Purification and Enzymic Properties of Mot1 ATPase, a Regulator
of Basal Transcription in the Yeast Saccharomyces
cerevisiae*
Joanne I.
Adamkewicz,
Christopher G. F.
Mueller
,
Karin E.
Hansen,
Wendy A.
Prud'homme§, and
Jeremy
Thorner¶
From the Department of Molecular and Cell Biology, Division of
Biochemistry and Molecular Biology, University of California,
Berkeley, California 94720-3202
Received for publication, March 28, 2000, and in revised form, April 26, 2000
 |
ABSTRACT |
The 1867-residue Mot1 protein is a
member of a superfamily of ATPases, some of which are helicases, that
interact with protein-nucleic acid assemblies. Mot1 is an essential
regulator of RNA polymerase II-dependent transcription
in vivo and dissociates TATA box-binding protein (TBP)-DNA
complexes in vitro. Mot1-(His)6 was purified to
apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP·TATA complexes, suggesting that no other protein or
cofactor is required. Mot1 behaved as a non-globular monomer in
hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was
stimulated about 10-fold by high ionic strength or alkaline pH, or by
deletion of the N-terminal TBP-binding segment, suggesting that the
N-terminal domain negatively regulates the C-terminal ATPase domain
(Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase
(but not Mot1C) was stimulated 
10-fold by yeast TBP, suggesting that
interaction with TBP relieves a conformational constraint in Mot1.
Double- or single-stranded TATA-containing DNA did not affect ATPase
activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit
detectable helicase activity in strand displacement assays using
substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed
dissociation of TBP from DNA was not prevented by a psoralen cross-link
positioned immediately preceding the TATA sequence. Thus, Mot1 most
likely promotes release of TBP from TATA-containing DNA by causing a
structural change in TBP itself, rather than by strand unwinding.
| |
INTRODUCTION |
Transcription by all three eukaryotic RNA polymerases requires
TBP.1 For initiation by yeast
Pol II, TBP binding to a TATA sequence present in most promoters
nucleates stepwise assembly of a large complex containing
TBP-associated factors (1), termed TFIID, as well as multiple general
transcription factors and Pol II (reviewed in Refs. 2-5). In an
alternative, but not mutually exclusive, model of initiation, TFIID
binding creates a platform for recruitment of a massive holoenzyme,
consisting of Pol II, the SRB proteins, and other cofactors (like
Gal11), as well as the general transcription factors, TFIIB, TFIIF, and
TFIIH (reviewed in Refs. 6-8). In both models, promoter binding by TBP
is the rate-limiting step in initiation complex formation and thus
provides a physiologically relevant target for transcriptional control
by both positive (9, 10) and negative (11-14) regulatory factors.
Factors that negatively regulate eukaryotic transcription utilize a
wide variety of mechanisms, including quenching of activators, promoting assembly of chromatin, recruiting chromatin-binding proteins
or histone-modifying enzymes, and interfering with the general
transcription machinery itself. In addition, several proteins are
thought to block Pol II transcription via interaction with TBP. Such
regulators include Even-skipped (eve gene product), a
Drosophila homeodomain protein (15, 16), and a general
repressor, NC2 (or Dr1-DRAP1) (17-19). NC2 binds to TBP·DNA
complexes and prevents the association of TFIIA and TFIIB, possibly by
altering conformation of the DNA (20-24).
Another kind of negative regulator of TBP action was first identified
both genetically and biochemically in the yeast Saccharomyces cerevisiae. A TBP-targeted repressing activity, termed ADI for "ATP-dependent inhibitor" of TATA binding, was detected
in yeast nuclear extracts (25). ADI removed TBP from the TATA element of the adenovirus major late promoter (AdMLP) in an
ATP-dependent manner. TFIIA, and to some extent TFIIB,
inhibited ADI activity by competing for binding to TBP or by
stabilizing the TBP·DNA complex, or both (25). It was subsequently
found that ADI corresponded to the product of the MOT1 gene
(26). The MOT1 locus had been previously identified by
temperature-sensitive mutations that increased basal expression of many
Pol II-specific genes (27, 28).
