanogaster TBP-related factor-1; DTT, dithiothreitol; GST, Schistosoma japonicum glutathione S-transferase; HA, influenza virus hemagglutinin; mAb, monoclonal antibody; Myc, product of the c-myc proto-oncogene; NLS, nuclear localization sequence; ORF, open reading frame; Raf, raffinose; Suc, sucrose; TAF, TBP-associated factor; TBP, TATA box-binding protein; UAS, upstream activating sequence; and, yTBP, Saccharomyces cerevisiae TATA box-binding protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair.
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