From the Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908
Received for publication, November 10, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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Mot1 is an essential Snf2/Swi2-related
Saccharomyces cerevisiae protein that binds the
TATA-binding protein (TBP) and removes TBP from DNA using ATP
hydrolysis. Mot1 functions in vivo both as a repressor and
as an activator of transcription. Mot1 catalysis of TBP·DNA
disruption is consistent with its function as a repressor, but the Mot1
mechanism of activation is unknown. To better understand the
physiologic role of Mot1 and its enzymatic mechanism, MOT1 mutants were generated and tested for activity in vitro and
in vivo. The results demonstrate a close correlation
between the TBP·DNA disruption activity of Mot1 and its essential
in vivo function. Previous results demonstrated a large
overlap in the gene sets controlled by Mot1 and NC2. Mot1 and NC2 can
co-occupy TBP·DNA in vitro, and NC2 binding does not
impair Mot1-catalyzed disruption of the complex. Residues on the
DNA-binding surface of TBP are important for Mot1 binding and the
Mot1·TBP binary complex binds very poorly to DNA and does not
dissociate in the presence of ATP. However, the binary complex binds
DNA well in the presence of the transition state analog
ADP-AlF4. A model for Mot1 action is proposed in which ATP
hydrolysis causes the Mot1 N terminus to displace the TATA box, leading
to ejection of Mot1 and TBP from DNA.
A critical step in the assembly of an active transcription complex
at an RNA polymerase II promoter involves recruitment of TATA-binding
protein (TBP)1 and
TBP-associated factors (1-3). TBP recruitment and activity are
influenced by a large number of transcription factors and components of
the general transcription machinery, many of which can interact
directly with TBP (3-6). MOT1 was uncovered in genetic screens for factors that repress transcription driven by a weak promoter (7-11). Consistent with its function as a repressor, Mot1 was
isolated independently as an ATP-dependent factor that disrupts the TBP·DNA complex (12). Mot1 binds the TBP·DNA complex in vitro (12) and contacts both TBP and about 17 bp of DNA
upstream of the TATA box (13). In the absence of DNA, Mot1 also
dimerizes with TBP (13-15). In this report, we refer to the Mot1·TBP
complex as the "binary" complex, and the Mot1·TBP·DNA complex
is referred to as the "ternary" complex.
Mot1 homologs have been identified in many eukaryotes. The human
homolog is BTAF1, which interacts with TBP (16, 17) and catalyzes
disruption of human TBP·DNA complexes (17). The insect homolog, the
89B helicase (18), may interact with TBP or TBP-related factor 1 (TRF1) in vitro (19). The Mot1 C terminus contains the conserved ATPase domain (7), whereas the Mot1 N terminus is
responsible for TBP binding (19-21). The structural basis for Mot1·TBP recognition is unknown, however, it was recently suggested that the Mot1 N terminus contains HEAT or ARM repeats, which compose a
class of structurally related leucine-rich repeats (22-24). Structural studies have shown that HEAT and ARM repeats form two Mot1 is a member of the Snf2/Swi2 ATPase family (25-27). It has
been suggested that at least some Snf2/Swi2 ATPases are
processive molecular motors, acting by driving DNA translocation or
rotation (28, 29). The Mot1·TBP·DNA system has been used to test
several theories about how these ATPases drive changes in protein·DNA interactions. Mot1 is not a helicase (13-15), nor does it travel long
distances on DNA after TBP is removed from the TATA box (30). Catalysis
of TBP·DNA disruption requires a grip by Mot1 on both upstream DNA
and TBP, although the upstream DNA and the TBP·DNA complex can be
conformationally uncoupled without impairing catalysis (13). These
results indicate that Mot1 does not dissociate TBP·DNA by propagation
of DNA twist or writhe through the TATA box. A similar result has been
reported for the Snf2/Swi2 family member ISWI (31). It is
possible that Mot1 interacts with the TATA box directly and in so doing
alters its structure or that Mot1 uses ATP hydrolysis to disrupt
TBP·DNA complexes via short-range tracking or ATP-driven insertion of
Mot1 into the TBP·DNA interface. Alternatively, Mot1 may mediate
TBP·DNA disruption by inducing a conformational change in TBP that
deforms the DNA-binding surface of TBP. Here we demonstrate that
residues on the DNA-binding surface of TBP impair the interaction of
TBP with Mot1, suggesting that Mot1 contacts the DNA-binding surface of
TBP, and explaining why the Mot1·TBP binary complex binds DNA poorly
compared with TBP alone. Binding of an ATP transition state analog
locks the binary complex into a form in which the Mot1·TBP complex
can bind DNA better than the nucleotide-free form of the Mot1·TBP
complex. These results suggest that ATP hydrolysis causes a change in
either the conformation of TBP or the interaction of Mot1 with the
DNA-binding surface of TBP and that these ATP-driven conformational
changes explain how Mot1 drives disruption of the TBP·DNA complex.
mot1 Library Construction and Screening--
Oligonucleotide
primers flanking the EcoRI site (bp position 1026 in the
MOT1 open reading frame (ORF)) and ClaI site
(position 2092) were used to amplify ~1 kb of the MOT1 ORF
using Taq polymerase under reduced fidelity conditions as
described previously (32). The PCR-amplified DNA was digested with
EcoRI and ClaI and cloned into an
EcoRI-ClaI-gapped plasmid containing the rest of
the MOT1 ORF under control of the GAL1 promoter
on a CEN ARS plasmid bearing the LEU2 gene (20).
Note that an additional ClaI site is present in the
MOT1 ORF, but this second site is blocked from
ClaI digestion by overlapping dam methylation.
Six independent transformants were picked at random from the bacterial
transformation of the primary ligation mix, and these were sequenced
and found to contain ~1-bp change per kilobase (kb) of amplified DNA.
Bacterial transformants containing the mutated DNA were then scraped
en masse from agar plates, inoculated at high density into
liquid media, and used in a large-scale plasmid purification prep. The
resulting purified plasmids were then used to transform yeast strain
AY29 (mot1 Site-directed MOT1 Mutants--
Site-directed mutagenesis was
performed using synthetic oligonucleotides and either overlapping PCR
or the Stratagene QuikChange kit, according to the instructions
provided by the manufacturer. Each mutation was engineered to encode a
change in a restriction site (either introduction of a new site or loss
of an existing site) to facilitate subcloning. Candidate transformants
containing the correct restriction sites were then sequenced completely
in a region that overlaps a DNA fragment with convenient restriction sites. The sequenced DNA fragment was then sub-cloned to LEU2 CEN
ARS plasmids derived from pRS315 (33) that contain the
MOT1 ORF driven by the GAL1 promoter or by a
448-bp fragment of the MOT1 promoter. All constructs encode
a Mot1 derivative with the Py tag (35) appended to the N terminus to
facilitate quantitation by Western blotting and purification using
antibody-coupled beads (20, 35). Additional details regarding plasmid
construction are available upon request. Plasmids containing the
site-directed alleles were transformed into AY29 yeast cells (see
above), and the ability of the constructs to support viability was
assessed by plasmid shuffling using standard techniques (34). Strains harboring alleles under control of the MOT1 promoter were
analyzed for growth defects on synthetic media without leucine and
containing raffinose, galactose, or glucose as the carbon source.
Strains harboring alleles under control of the GAL1 promoter
were streaked to galactose-containing plates to induce expression prior
to plasmid shuffling. Growth of strains was compared with congenic
wild-type cells by incubation at 16 °C, 30 °C, 32 °C, and
35 °C.