The MOT1 gene was isolated by complementation of the
temperature-sensitive lethality of the mot1-1 allele and
encodes an 1867-residue protein (calculated at 210 kDa), which is
essential for yeast cell viability (27). The C-terminal third of the
polypeptide (residues 1270-1747) bears strong sequence similarity to a
domain conserved in a large superfamily of ATPases (reviewed in Ref. 29) and is an efficient ATPase in vitro (26). Mutants
defective in ATP hydrolysis in vitro are non-functional
in vivo (26). This domain is characterized by seven
signature motifs, including a nucleotide-binding element. Some of the
members of this superfamily are demonstrated DNA or RNA helicases
(reviewed in Refs. 30-32). Mot1 is the founding member of a distinct
class of these proteins (27), now generally referred to as the
"Snf2 family" after its most well known member, S. cerevisiae Snf2/Swi2 (reviewed in Refs. 33 and 34). The
proteins in this family, which includes representatives from bacteria,
yeast, Drosophila, and man, are significantly more closely
related to each other than to any other member of this ATPase
superfamily. Thus, Mot1 is a novel type of TBP inhibitor, one that
requires the energy of ATP hydrolysis for its action.
Because of the effects of Mot1 and Snf2 on transcription and
because more distantly related members of the ATPase superfamily are
well characterized helicases (35-37), it was originally proposed that
Mot1 (27), Snf2 (38), and other members of that family (31)
might be DNA-dependent helicases. To date, however, no member of the Snf2 family has been shown to possess helicase
activity. To assess the properties of Mot1 and to begin to explore the
mechanism by which Mot1 removes TBP bound to a TATA sequence, we
undertook the purification of native Mot1 from yeast cells and
investigated its solution structure and catalytic properties in
vitro. Here we describe this purification and analysis of Mot1 and
the cumulative evidence indicating that Mot1-dependent ATP
hydrolysis does not act to release TBP from DNA by a strand-unwinding mechanism.
| |
EXPERIMENTAL PROCEDURES |
Materials--
All reagent chemicals were purchased from Sigma
or other appropriate commercial suppliers, as specified. Restriction
enzymes were purchased from New England BioLabs, Roche Molecular
Biochemicals, or other commercial sources.
Plasmid Construction--
To facilitate purification of Mot1, a
sequence encoding a (His)6 tract was fused to the 3'-end of
the MOT1 open reading frame using a simple method for
precise gene fusion based on the polymerase chain reaction (PCR) (39).
For this purpose, amplification was carried out under standard
conditions using Taq polymerase, 10 fmol of
NotI-linearized pRSMOT1 (27) as template, and primers CM7
(5'-AAA TTT CCA CCG CGG TGG ATT AAT GAT GAT GAT GAT GAT GAC CTC GTA AAG
TTT TGA TGA-3') and CM2 (5'-CTA GAC ATG GTT GAA AAT GA-3'). The
resulting 670-bp PCR product was digested with BstXI and
inserted into the BstXI sites of plasmid pKH6 (26), yielding pCM112, a URA3-marked 2 µm DNA-containing plasmid that
carries MOT1-(His)6 under control of
the galactose-inducible GAL1 promoter. The construction of
MOT1-(His)6 was verified by direct
nucleotide sequence analysis of the PCR-derived DNA. The deduced
C-terminal sequence of Mot1-(His)6 is
-KTLR1867GHHHHHH-COOH (authentic Mot1 residues
are in boldface).