Purification of Recombinant TBP and TBP Mutants--
Recombinant
full-length TBP and TBP mutants expressed under the control of the T7
promoter as a fusion with N-terminal six-histidine tag were obtained by
transformation of BL21(DE3) Escherichia coli cells with the
appropriate plasmid expression vectors (13, 36). Cells were inoculated
into 1 liter of yeast extract Tryptone (YT) media containing 100 µg/ml ampicillin or 30 µg/ml kanamycin at 37 °C and were grown
to an optical density at 600 nm of 0.7-1.0. Isopropyl- Native Gel Electrophoresis--
For detection of TBP by native
gel electrophoresis (Figs. 6C, 6D, and
7B), full-length TBP and Mot1 were incubated in binding buffer (13) containing 120 mM KCl and 12 mM
HEPES buffer, pH 7.6 (37), using proteins at the concentrations
indicated in figure legends. The gels in Figs. 6C and
7B were run with the electrodes reversed: samples were
loaded on the side of the positive electrode, and run toward the
negative electrode. The gel was, however, pre-run for >50 min with the
electrodes connected in the usual fashion before loading. Following
electrophoresis, the gels were boiled in 1% SDS for 1 min, then
transferred to Immobilon and TBP was detected using TBP antiserum. The
TBE gel shift assay in Fig. 8A was performed as described
previously (38). Gel shift assays were otherwise performed as described
previously (13) using 5 nM core domain TBP (gift of J. Geiger) or full-length TBP with minor modifications as indicated.
Synthesis and labeling of the 36- and 17-bp DNAs, and preparation of
the radiolabeled 100-bp adenovirus major late promoter fragment, was as
previously described (13). DNA concentration was about 0.5 nM in the reactions. The concentration of Mot1 needed to
bind 50% of the TBP·DNA complex is ~5 nM (13). The
concentration of Mot1 used was estimated from this activity and is
indicated in the figure legends. ATP was used at between 5 and 100 µM. ADP was used at 100 µM. NaF was used at
2.5 mM. AlCl3 was used at 10 µM
(39). Bur6 was used at 13 nM and Ydr1/Ncb2 at 60 nM (38); both proteins were a gift of G. Prelich.
Purification of Mot1 and Pull-down Assays--
Mot1 was
expressed and purified from yeast using antibody-coupled beads exactly
as described previously (20). The antibody-coupled beads were prepared
using Py monoclonal antibody that recognizes the Mot1 epitope tag (20),
which was prepared at the University of Virginia Lymphocyte Culture
Center. For detection of TBP binding to immobilized Mot1, Mot1-coupled
beads were equilibrated with buffer T-60 (30 mM Tris (pH
8.0), 5 mM magnesium chloride, 0.1% Brij-58, 1 mM dithiothreitol, protease inhibitors plus 60 mM potassium chloride). One hundred nanograms of
full-length recombinant yeast TBP (or TBP mutant) was added in 500 µl
of buffer T-60, and the reaction was incubated for 30 min at room
temperature. After binding, the unbound material was collected and the
beads were washed with buffer T containing increasing concentrations of
KCl; samples marked "Eluate" were collected in T-1000. The eluted
proteins were precipitated with acetone, and TBP present in the eluates was detected by Western blotting using rabbit polyclonal anti-TBP antisera.
Preparation of GST-TBP and Mot1 Binding to
GST-TBP--
One-liter cultures of DH10B bacterial cells containing
plasmid pGEX-1 (Amersham Biosciences) or a plasmid expressing GST fused to full-length yeast TBP (kindly provided by Ron Reeder) were grown in
YT medium at 37 °C to an optical density at 600 nm of 0.7-1.0.
Isopropyl- Sequence Analysis--
Blocks of conserved sequences in the Mot1
N terminus were identified with a set of Mot1 homologs found in Entrez
protein sequence data bank (available at
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein) (from Homo
sapiens (accession number AAC04573), Arabidopsis thaliana (T47857), Saccharomyces cerevisiae (P32333),
Schizosaccharomyces pombe (T40642), and Drosophila
melanogaster (AAF55260)) using the MACAW (40) and ClustalW
algorithms (available at www.ibc.wustl.edu/service/msa/index.html). HEAT repeats in Mot1 reported by previous authors (23, 24) included
five in the B block. We observed sequence similarity of the second B
block HEAT repeat to sequences immediately downstream using MACAW. This
had not been found in the original sequence analysis, and Fig.
1B therefore includes an additional HEAT repeat in the B
block, for a total of six.
Four Conserved Regions in Mot1 N Terminus--
Alignment of Mot1
homologs revealed conserved blocks outside of the ATPase, which we
designate A-D (Fig. 1A).
These blocks were not found in any protein except Mot1 or its homologs.
The A and B blocks in the human and yeast proteins are about 40%
identical. For example, Fig. 1B shows the sequence of the
S. cerevisiae Mot1 A block; the asterisks
indicate residues that are identical in the yeast and human proteins.
Mot1 contains a series of HEAT repeat sequences dispersed throughout
the N terminus (23, 24). Remarkably, the four conserved N-terminal
domains of Mot1 coincide with the positions of the HEAT repeats (Fig.
1, A and B; the brackets in Fig.
1B indicate where two HEAT repeats fall within the A
block).
Temperature-sensitive Alleles of mot1--
PCR-based mutagenesis
was used to introduce random changes in the MOT1 open
reading frame in the region between codons for Arg-345 and
Asn-697. This region was chosen because previous deletion analysis
indicated an important role for residues in this region in TBP
recognition (19, 20). A CEN ARS plasmid library expressing mutagenized mot1 under GAL1 control was used,
because genes that express catalytically defective mot1 were expected
to be dominant inhibitors of cell growth (20). Library construction and
screening are described under "Materials and Methods." Note that
35 °C was chosen for the non-permissive temperature, because the
wild-type MOT1+ strain used in these studies is itself
somewhat growth-impaired at temperatures above 35 °C (not shown).
The alleles isolated are recessive, and most of these contain multiple
base pair changes (Table I). A single
amino acid change, L383P, is responsible for the temperature-sensitive
(ts) growth phenotype of a strain harboring mot1-41, because
the same mutation in mot1-42 conferred the same phenotype.
However, more than one amino acid change is required for the ts
phenotypes conferred by mot1-71 and mot1-81, because no single amino acid change encoded by these alleles resulted in the conditional phenotype; several pairwise combinations of mutations in conserved residues also failed to confer a ts phenotype (not shown). The mot1-14 phenotype likely results from a low
level of translational by-pass of the premature stop codon substituted for Trp-496, because deletion of the mot1 open reading frame
downstream of this stop codon is lethal (not shown).
Growth phenotypes of strains carrying the GAL1-driven
alleles are summarized in Table I. Comparison of strain growth by
serial dilution spot assay demonstrated that, compared with wild-type cells, the mot1 strains displayed growth defects of
~100-1000-fold when incubated at 35 °C (not shown). Growth
phenotypes of these mot1 strains were similar regardless of
whether the alleles were expressed under control of the GAL1
or MOT1 promoters. Western blot analysis of whole cell
extracts from cells grown at 30 °C, using an antibody that
recognizes epitope-tagged versions of these proteins, demonstrated that
proteins encoded by mot1-41, mot1-71, and
mot1-81 were expressed at wild-type levels, whereas
full-length protein encoded by mot1-14 was nearly
undetectable (Fig. 2A). Mot1
protein level in the mot1-42 strain is intermediate (Fig. 2A, lane 8).
Wild-type and mutant Mot1 proteins were purified from yeast
overexpression strains using antibody-coupled beads (20). The purified
proteins were then tested in gel mobility shift assays for the ability
to bind TBP·DNA complexes and for ATP-dependent TBP·DNA
disruption activity (20). As shown in Fig. 2B (lanes 1-7), addition of wild-type Mot1 led to formation of
Mot1·TBP·DNA ternary complexes that were disrupted in the presence
of ATP. The Mot1-41, Mot1-42, Mot1-71, and Mot1-81 proteins did not
stably bind to TBP·DNA complexes. These proteins also failed to
disrupt TBP·DNA complexes in the presence of ATP even when
severalfold more protein was used than was required for wild-type Mot1
to quantitatively super-shift and disrupt the TBP·DNA complexes
formed under these conditions (Fig. 2B, lanes
8-13, and Fig. 2C). Thus, the defects in cell growth
resulting from Mot1-41, Mot1-42, Mot1-71, or Mot1-81 can be explained
by general defects of these proteins in TBP·DNA recognition.
Alanine Scanning Mutations in the MOT1 A Block--
To map the
surface of Mot1 required for TBP binding, extensive mutagenesis of the
N-terminal conserved regions was undertaken. While this work was in
progress, it was reported that the A and B blocks contain HEAT (or ARM)
repeats (22-24). These repeats stack via hydrophobic interactions to
form an extended, helical structure (22); thus, alanine scanning of
polar residues should not affect the overall fold but could inhibit
polar interactions. Mot1 missing the entire A block (Mot1-260; deletion
of amino acids 1-98) does not support cell viability (Table
II) even though this N-terminally truncated protein is expressed at wild-type levels (not shown). The A
block is thus essential for Mot1 function in vivo. The
Mot1-260 protein is also defective for formation of Mot1·TBP·DNA
ternary complexes and ATP-dependent disruption of TBP·DNA
in vitro (Fig. 3A,
lanes 3 and 4). Similarly, deletion of the
"linker" connecting A and B blocks generated a non-functional
protein in vivo and in vitro (mot1-274; Fig.
3A and Table II) suggesting that important residues are
located within the linker or that the A and B blocks must be
appropriately positioned for Mot1 to function.
Site-directed A block mutant alleles were constructed on low copy
plasmids under control of the MOT1 or GAL1
promoter and introduced into a yeast strain containing a deletion of
the chromosomal copy of MOT1. All of the conserved charged
and polar residues within the A block were mutated to alanine either
singly or in clusters, and, remarkably, none of these residues were
found to be essential for Mot1 function in vivo, even though
many of these residues are conserved across species (see Fig.
1B, data are summarized in Table II). Strains carrying each
of the MOT1-driven A block alleles were also screened for
growth defects at 16 °C, 32 °C, and 35 °C but no ts or
cold-sensitive phenotypes were observed (not shown). These strains also
display no growth defects on synthetic or rich media with glucose,
galactose, or raffinose as the carbon source (not shown).
In contrast to the normal growth observed with the A block alanine
mutations when expressed under control of the MOT1 promoter, several A block mutant alleles displayed a severe dominant-negative phenotype when expressed from the GAL1 promoter.
GAL1-driven MOT1 is expressed at a 20-50-fold
higher level than MOT1 under control of the MOT1
promoter (Fig. 3B, lane 1 versus
2). As summarized in Table II, GAL1-expressed
alleles of MOT1-encoding mutations at the extreme N terminus
severely inhibited cell growth when cells were grown on galactose in
the presence of wild-type Mot1. The most severe defect was seen in
MOT1-101 cells (Table II). MOT1-101 and
wild-type MOT1 were expressed at equivalent levels under
GAL1 control (Fig.
4B), and severe growth defects
were observed on plates containing galactose or galactose plus
raffinose (not shown), indicating that these alleles do not simply
confer an inability of cells to metabolize galactose. In the absence of the wild-type MOT1 gene, cells expressing
MOT1-101 were inviable on galactose-containing media, cells
expressing mot1-204, mot1-205, or
mot1-206 grew more slowly than wild-type cells at 30 °C,
and mot1-204 and mot1-205 conferred ts growth at
35 °C (not shown). The lethality induced by GAL1-driven
MOT1-101 is due to elevated expression levels of this
protein, because cells grew well with MOT1-101 as the sole
source of Mot1 when the allele was expressed under control of the
normal MOT1 promoter (Table II).
Alleles of MOT1 that encode proteins that recognize TBP but
are defective in ATP-dependent TBP·DNA disruption exert
dominant-negative effects on cell growth (41). This is due to
interference with TBP function, because these dominant-negative
phenotypes can be suppressed by overexpression of SPT15,
which encodes TBP (41). To determine if the dominant-negative A block
mutants interfere with TBP function in vivo, high copy
plasmids expressing SPT15 were introduced into strains
expressing the dominant-negative A block allele MOT1-101.
As shown in Fig. 3C, the lethality induced by
GAL1-driven MOT1-101 can be suppressed by
SPT15 overexpression. Side-by-side comparisons (not shown)
demonstrate that overexpression of SPT15 does not fully
restore growth of these strains to wild-type rates, but these results
suggest that the lethality induced by these A block mutations can be
explained, at least in part, by interference with normal TBP function
in vivo. SPT15 overexpression was unable to
suppress the growth defect in the GAL1-MOT1-101 cells in
which MOT1-101 was the only source of Mot1 (not shown). This suggests that elevated levels of Spt15 suppress
MOT1-101 by interacting with the encoded mutant protein and
thereby allowing wild-type Mot1 to function. As shown in Fig.
3D, Mot1-101 does recognize and dissociate TBP·DNA
complexes in vitro, but the affinity of Mot1-101 for
TBP·DNA complexes is reduced at least 16-fold compared with wild-type
Mot1. Possible molecular explanations for the growth defects of
MOT1-101 cells are discussed below.
Alanine Scanning Mutations in the MOT1 B Block--
Mutations were
also engineered in conserved clusters of charged or polar amino acids
in the Mot1 B block (residues 289-583, Fig. 1A). The B
block overlaps significantly with the region of the open reading frame
mutagenized for the ts screen described above. Mutant alleles were
expressed in yeast cells on low copy plasmids under control of the
MOT1 promoter or the GAL1 promoter as for the A
block mutants, and the results are summarized in Table II. Mutation of
Glu-308, Arg-310, and His-311 resulted in a recessive loss-of-function
allele (mot1-102; Table II). mot1-102 is
expressed at wild-type levels (Fig. 4B), and this protein
does not detectably recognize or disrupt TBP·DNA complexes (Fig.
4A). Mutation of conserved residues Asp-361 or Asp-365 also
results in complete loss of Mot1 function in vivo (Table
II). The Mot1-104 protein, which contains both of these amino acid
changes is expressed at wild-type levels (Fig. 4B), and,
like Mot1-102, does not detectably bind or disrupt TBP·DNA complexes
in vitro (Fig. 4A). Two alleles with wild-type
in vivo function that encode changes in highly conserved
residues were analyzed biochemically and found to recognize TBP·DNA
complexes and support ATP-dependent TBP·DNA disruption equivalently to wild-type Mot1 (Mot1-103 in Fig. 4A,
Mot1-216 in 4C). Thus, the ability of the MOT1
alleles to support growth and the abilities of the encoded proteins to
support TBP·DNA disruption are correlated.
TBP Residues That Participate in Mot1 Binary and Ternary Complex
Formation--
The Mot1·TBP binary complex binds DNA poorly (13).
This suggests either that Mot1 interacts with the DNA-binding surface of TBP or that Mot1 induces a conformational change in TBP that affects
the ability of TBP to bind to DNA. These results also suggest that Mot1
contacts TBP differently depending on whether TBP is bound to DNA. The
N terminus of a human Mot1 homolog, BTAF1, also binds to TBP and can
inhibit TBP binding to DNA (21). To better define how Mot1 recognizes
TBP, TBP mutants were tested in vitro for the ability to
interact with Mot1 in the absence of DNA. TBP mutants that retain DNA
binding activity were also tested to determine if Mot1 could catalyze
disruption of their interaction with DNA. Mot1 was loaded onto
antibody-coupled beads as previously described (20), the beads were
incubated with full-length TBP and washed, and the TBP association with
the beads was assayed by Western blotting using TBP antibodies. TBP was retained on beads loaded with Mot1, whereas TBP binding to beads alone
was nearly undetectable (Fig.
5A, lanes 5 and
9 versus lane 3). Control experiments
established that the Mot1·TBP binary interaction was insensitive to
ethidium bromide and DNase I, indicating that the association was not
mediated by contaminating DNA (Fig. 5A, lanes 5 versus 7 and 9 versus
11). Because Mot1 used in these experiments was obtained
from a yeast overexpression system, we also established that there was
no contaminating TBP in the affinity-purified Mot1 preparation, and the
TBP retained by the Mot1 beads therefore resulted from the interaction
of Mot1 with the recombinant TBP added to the reactions (Fig.
5A, lanes 12-15).
Two mutants with solvent-exposed amino acid changes in the same
Three TBPs with mutations on the DNA-binding surface were also tested
for interaction with Mot1. As shown in Fig. 5B, TBP N159D
retained the ability to interact with Mot1, whereas TBP V71E and TBP
V161E do not interact detectably with Mot1 in this assay. The Mot1 N
terminus binds weakly to TBP·DNA (20), and the Mot1 N terminus is
also sufficient for formation of the Mot1·TBP binary complex (Fig.