Large Scale Yeast Cell Growth--
S. cerevisiae
strain SC295 (MATa ura3-52 leu2-3, 112 reg1-501
gal1 pep4-3) (40), which is both vacuolar protease-deficient and
unable to impose glucose-mediated repression, was transformed with
pCM112 using a lithium acetate procedure (41), and transformants were
selected on synthetic-complete medium containing 2% glucose (dextrose)
and lacking uracil (SCD-Ura) (42). The plasmid-bearing cells were grown
to saturation in 5 ml of liquid SCD-Ura at 30 °C and used to
inoculate a larger culture (250 ml) of SCD-Ura + 25 mg/ml kanamycin,
which was also grown to saturation and then used as the inoculum for an
110-liter culture of SCD-Ura + 25 mg/ml kanamycin in a 200-liter
fermenter (New Brunswick Scientific). Cells were grown in the fermenter
at 30 °C with vigorous aeration and stirring to an
A600 nm = 0.6, whereupon solid galactose was
added to a final concentration of ~2%, and the culture was incubated
for additional 6 h. Cells were then harvested using a compressed
air-driven super-centrifuge (Sharples), washed by resuspension in 500 ml of Lysis Buffer (50 mM HEPES, pH 7.8, 0.5 M
NaCl, 1 mM DTT, 0.1 mM EDTA), and recollected
by centrifugation for 10 min at 5000 × g. The washed
cells were resuspended in Lysis Buffer (2 ml/g wet weight of cell
paste), frozen dropwise by dripping into liquid N2, and
stored at 
70 °C until used.
Purification of Mot1-(His)6 from S. cerevisiae--
For a typical preparation, 270 g of frozen cell
beads were thawed in a water bath at room temperature and then
immediately placed on ice. All subsequent operations were performed at
4 °C. A battery of protease inhibitors were added to the cell
suspension (final concentration indicated) as follows: EDTA (1 mM), phenylmethylsulfonyl fluoride (PMSF) (1 mM), benzamidine (1 mM), aprotinin (5 µg/ml), leupeptin (5 µg/ml), and pepstatin A (5 µg/ml). The cell suspension was lysed by cavitation with glass beads (0.45 mm) using 10 30-s pulses, separated by 1-min intermittent periods of cooling, in a
stainless steel Bead-Beater (Biospec Products) surrounded by an
ice-water jacket. The lysate was decanted, and the beads were rinsed
with a small volume (100 ml) of Lysis Buffer containing 1 mM PMSF and 1 mM EDTA (Lysis Buffer + PMSF + EDTA). The lysate and rinse were pooled and clarified by centrifugation
at 19,600 × g for 20 min. The supernatant fraction was
decanted into fresh tubes and clarified again in the same manner. The
clarified material (crude extract) was pooled, and the protein
concentration was determined by the dye-binding method of Bradford (43)
using a commercial kit (Bio-Rad) and bovine serum albumin (BSA) as the standard. The crude extract was diluted to 15 mg/ml using Lysis Buffer + PMSF + EDTA and slowly adjusted to a final ammonium sulfate concentration at 25% of saturation by addition of the finely ground solid salt with stirring on ice over the course of 15 min. After equilibration with stirring for an additional 30 min, the precipitated material was removed by centrifugation at 16,300 × g
for 15 min. The supernatant solution was decanted to a fresh container,
additional ammonium sulfate added in a similar manner to a final
concentration at 50% of saturation, and the precipitated material
again collected by centrifugation. The 25-50% precipitate was
redissolved in 1 liter of Binding Buffer (50 mM HEPES, pH
7.8, 0.5 M NaCl) containing 5 mM imidazole and
protease inhibitors, as above, and loaded at a flow rate of 1 ml/min
onto a column containing a bed (10 ml) of
Ni2+-iminodiacetate-agarose (His-BindTM, Novagen) that had
been pre-equilibrated with Binding Buffer containing 5 mM
imidazole. The flow-through was reloaded at the same flow rate and then
collected. The column was washed and eluted with Binding Buffer plus
increasing concentrations of imidazole in a stepwise fashion, as
follows: four 20-ml washes of 5 mM imidazole, five 20-ml
fractions of 20 mM imidazole, two 20-ml fractions of 40 mM imidazole, and three 10-ml fractions of 500 mM imidazole. The majority of the Mot1-(His)6
eluted in the 40 and 500 mM imidazole fractions. The 40 and
500 mM eluates were pooled separately, placed in dialysis
tubing (SpectraPor), and dialyzed exhaustively against 20 mM HEPES, pH 7.8, containing 0.5 M NaCl, and
then against Buffer A (20 mM HEPES, pH 7.8, 0.5 M NaCl, 1 mM DTT, 1 mM EDTA). The
dialysates were pooled and concentrated by ultrafiltration under
N2 gas in a stirred pressure cell (Amicon) fitted with a
YM30 Diaflo membrane (Amicon) to a protein concentration of 0.64 mg/ml.