5C). As was observed with the full-length Mot1 protein,
there was no detectable binding of the Mot1 N terminus to TBPs with
mutations in critical residues on the convex (K145L) or concave, DNA
binding (V71E) surface of TBP. These results are consistent with and
extend previously published results (21) and suggest that the inability
of Mot1·TBP complexes to bind to DNA (13) is due to a direct
interaction between Mot1 and the TBP binding surface. Remarkably, this
would imply that the Mot1 N terminus embraces TBP via an extensive
surface, making specific contacts with TBP simultaneously on opposite
sides of the molecule.
Conformational Change of Mot1·TBP Binary Complex Induced by
ATP--
The Mot1·TBP binary complex does not detectably bind DNA
in vitro, but it does hydrolyze ATP (20). Addition of ATP to
pre-formed Mot1·TBP complexes allows TBP·DNA complexes to assemble
on a DNA template that is too short to support Mot1-catalyzed
disruption (13). One interpretation of these results is that Mot1
dissociates from TBP in the presence of ATP. Other experimental
approaches have led to the conclusion that ATP does not induce the
Mot1·TBP complex to dissociate (15), suggesting that disruption of
Mot1·TBP binary complexes requires both ATP and DNA. To test this
idea, three different experimental approaches were compared directly. In the first experiment, ATP and a 17-bp TATA DNA sequence (too short
to support Mot1 binding, see "Materials and Methods" and Ref. 13)
were added to a reaction containing the pre-formed Mot1·TBP binary
complex. As shown in Fig. 6A
(lanes 7 and 8), ATP induced the dissociation of
Mot1·TBP binary complexes and formation of TBP·DNA complexes when a
short DNA template was added to the reaction. Using a standard 36-bp
DNA template that does support Mot1 action (Fig. 6A,
lanes 1-6), Mot1 can load onto pre-formed TBP·DNA
complexes and disrupt them using ATP, but, as expected, no TBP·DNA
complexes were detected when the DNA and ATP were added to Mot1·TBP
complexes, because any TBP loaded onto this DNA template was
dissociated by Mot1. Consistent with these and previously published
results (15), Mot1 bound to GST-TBP beads was not released in the
presence of ATP alone (Fig. 6B). In contrast, however, ATP
and DNA catalyzed release of less than half the Mot1 from GST-TBP beads
(Fig. 6B, lane 5). The simplest interpretation of
these results is that ATP and DNA can induce dissociation of the
Mot1·TBP binary complex, but that tethering TBP to agarose beads
impairs the catalytic activity of Mot1. Similar results were obtained
in a reciprocal experiment using Mot1 bound to agarose beads and
TBP in solution (not shown).
To better define the effect of ATP on the Mot1·TBP binary complex, a
non-denaturing gel electrophoresis assay was used, but TBP and Mot1
were monitored by Western blotting rather than using radiolabeled DNA
as in a conventional gel shift experiment. Under these conditions, free
TBP was positively charged and entered a gel run toward the negative
electrode (37). As shown in Fig. 6C (lanes 1-6),
addition of Mot1 diminished the amount of free TBP that entered the
gel. Mot1 was also incubated with TBP K138L, a TBP mutant that is not
recognized by Mot1 (see Fig. 5). As shown in Fig. 6C
(lanes 9-14), the amount of free TBP K138L was not diminished by addition of Mot1 so the decrease in the amount of TBP
detected when Mot1 was added is not trivially due to degradation of
TBP. Although TBP is slightly positively charged, Mot1 is predicted to
have a slight negative charge under these conditions and the bulky
Mot1·TBP binary complex is apparently nearly uncharged and did not
enter gels run toward either the positive or the negative electrode.
The negatively charged TBP·DNA complex could be detected, however
(Fig. 6D, lane 2). As expected, the
Mot1·TBP·DNA ternary complex was not formed on the 17-bp DNA used
(Fig. 6D, lane 3). Importantly, addition of ATP
to Mot1·TBP binary complexes did not result in release of free TBP
(Fig. 6C, lane 6 versus 5). However, addition of ATP and DNA to pre-formed Mot1·TBP complexes resulted in the appearance of the TBP·DNA complex (Fig.
6D, lane 6), consistent with the results in Fig.
6A. We conclude that while the Mot1·TBP binary complex
hydrolyzes ATP, addition of ATP alone does not induce the complex to
fall apart. However, the Mot1·TBP binary complex can be dissociated
in a reaction that contains both ATP and DNA. Mot1·TBP complexes
bound to agarose beads do not support Mot1 catalytic activity.
A Transition State ATP Analog Facilitates Loading of Mot1·TBP
Complexes onto DNA--
Because ATP does not induce Mot1·TBP binary
complex dissociation but does facilitate TBP binding to DNA, we
considered the possibility that locking the Mot1 ATPase into a
conformational state somewhere along the catalytic path could generate
a Mot1·TBP binary complex with enhanced DNA binding activity. This
was tested using ADP aluminum fluoride (ADP-AlF4), which
binds to ATP-binding sites and mimics the presumed transition state of
ATP during hydrolysis (39). ADP-AlF4 does not cause
disruption of the Mot1·TBP·DNA ternary complex (Fig.
7A, lane 5 versus 3), although ADP-AlF4 appears
to bind to the Mot1 ATP-binding site, because preincubation of Mot1
with ADP-AlF4 prevents Mot1 from utilizing ATP added
subsequently (Fig. 7A, lanes 11-14). Disruption
of TBP·DNA complexes by Mot1 therefore requires ATP hydrolysis or
perhaps multiple rounds of ATP hydrolysis. Consistent with the results
in Fig. 6A, little Mot1·TBP·DNA ternary complex was
detected when Mot1 and TBP were preincubated prior to addition of DNA
(Fig. 7A, lanes 6 versus 3). However, addition of ADP-AlF4 to pre-formed
Mot1·TBP binary complexes allowed the binary complexes to load onto
DNA (Fig. 7A, lanes 8 versus
3 and 6). Interestingly, addition of
ADP-AlF4 does not cause the Mot1·TBP binary complex to
dissociate (Fig. 7B), suggesting that the
Mot1·TBP·ADP-AlF4 complex does not require interactions
between Mot1 and the DNA-binding surface of TBP for stability or that
Mot1 modulates the DNA binding activity of TBP exclusively by directing
conformational changes in TBP. Thus, ADP-AlF4 can convert
the conformation of the Mot1·TBP binary complex into a form capable
of binding DNA. Because ATP hydrolysis does not cause the binary
complex to dissociate, these results support the hypothesis that one
stage of the Mot1 ATP hydrolysis cycle opens or activates the binary
complex to DNA binding.
Interaction of Mot1 with NC2·TBP·DNA--
Because mutations on
the DNA-binding surface of TBP impair interaction with Mot1, and ATP or
ADP-AlF4 can cause the TBP DNA-binding surface in the
binary complex to become accessible to DNA, one simple model is that a
portion of the Mot1 N terminus contacts the TBP DNA-binding surface and
this interaction is transiently disrupted during the ATP hydrolysis
cycle. NC2 specifically recognizes the "underside" of the TBP·DNA
complex (42), so NC2 might block loading of or catalysis by Mot1 by
preventing Mot1 from interacting with the concave surface of TBP. There
is also a striking overlap of the gene sets whose transcription is
controlled by NC2 and Mot1, suggesting a mechanistic interplay between
them at specific promoters (11, 43-45). To address the biochemical
interplay of Mot1 and NC2 in vitro, gel mobility shift
experiments were performed. The gel mobility shifts of the
Mot1·TBP·DNA and NC2·TBP·DNA complexes were readily
distinguished with the NC2·TBP·DNA complex migrating just slightly
more slowly than the TBP·DNA complex (Fig.
8A). Addition of both Mot1 and
NC2 resulted in the appearance of a new species (Fig. 8A,
lane 6). The same results were observed in both TBE gels
(Fig. 8A), which favor the NC2·TBP·DNA shift, and in TG
gels (Fig. 8B, compare band marked by the bracket
with that marked by the asterisk), which stabilize the
TBP·DNA shift but destabilize the NC2·TBP·DNA shift. These
results show that Mot1 and NC2 do not compete for TBP binding; rather,
they cooperate to form Mot1·NC2·TBP·DNA quaternary complexes.
In addition, ATP caused disruption of the quaternary complex (Fig.
8A, lane 6 versus 7; Fig.
8B, lane 6 versus 7),
indicating that NC2 also provides no barrier to Mot1-catalyzed
TBP·DNA disruption.
Mot1 is an essential, conserved yeast protein (7) that interacts
genetically and biochemically with TBP (14, 41). Mot1 catalysis of
TBP·DNA disruption can explain its role as a repressor of
transcription (43), but how Mot1 activates transcription and the
mechanism of the TBP·DNA disruption reaction are unknown. The results
in this paper provide mechanistic insight into how the Mot1 ATPase is
used to drive TBP·DNA disruption in vitro, and in
vivo analysis of Mot1 mutants demonstrates a close correlation between the ability of Mot1 to catalyze TBP·DNA disruption and the
ability to provide the essential function of Mot1 in vivo. These results also explain previous data demonstrating inhibition of
TBP DNA binding by Mot1. Furthermore, the role of ATP in opening the
Mot1·TBP binary complex to DNA binding suggests a model, discussed below, for how Mot1 catalyzes TBP·DNA disruption.
Leucine Repeats in the Mot1 N Terminus--
The leucine repeats of
the Mot1 N-terminal domain have been identified as either HEAT (24) or
ARM (22) repeats. In either case, the leucine repeats of Mot1 coincide
with the blocks of conserved sequence in the N terminus (Fig. 1). There
is no structural information about them, but these results suggest that
the Mot1 N terminus probably adopts an extended conformation, similar
to importin
A direct test of the ARM/HEAT repeat model for Mot1 is not possible
because of the limited structural information on Mot1 and the large
size of the Mot1 N terminus. Hydrophobic residues are also predicted to
play important roles in both stabilization of interactions between
leucine repeats and interaction with TBP, but Mot1 proteins with
mutations in hydrophobic residues would also be expected to be
defective, because mutation of residues in the hydrophobic core of the
protein could lead to instability of the native structure. A deeper
understanding of the structural basis of the Mot1 defects reported here
awaits future structural analysis.
Function of the Mot1 A Block--
The A block is required for the
Mot1·TBP interaction (Fig. 3A), yet most of the conserved
polar residues of the A block can be changed to alanines without
affecting cell viability (Table II). Only the mutation of a few of the
first ten amino acids of the protein had any effect. In particular,
mutation of Arg-7, Asp-9, and Arg-10 caused dominant inhibition of cell
growth when the mutant gene was expressed from the GAL1
promoter (MOT1-101), and GAL1-controlled alleles
encoding mutations in Arg-7 or Asp-9 conferred temperature-sensitive
growth. Overexpression of SPT15 rescued cells from
overexpression of MOT1-101 (Fig. 3C), supporting the idea that the dominant negativity is due to an altered interaction with TBP. Mot1-101 protein is defective for binding TBP·DNA but has
no obvious catalytic defect (Fig. 3D). One possibility is that the Mot1-101·TBP binary complex may be unusually stable: recycling of TBP after Mot1 action may be required in vivo.
Formation of Mot1-101·TBP binary complexes could not be assessed
in vitro using the pull-down assay, because purified
Mot1-101 was found to interact nonspecifically with agarose beads (not
shown), perhaps suggesting that the N terminus of Mot1-101 is not
stably folded. Alternatively, the polar residues at the Mot1 N terminus
may be important for an interaction with another protein that modulates the catalytic activity of Mot1 in vivo.
Two Putative Mot1-binding Sites on TBP--
Previous results (19,
38) and those in Fig. 5 demonstrate that the interaction of Mot1 with
TBP requires lysine residues in TBP helix 2, on the convex surface of
TBP opposite the DNA-binding site (Fig.
9A). These residues are
required for both binary and ternary complex formation. A second
putative Mot1-binding site is located on the concave DNA-binding
surface of TBP and is defined by valine 71 and valine 161 (Fig.
9A). Mutation of these residues disrupts interaction with
both DNA and Mot1, but these altered TBP molecules are unlikely to be
simply misfolded, because they are expressed in soluble form at normal
levels (not shown) and these same TBPs can stimulate transcription
in vivo (51). A direct interaction between the
DNA-binding surfaces of Mot1 and TBP can explain why the Mot1·TBP
binary complex does not bind DNA. Interestingly, although Mot1 does not
form a binary complex with TBP K127L, Mot1·TBP K127L·DNA ternary
complexes were detectable, and these ternary complexes were
disrupted in the presence of ATP (Fig. 5, B and
E). Similarly, a TBP with altered specificity for DNA
binding supports Mot1·TBP·DNA ternary complex formation and
ATP-dependent disruption (30) but is defective for binary interaction with BTAF1, a human Mot1 homolog (21). We suggest that Mot1
contacts the convex surface of TBP in both binary and ternary complexes
and that Mot1 interaction with TBP alone requires a direct interaction
between Mot1 and the TBP DNA-binding surface. In contrast, Mot1 does
not directly interact with the TBP DNA-binding surface in
Mot1·TBP·DNA ternary complex, but instead contacts the DNA upstream
of the TATA box (13). Recent data (52) demonstrate that human TBP
Lys-138 can affect DNA binding despite being located on the opposite
side of the TBP DNA-binding surface. Therefore, an alternative
possibility is that the effects of TBP DNA-binding surface mutations on
Mot1 interaction result from reciprocal changes in TBP conformation
rather than a direct interaction with Mot1. This possibility remains to
be tested.
ATP Switches Mot1·TBP Binary Complex Affinity for DNA--
The
inability of Mot1·TBP binary complexes to bind DNA can be overcome by
addition of ATP and the use of a DNA probe that is too short to allow
Mot1 binding (13). We interpreted this result to indicate that the
binary complex dissociates in the presence of ATP. On the other hand,
an immobilized binary complex does not dissociate in the presence of
ATP (19), suggesting that both DNA and ATP are required for
dissociation. Here we report that ATP hydrolysis by the binary complex
does not cause the binary complex to dissociate but, rather, that ATP
induces a change in Mot1·TBP conformation that allows TBP to bind to
DNA and Mot1 to be released. Experiments with the non-hydrolyzable ATP
analog ADP-AlF4 provided additional support for this model.
Although binding of ADP-AlF4 by Mot1 is not sufficient to
drive Mot1·TBP·DNA ternary complex dissociation,
ADP-AlF4 did allow the binary complex to load onto DNA
(Fig. 7A).
Mechanism of Disruption--
Several mechanisms have been proposed
for protein·DNA disruption by the Snf2/Swi2-related ATPases,
including Mot1 (13, 28, 53, 54). In contrast to mechanisms employed by
at least some chromatin remodeling enzymes, Mot1 does not use ATP
hydrolysis to propagate DNA bending, twisting, or strand separation
through the TATA box (13). Mot1 also does not use ATP hydrolysis to track processively along DNA (30). Mot1-mediated changes in TBP
conformation have been proposed to explain how Mot1 regulates the
interaction between TBP and DNA (15). In support of the TBP
conformational change model, human TBP·K138 modulates DNA binding
affinity (52), a result suggesting that amino acids distal to the
DNA-binding surface can affect DNA binding by directing a change in TBP
conformation. Because this residue is critical for the interaction of
yeast TBP and Mot1 (Fig. 5), it is possible that Mot1 interaction with
the convex surface of TBP causes a change in the conformation of the
TBP DNA-binding surface that is modulated by ATP.
The effects of TBP DNA-binding surface mutations on Mot1 interaction
are most simply explained, however, by proposing a direct interaction
between Mot1 and the DNA-binding surface of TBP. Combining this with
previous observations, we propose the mechanism shown in Fig.