The protein was quantitatively precipitated by addition of ground
ammonium sulfate to a final concentration at 85% of saturation and
collected by centrifugation for 15 min at 17,300 × g.
The pellet was redissolved in 2.5 ml of Buffer A, and the insoluble
particulate material was removed by centrifugation for 2 min at 14,000 rpm in a microcentrifuge (Eppendorf). The redissolved material was
loaded at a flow rate of 0.5 ml/min using an FPLC apparatus (Waters)
onto a column (Hi-Prep 16/60) containing a bed of a size-exclusion
resin (Sephacryl S-300HR; Amersham Pharmacia Biotech) that had been
pre-equilibrated with Buffer A. The column was eluted with Buffer A,
and fractions (2 ml) were collected. The elution position of
Mot1-(His)6 was detected using a flow-cell, UV monitor, and
chart recorder and confirmed by SDS-PAGE followed by silver staining
and by immunoblotting using specific rabbit polyclonal anti-Mot1
antibodies (26). Subsequent experiments were performed with the peak
fractions eluted from the size-exclusion column, unless otherwise indicated.
Estimation of Native Molecular Mass by Gel Filtration
Chromatography--
Globular protein standards (Amersham Pharmacia
Biotech) were purchased as lyophilized powders and resuspended
individually, or as a mixture, at a concentration of 1 mg/ml each in
either Buffer A or Buffer A containing 10 mM
MgCl2 and 1 mM ATP. The standards were loaded
in a volume of 2 ml at a flow rate of 0.5 ml/min using an FPLC
apparatus (Waters) onto a column (Hi-Prep 16/60) containing a bed of a
size-exclusion resin (Sephacryl S-300-HR; Amersham Pharmacia Biotech)
that had been pre-equilibrated with the same buffers. The column was
eluted with the same buffers, and 2-ml fractions were collected.
Elution position of each protein standard was monitored both by an
in-line UV detector and by spectrophotometric analysis of corresponding
fractions. Elution volumes of the standards were used to construct a
selectivity curve (Kav versus log
Mr). After calibration with the standards in
each of the two different buffers, purified Mot1-(His)6
(Ni2+-iminodiacetate-agarose eluate) was loaded onto the
calibrated column in the same volume at the same flow rate and eluted.
The fractions obtained were analyzed by SDS-PAGE with silver staining and by immunoblotting to determine the elution volume of
Mot1-(His)6.
Estimation of Native Molecular Mass by Rate Zonal
Sedimentation--
Solutions of 20, 15, 10, and 5% sucrose (w/v) in
Buffer B (20 mM HEPES, pH 7.8, 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA) were layered sequentially
into upright 13 × 51-mm Ultra-Clear centrifuge tubes (Beckman),
then equilibrated at 4 °C for 4 h in a horizontal position, and
returned to an upright position, thereby generating a linear 5-20%
sucrose gradient. Fractions from representative tubes were collected
and analyzed using a refractometer to confirm the linearity of the
gradients (data not shown). Lyophilized globular protein standards were
resuspended in Buffer B at 1 mg/ml and mixed with purified Mot1
(Sephacryl S-300-HR eluate), either individually, in pairs, or all
together. Samples (150 µl) of the protein solution were layered on
top of four separate gradients and then subjected to centrifugation in
an SW-55Ti rotor at 50,000 rpm (250,000 × g at
ravg) for 6 h at 4 °C in an
ultracentrifuge (Beckman L8-80M), which was allowed to decelerate
without braking. Fractions (100 µl) were collected from each tube
using an automated apparatus (model 640 Density Gradient Fractionator,
ISCO), and the positions of the proteins were determined by UV
spectrometry (for hemoglobin, 
max = 555 nm), by protein
assay (for catalase and aldolase), and SDS-PAGE and immunoblot analysis
(for Mot1-(His)6).