9B. The catalytic cycle then involves an ATP-driven
insertion of the Mot1 N terminus into the TBP·DNA interface. This
"power stroke" results in disruption of TBP·DNA contacts and the
formation of new interactions between Mot1 and the DNA-binding surface
of TBP. Once separated from DNA, the binary complex can hydrolyze ATP
in a process that involves dramatic conformational changes in which the
Mot1 N terminus alternates position in and out of contact with the
DNA-binding surface of TBP. The conformation in which the DNA-binding
surface of TBP is "open" can be trapped with ADP-AlF4,
a state in which TATA-containing DNA can bind to TBP. Note that in the
model (Fig. 9B), the conformation of TBP is different in the
Mot1·TBP binary complex than when TBP is free, reflecting the
possibility that Mot1 may induce a conformational change in TBP as part
of its catalytic mechanism.
Mechanistic Insights Provided by Mot1-NC2
Interaction--
Chromatin immunoprecipitation experiments established
that both Mot1 and the NC2 subunit Bur6 are localized to the promoters that they regulate (43, 55). Microarray experiments have shown that
Mot1 and Bur6 regulate many of the same genes (43, 45). The results in
Fig. 8 demonstrate that Mot1 and NC2 can occupy the same promoter at
the same time and show that NC2 does not impede the catalytic activity
of Mot1. Thus, if such catalytic activity is altered at some promoters,
as has been suggested to explain how Mot1 can activate the expression
of some genes (43), this putative change in biochemical activity must
depend on promoter-associated factors other than the NC2 complex. In
addition, because Mot1 does not interfere with access of NC2 to
TBP·DNA, Mot1 is unlikely to contact the underside of the TBP·DNA
complex and the catalytic action of Mot1 is unlikely to require contact
with the major groove of the TATA box.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
helices joined by a short loop (ARM repeats have a short additional
helix),
and these can stack upon each other to form a "superhelix" that
provides an extensive surface for macromolecular interaction (22).
Previous analysis of Mot1 deletion mutants indicated that an extended
portion of the Mot1 N terminus is responsible for recognition of TBP
(19, 20). It has also been reported that, in solution, Mot1 is a
non-globular monomer (15). Taken together, these data suggest a model
in which Mot1 adopts an extended conformation that provides a large
surface for interaction with TBP. To test the model, mutations were
made in both Mot1 and TBP, and the effects on the Mot1·TBP
interaction were determined. Because HEAT and ARM repeats are based
mostly on hydrophobic interactions (22), it was expected that most
polar residues in the N-terminal domain would not be essential, which
we have found to be the case.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
::TRP1, carrying plasmid
pMR13 (MOT1+ URA3+)) (20), which is otherwise congenic to
YPH499 (33) by selection on synthetic complete media containing glucose
but without leucine using standard techniques (34). Approximately
13,000 transformants were replica-plated to synthetic glucose- or
galactose-containing media lacking leucine and incubated at 30 °C
for 3-5 days. Comparison of the glucose and galactose-containing
plates did not reveal any GAL1-inducible alleles of
MOT1, which caused slow growth in the presence of wild-type MOT1. Colonies were then replica-plated from
galactose-containing media to media containing galactose and
5-fluoroorotic acid (34) to select for loss of the
URA3-marked plasmid containing the wild-type MOT1
gene. Approximately half of the transformants did not survive the
5-fluoroorotic acid selection, indicating that these strains harbored
alleles of MOT1, which do not support growth in the absence of wild-type MOT1. The remaining viable strains were
screened for temperature-sensitive growth defects by replica plating to synthetic galactose plates minus leucine and incubation at 30 °C and
35 °C. Temperature-sensitive strains were re-streaked, and the
plasmids were isolated and re-transformed to the MOT1 deletion strain to confirm the plasmid-linked temperature-sensitive (ts) phenotype. Candidate genes were then sequenced through the entire EcoRI-ClaI region of the ORF, and
the mutant fragments were subcloned to a new plasmid backbone
containing the remainder of the MOT1 gene to be sure that
mutations in the EcoRI-ClaI DNA fragment were
responsible for the phenotypes observed.
-D-thiogalactopyranoside was added (0.5 mM final concentration), and the cells were incubated at
37 °C for 3 h to allow protein expression. Cells were harvested
and resuspended in buffer I (40 mM sodium phosphate, pH
8.0, 300 mM NaCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 2 µM benzamidine, 2 µM pepstatin, 0.6 µM leupeptin, and 2 µg/ml chymostatin) containing 5 mM imidazole and lysed by sonication. After sonication, the cleared lysate was incubated at
4 °C for 1 h with 0.2 ml of nickel-nitrilotriacetic
acid-agarose (Qiagen) pre-equilibrated with buffer I, which included 5 mM imidazole. The mixture was then loaded into a column,
and the resin was washed with 10 ml buffer I plus 5 mM
imidazole and subsequently with 5 ml of buffer I containing 20 mM imidazole. Finally, the bound protein was eluted with
buffer I containing 200 mM imidazole. The yield was
quantitated by Bradford assay (Bio-Rad), and the purity was assessed by
Coomassie Blue staining of 10% protein gels. Based on the Coomassie
Blue staining, the proteins were estimated to be about 90% pure.
-D-thiogalactopyranoside was added (1.0 mM, final concentration), and the cells were incubated at
37 °C for an additional 3 h. Cells were harvested by
centrifugation and resuspended in 20 ml of buffer T (30 mM
Tris-HCl, pH 8.0, 2 µM pepstatin A, 1 mM
phenylmethylsulfonyl fluoride) containing 150 mM KCl (T-150
buffer). Cells were lysed by sonication, and debris was removed by
centrifugation. After centrifugation, 0.5 ml of GST lysate or 1.5 ml of
GST-TBP lysate was incubated at 4 °C for 1 h with 20 µl of
glutathione-agarose equilibrated with buffer T containing 60 mM KCl (T-60). The agarose was washed three times with 1 ml
of T-150 buffer, and the entire 20-µl sample of agarose-bound material was used for testing the binding of Mot1. 10 ng of the eluted
Mot1 protein obtained from yeast overexpression strains (see above) was
added to a 20-µl suspension of GST or GST-TBP agarose in T-60 buffer
and incubated on a roller for 1 h at 4 °C. The agarose was
washed once with 0.6 ml of T-60 buffer, then an elution step was
carried out with 0.6 ml of T-60 buffer with 5 mM
MgCl2, with or without 50 µM ATP and with or
without 1 nM TATA sequence DNA. Eluted proteins were
precipitated with acetone for analysis by Western blotting using the Py
antibody (35), which recognizes the N-terminal epitope tag.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conserved sequence motifs in Mot1.
A, the top schematic shows the position of
conserved blocks in the Mot1 amino acid sequence identified by
comparing Mot1 homologs using the programs ClustalW and MACAW (see
"Materials and Methods"). Conserved Mot1 blocks are designated
A, B, C, and D, and the
conserved Snf2/Swi2-like ATPase is shaded black.
Numbers indicate boundaries of the conserved regions in
S. cerevisiae Mot1. Note that these programs identified
three conserved blocks of sequence connected by short linker sequences
in the region spanning residues 289-583, and the entire region is
referred to as the B block. The bottom schematic
shows the positions of HEAT repeats in gray (23, 24).
B, sequence of the Mot1 A block. Brackets
indicate the positions of the two hydrophobic HEAT repeats. Residues
mutated in this study appear in boldface, and
asterisks mark residues identical in yeast Mot1 and human
BTAF1. Yeast Mot1 and human BTAF1 are 40% identical over the A
block.
Growth of strains harboring temperature-sensitive alleles of mot1 under
GAL1 control
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Fig. 2.
Analysis of temperature-sensitive
mot1 alleles. A, Western blot (Py
monoclonal antibody) analysis of Mot1 protein levels present in whole
cell extracts prepared from strains carrying the indicated
mot1 allele. "wt" refers to wild-type Mot1.