Co-expression of Differentially Tagged Functional Variants of
Mot1--
Yeast strain BJ2168 (MATa ura3-52
leu2 trp1 prb1-1122 pep4-3 prc1-407) (44) was transformed with 2 µm DNA-containing plasmids expressing MOT1-myc (pKH7) (45)
and either MOT1-(His)6 (pCM112) or untagged
MOT1 (pKH6) (45). Cells were grown in an appropriate
selective medium and induced for 2 h by the addition of galactose
(2% final concentration). Cells were harvested by centrifugation,
washed, and resuspended in Binding Buffer. Extracts were prepared
immediately thereafter by vigorous vortex mixing with glass beads and,
after clarification, were loaded in parallel onto
Ni2+-iminodiacetate-agarose columns. The columns were
washed with the same buffer, then eluted using the same buffer
containing increasing concentrations of imidazole (20, 40, 60, and 500 mM), and then stripped with Binding Buffer + 100 mM EDTA. Proteins present in the eluted fractions were
analyzed by SDS-PAGE and immunoblotting with an appropriate antibody.
Purification of Bacterially Expressed TBP and Bacterially
Expressed Mot1C--
The construction of a plasmid that expresses the
catalytically active C-terminal ATPase domain of Mot1 (Mot1C) in
Escherichia coli and purification of this fragment of the
enzyme from this source have been described previously (26). Likewise,
full-length yeast TBP (SPT15 gene product) was expressed in
E. coli and purified by a procedure described in detail
elsewhere (46).
Assay of ATPase Activity--
Samples of Mot1-(His)6
(765 ng) or Mot1C (700 ng) in Buffer A containing 10% glycerol, or an
equal volume of the same buffer, were incubated, in duplicate, in a
total volume of 50 µl containing 4 mM MgCl2,
0.1 mg/ml BSA, 0.25 mM [
-32P]ATP (20 µCi/mmol), and 50 mM HEPES (and/or other buffering
species adjusted to the pH value indicated in the experiments shown)
with or without added salt at the concentrations indicated in the
experiments shown. Reactions were initiated by the addition of enzyme,
incubated for 30 min at 30 °C, and terminated by addition of an
equal volume of ice-cold 100 mM EDTA. Products were
analyzed by thin layer chromatography and scintillation counting as
described before (26). When present, yeast TBP purified from E. coli was added in the amounts indicated; likewise, when present,
"TATA DNA" was added as a 28-base oligonucleotide containing the
sequence of the TATA element of the AdMLP (5'-CCT GAA GGG GGG CTA TAA
AAG GGG GTG G-3'), which was generated by chemical synthesis, and used
either in single-stranded (ssDNA) or double-stranded (dsDNA) form, the
latter of which was prepared by annealing to a perfectly complementary oligonucleotide.
Assays of Helicase Activity--
The universal template for each
substrate was a circular single-stranded phage DNA containing the
complement of a TATA box. To prepare this molecule, a 101-bp
EcoRI-BamHI fragment from plasmid pRW2 (47) that
contains the AdMLP TATA sequence was inserted into the corresponding
sites of the replicative form of M13mp18, yielding plasmid pKH50.
Progeny single-stranded phage DNA was isolated from E. coli
cells infected with pKH50 using precipitation with polyethylene glycol
according to standard procedures (48). A helicase substrate with a
flush-ended double-helical region was prepared by annealing the
circular phage DNA with the perfectly complementary 28-base TATA DNA
oligonucleotide. Helicase substrates with 5'- and 3'-single-stranded
tails were prepared by annealing the circular phage DNA with
oligonucleotides "5'-TATA" and "3'-TATA", which were otherwise
identical to the TATA DNA oligonucleotide, except that each contained
an additional 10-base, non-complementary, overhanging sequence at its
5'- or 3'-end, respectively. The oligonucleotides were labeled using
two different strategies. The TATA DNA and 5'-TATA oligonucleotides
were labeled at their 3'-ends, as follows. The single-stranded template
(1 pmol) was mixed with a 10-fold molar excess of the oligonucleotide,
heated to 85 °C, and cooled slowly to room temperature on the
benchtop. To the annealed substrate, 20 µCi of
[-32P]dGTP (3 Ci/µmol; NEN Life Science Products)
and 10 units of the Klenow fragment of E. coli DNA
polymerase I (New England BioLabs) were added in a buffer (60 µl
total volume) containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, and 6.67 mM DTT. The mixture was incubated at room temperature for
30 min, and then reaction was terminated by extraction with an equal
volume of phenol:chloroform:isoamyl alcohol (25:24:1). The
"3'-TATA" oligonucleotide was labeled at its 5'-end using
[-32P]ATP (3 Ci/µmol; NEN) and T4 polynucleotide
kinase (New England Bio-Labs), as recommended by the manufacturer, and
the reaction was terminated by extraction with
phenol:chloroform:isoamyl alcohol (25:24:1). Thereafter, a 10-fold
molar excess of the labeled 3'-TATA oligonucleotide was mixed with 1 pmol of the single-stranded template, heated to 85 °C, and cooled
slowly to room temperature. All three annealed substrates were
separated from unincorporated label by gel filtration over a bed (1 ml)
of Sephadex G-25 (Amersham Pharmacia Biotech) equilibrated in TE Buffer
(10 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing
100 mM NaCl, and then further purified before use by
centrifugation through a Microspin S-400 column (Amersham Pharmacia Biotech).