In extract from the strain labeled "vector," Mot1 is
present but is untagged. In lanes 1-6, the indicated
mot1 alleles were expressed from the GAL1
promoter. Similarly, in lanes 7 and 8, the level
of wild-type Mot1 was compared with the level of Mot1-42. B,
TBP and radiolabeled TATA-containing DNA were combined and wild-type or
mutant Mot1 proteins were added subsequently in the presence or absence
of ATP. The positions of the free DNA, TBP·DNA complex, and
Mot1·TBP·DNA ternary complex are indicated by the
arrows. DNA (0.5 nM) and TBP (5 nM)
were present in all of the reactions. Lanes 2-7 contained
wild-type Mot1 as follows: reactions in lanes 2 and
3 contained 0.75 nM Mot1, lanes 4 and
5 contain 2.25 nM Mot1 and lanes 6 and 7 contain 3.75 nM Mot1 (estimated by
activity, see "Materials and Methods"). Lanes 8-13
contained 2-3 units of the mot1 mutant proteins (based on
concentration as determined by Western blotting). C, gel
shift assay comparing activity of wild-type Mot1 to Mot1-42. Wild-type
Mot1 was used at 10 nM in lanes 3 and
4, and Mot1-42 at 100 nM in lanes 5 and 6.
Growth of strains harboring site-directed mutations in mot1
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Fig. 3.
Mutational analysis of the Mot1 A block.
A, gel mobility shift analysis of purified wild-type Mot1
(lanes 1 and 2) versus mot1
derivatives missing the entire A block (residues 1-98; lanes
3 and 4) or missing residues between the A and B blocks
(98-274; lanes 5 and 6). Core domain TBP was
used at 5 nM, Mot1 and derivatives at 10 nM,
and the DNA concentration was 0.5 nM. The abundance of the
TBP·DNA complex was unaffected by addition of 3-10-fold more of
either mot1 derivative (not shown). ATP (5 µM) was added
where indicated. B, the levels of Mot1 protein expressed
from the MOT1 promoter versus the GAL1
promoter were compared by Western blotting. The levels of Mot1 were
20-50-fold higher in cells with MOT1 under GAL1
control than when the MOT1 gene was under MOT1
promoter control. C, suppression of the
GAL1-MOT1-101 dominant-negative growth defect by
overexpression of SPT15, the gene encoding TBP. Yeast strain
YPH499 (33) was transformed with a CEN ARS plasmid
containing GAL1-MOT1-101 and a 2-µm vector carrying
SPT15 or the 2-µm vector without SPT15
("vector"). Four independent transformants from each
transformation were re-streaked to glucose-containing plates
(left panel) or galactose-containing plates (right
panel). On the glucose-containing plate, the
GAL1-driven MOT1-101 gene is not
expressed, and all strains grew equivalently; on galactose,
GAL1-MOT1-101 expression inhibited cell growth (right
panel), but this defect is suppressed by overexpression of
SPT15 (right panel). 2-µm SPT15
alone does not affect cell growth on glucose- or galactose-containing
media (Ref. 41 and data not shown). D, gel mobility shift
analysis as in Fig. 2 (B and C) using wild-type
Mot1 (lanes 1-4) or Mot1-101 (lanes 5-14) plus
or minus ATP as indicated. The relative amounts of Mot1 or Mot1-101
added are indicated. 1 unit of Mot1 (~5 nM, see
"Materials and Methods") shifts a fraction of the TBP·DNA complex
to the Mot1·TBP·DNA ternary complex (lane 1) and nearly
completely disrupts TBP·DNA in the presence of ATP (lane
3). In contrast, 64-fold more Mot1-101 is required to obtain
ternary complex formation similar to 1 unit of wild-type Mot1
(lane 11); ~32-fold more Mot1-101 is required to disrupt
TBP·DNA complexes to the extent seen with 1 unit of wild-type Mot1
(lane 13).
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Fig. 4.
Mutational analysis of the Mot1 B block.
A, gel mobility shift analysis was performed using purified
wild-type Mot1, mot1-102, mot1-103, or mot1-104 as indicated, with
TBP and radiolabeled DNA (as in Fig. 2B). Ten micromolar ATP
was added as indicated. Mot1 (or mutants) was present at about 5 nM. Titrations of Mot1 proteins demonstrated that Mot1-103
functions as well as wild-type Mot1 in this assay, whereas Mot1-102 and
Mot1-104 have no detectable TBP·DNA binding or disruption activity.
Reactions in lanes 3-6 contained about 2-fold more Mot1
protein (judged by Western blotting) than the amount of wild-type Mot1
protein required for full activity in this assay. B, Western
analysis (Py monoclonal antibody) of whole cell extracts prepared from
cells containing the indicated GAL1-driven alleles of
MOT1. Cultures of cells were grown to mid-log in
raffinose-containing medium then induced with the addition of galactose
to 2% for 2 h prior to harvest. "Vector" refers to
extract from cells harboring plasmid with no epitope-tagged
MOT1 gene. C, gel mobility shift analysis as in
A using purified Mot1-216, which displays ternary complex
formation and TBP·DNA disruption activity equivalent to wild-type
Mot1.
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Fig. 5.
TBP DNA-binding surface is critical for Mot1
binding to TBP. A, TBP interaction with Mot1 bound to
agarose beads. Agarose beads with or without Mot1 (as indicated) were
incubated with recombinant full-length TBP. The unbound flow-through
(FT) and bead-bound materials (Eluate) were
analyzed by Western blotting using a rabbit polyclonal antibody
directed against TBP. DNase I or ethidium bromide (EtBr)
were included in the wash buffers as indicated. In the reaction
analyzed in lanes 14 and 15, no recombinant TBP
was added; the absence of detectable TBP signal indicates that TBP did
not contaminate the Mot1 preparation used for these studies.
B, interaction of recombinant TBP or TBP mutants with
agarose beads or Mot1 beads. The analysis was performed as in
A using wild-type TBP or mutants as indicated. Note that
wild-type TBP and TBP N159D are the only proteins that bound detectably
to Mot1 beads (lane 5). C, binding of wild-type
TBP or TBP mutants to beads alone (vector) or Mot1
N-terminal fragments bound to beads. Mot1-1280 is a Mot1 fragment with
residues 1-1280, and Mot1-800 has the first 800 residues of Mot1 (see
Fig. 1A). Analysis was performed as in A and
B of this figure. D, gel mobility shift analysis
was performed using radiolabeled DNA, Mot1, and the indicated TBPs as
in Fig. 2C. Lane 1 shows position of free DNA.
Lanes 2-8 each contain 2.5 nM purified
recombinant full-length wild-type TBP. Lanes 9-15 each
contain 2.5 nM purified recombinant TBP K138L. Relative
amounts of purified Mot1 were added as indicated, where 1 unit
(lane 3) is ~5 nM. Note that TBP K138L·DNA
complexes are unaffected by Mot1. E, gel mobility shift
analysis as in D but using TBP K127L where indicated.
Lane 1 shows position of free DNA. Reactions in lanes
2-8 each contained 2.5 nM purified recombinant
full-length wild-type TBP, and reactions in lanes 9-15 each
contained 25 nM purified recombinant TBP K127L (a longer
exposure is shown than in panel D). Relative amounts of
purified Mot1 were added as in D. TBP K127L is defective for
DNA binding, and the TBP K127L·DNA complex was barely detectable
under these conditions. However, Mot1 stabilized TBP K127L binding to
DNA (note ternary complex in lanes 10-12), and the complex
dissociated in the presence of ATP (lanes 13-15).
helix (helix 2) on the "top" convex surface of TBP were tested for
interaction with Mot1 in this assay. As shown in Fig. 5B,
TBP K138L and K145L were both defective for interaction with Mot1. TBP
Lys-145 was previously shown to be critical for Mot1 recognition of
TBP·DNA complexes (38), and TBP K133L,K138L was shown to be
defective for Mot1-catalyzed disruption (12). TBP K138L·DNA complexes
are not stably bound Mot1 (Fig. 5D), demonstrating that
mutation of either Lys-138 or Lys-145 alone is sufficient to block
recognition by Mot1. Thus, these residues on the convex surface of TBP
are required for interaction with Mot1 in both the presence and absence
of DNA. TBP Lys-127 is located at the extreme N terminus of helix 2 near the upstream edge of the TBP DNA-binding surface (see Fig.
9A). Whereas TBP K127L is defective for interaction with
Mot1 in the absence of DNA (Fig. 5B), Mot1 can stabilize the
interaction of TBP K127L with DNA to some extent, and the Mot1·TBP
K127L·DNA ternary complex dissociates in the presence of ATP (Fig.
5E). This residue may define a difference in the
architecture of the Mot1·TBP and Mot1·TBP·DNA complexes, or
alternatively, this ternary complex may fall apart during ATP hydrolysis simply because TBP K127L binds DNA poorly.
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Fig. 6.
DNA and ATP together facilitate Mot1·TBP
dissociation. A, gel mobility shift assay using
radiolabeled TATA-containing DNAs of different lengths. 5 nM TBP, 0.5 nM DNA, 5 µM ATP, and
40 nM Mot1 were added where indicated. Positions of free
DNA, TBP·DNA, and Mot1·TBP·DNA ternary complex are shown. TBP
core domain was incubated with either Mot1 or DNA for 30 min then
either loaded on the gel or incubated with DNA or Mot1 with or without
ATP for 30 min before loading. The order in which components were
incubated for each reaction is shown above the lanes.
Reactions were loaded at 2-min intervals, accounting for the gradual
upward trend of the free DNA and TBP·DNA bands as the reactions were
loaded from left to right. Mobility of the
TBP·DNA complex is affected by DNA length (not shown). B,
bead-bound Mot1·TBP complexes were incubated with ATP and/or DNA (as
indicated), and the beads were washed to remove unbound material. The
beads were then boiled in SDS sample buffer, and the bead-bound
material was analyzed for Mot1 by Western blotting using the Py
monoclonal antibody (see "Materials and Methods"). Reaction in
lane 1 was performed with GST beads, whereas reactions in
lanes 2-5 were performed using GST-TBP. Note that no
detectable Mot1 bound to GST-Sepharose beads (lane 1),
whereas ATP and DNA caused less than half of the bound Mot1 to be
dissociated from GST-TBP beads (lane 2 versus
lane 5). C, TBP alone (lane 1) or
full-length TBP plus Mot1 (lanes 2-7) were incubated in the
absence (lanes 1-6) or presence (lane 7) of ATP.
An identical series of reactions were run in parallel using TBP K138L
rather than wild-type TBP (lanes 9-14). The reactions were
loaded at the top onto non-denaturing polyacrylamide gels and
electrophoresed with electrodes connected as shown. Following
electrophoresis, Western analysis was performed to detect TBP or TBP
K138L. The band represents monomeric TBP (37). The reaction
in lane 1 contained no Mot1, 2.5 nM Mot1 was
used in the reaction in lane 2, 10 nM Mot1 in
lane 3, 30 nM Mot1 in lane 4, and 80 nM Mot1 in lanes 5-6. Lane 10 contains 5 nM Mot1, and the Mot1 concentration doubles in
each of the next three lanes to 40 nM in lanes
13-14. D, the experiment was performed as in
C except that the gel was run with the electrodes reversed,
to visualize negatively charged species. The position of the TBP·DNA
complex is shown. The asterisk indicates a TBP-containing
species (likely an aggregated form of TBP) that is present in the TBP
preparation but does not affect the interpretation of the
results.
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Fig. 7.
ATP transition state analog facilitates
binding of Mot1·TBP complex to DNA. A, gel mobility
shift assay using radiolabeled 0.5 nM TATA DNA and 5 nM TBP. Approximately 40 nM Mot1, 5 µM ATP, and ADP-AlF4 (see "Materials and
Methods") were added where indicated. Orders of addition of DNA, TBP,
Mot1, ATP, and ADP-AlF4 are indicated. In the first
incubation ("Added 1st"), components were incubated for
30 min. Additional components were then added as indicated
("Added 2nd"), and the reactions were incubated for 30 min and loaded onto the gel. ATP was added 10 min after
AlCl3 in lane 13. The position of the
Mot1·TBP·DNA ternary complex is marked "3o."
Phosphorimaging analysis showed that the ternary complex band in
lane 6 obtained after preincubation of Mot1 and TBP is only
10% the intensity of the ternary complex band in lane 3.
The ternary complex bands in lanes 5 and 8 are
both 50% the intensity of the ternary complex band in lane
3, indicating 50% loss of the ternary complex in the presence of
ADP-AlF4, but that ADP-AlF4 completely restores
the ability of pre-formed Mot1·TBP complexes to bind to DNA.
B, Mot1 alone (lane 1), TBP alone (lanes
2, 3, and 7) or TBP plus Mot1 (lanes
4-6) were incubated in the absence (lanes 1,
4, and 7) or presence of ATP (lanes 3 and 6) or ADP-AlF4 (lanes 2 and
5). Proteins were incubated with ATP or ADP-AlF4
for a total of 30 min. In reactions with both Mot1 and TBP, Mot1 was
incubated with ATP or ADP-AlF4 for 20 min, followed by
addition of TBP for 10 min. The reactions were loaded onto
non-denaturing polyacrylamide gels and electrophoresed as in Fig.
6C. Following electrophoresis, Western analysis was
performed to detect TBP. The position of TBP is shown. Mot1 and TBP
were used at 50 nM, ATP was 100 µM, and
ADP-AlF4 was used as described under "Materials and
Methods."
View larger version (36K):
[in a new window]
Fig. 8.
Mot1 binds and disrupts the TBP·NC2·DNA
complex. A, gel mobility shift analysis using a Tris
borate-EDTA (TBE) native gel, 0.5 nM
radiolabeled TATA DNA, and 5 nM TBP. The positions of the
various complexes are indicated by the arrows. Mot1 (5 nM) and NC2 (composed of Bur6, 13 nM, and
Ydr1/Ncb2, 60 nM) were added where indicated. B,
gel mobility shift analysis using a Tris-glycine (TG) native
gel was performed using radiolabeled TATA DNA, TBP, Mot1, and NC2 as in
A. The bracket in lane 3 marks the
Mot1·TBP·DNA shift, which was not discrete in this experiment. The
asterisk indicates the distinct Mot1·NC2·TBP·DNA
shift.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(46, 47) or
catenin (48). The Mot1 N terminus is
sufficient for TBP binding (Fig. 5C) and is necessary for
activation of the ATPase (20). Karyopherin 114, an importin
family
member, binds TBP (49, 50), so there may be other TBP-hydrophobic repeat interactions.
View larger version (34K):
[in a new window]
Fig. 9.
Model for Mot1 catalytic cycle and the role
of ATP. A, S. cerevisiae TBP·DNA
structure (56). Residues required for Mot1·TBP binary complex
formation are shown in red. Residues required for
Mot1·TBP·DNA ternary complex formation are shown in
orange. DNA is black. B, Mot1 binds
TBP·DNA reversibly via interaction with the convex surface of TBP and
upstream DNA. The data support a model in which the Mot1·TBP complex
is stabilized by interactions between Mot1 and both the convex and
concave surfaces of TBP, and the binary complex does not readily
dissociate or bind DNA. An alternative possibility that is consistent
with the data is that Mot1 binding induces a conformational change in
TBP (shown as a distorted TBP in the Mot1·TBP binary complex), and
the altered conformation of TBP binds DNA poorly. At one step in the
ATP hydrolysis cycle, mimicked by binding of ADP-AlF4, the
Mot1·TBP complex has an altered conformation in which the DNA-binding
surface of TBP is either transiently accessible to DNA or TBP assumes
its high affinity DNA binding conformation. ATP hydrolysis induces
dissociation of the Mot1·TBP binary complex from DNA by weakening
interaction of TBP with DNA, possibly through formation of contacts
between Mot1 and the TBP DNA-binding surface.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Kai Post for construction of the MOT1 mutant plasmid library, Ron Reeder for the GST-TBP expression plasmid, Bob Roeder, Frank Pugh, and Tetsuro Kokubo for yeast TBP expression plasmids, and Greg Prelich for TBP K145E and the purified NC2 components. We are also grateful to Tatsuya Hirano for communicating results prior to publication, to Tony Weil for advice on the non-denaturing gel-electrophoretic analysis of protein·protein complexes, and to Dan Engel, Patrick Grant, Tsuyoshi Miyake, and members of the Auble laboratory for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM55763 (to D. T. A.).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.
§ Present address: Division of Biology, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Virginia Health System, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0733. Tel.: 434-243-2629; Fax: 434-924-5069; E-mail: dta4n@virginia.edu.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M211445200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TBP, TATA-binding protein; ORF, open reading frame; ts, temperature-sensitive; GST, glutathione S-transferase; ARM, armadillo.
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