Helicase reactions (20 µl total volume) contained 0.2 mM
substrate (in nucleotides), 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mg/ml BSA, 1 mM DTT, 5 mM MgCl2, and 1 mM ATP. Reactions
were initiated by the addition of enzyme, either
Mot1-(His)6 (765 ng), Mot1C (700 ng), or, as a positive
control, purified SV40 T antigen (500 ng; gift of E. Fouts and M. Botchan, University of California, Berkeley) in the presence or absence
of purified yeast TBP (1 µg). Reactions were incubated at 30 °C
for 1 h and terminated by addition of 2 µl of 10× Stop Buffer
(40% glycerol, 50 mM EDTA, 1% SDS, 0.1% bromphenol
blue). To separate displaced oligonucleotide from unreacted substrate,
samples were resolved by electrophoresis through a 5% non-denaturing
polyacrylamide gel (48). Gels were fixed in 10% methanol, 10% acetic
acid, dried under vacuum, and analyzed by autoradiography using x-ray
film (Kodak Biomax-MR).
SDS-PAGE and Immunodetection of Mot1--
Protein samples were
diluted in SDS-PAGE loading buffer, heated to 100 °C for 1 min, and
resolved by discontinuous SDS-PAGE on 6.5% polyacrylamide slab gels
(49). Proteins were visualized by staining with either Coomassie
Brilliant Blue dye or silver nitrate (50). For immunodetection,
proteins were transferred electrophoretically to a nitrocellulose
membrane (BAS3, pore size 0.2 µm; Schleicher & Schuell) using a
Trans-Blot-SDTM apparatus (Bio-Rad), as directed by the manufacturer.
The membrane was incubated at room temperature for 1 h in Blocking
Buffer (5% non-fat dry milk, 500 mM NaCl, 25 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40) and then incubated
with an appropriate primary antibody in Blocking Buffer for 1 h
(or, occasionally, overnight). For detection of Mot1, a 1:10,000
dilution of a rabbit polyclonal anti-Mot1 antiserum that was raised
against the C-terminal 612 residues of Mot1 (45) was used. For
detection of c-Myc-tagged Mot1, a 1:5,000 dilution of ascites fluid
containing anti-c-Myc monoclonal antibody 9E10 was used. After thorough
washing and incubation with an appropriate horseradish
peroxidase-labeled secondary antibody (Santa Cruz Biotechnology or
Babco), immune complexes were visualized using a commercial
chemiluminescence detection system (RenaissanceTM, NEN Life Science
Products), as recommended by the manufacturer.
Gel Mobility Shift Assay--
A double-stranded DNA probe
spanning the AdMLP was prepared by digesting pRW2 (47) with
EcoRI (at position 50) and XbaI (at position
+33). The resulting 83-bp fragment was end-labeled by filling in the
overhangs using the Klenow fragment of E. coli DNA
polymerase I in the presence of [-32P]dATP (NEN Life
Science Products). The radiolabeled DNA probe was separated from
unincorporated radioactivity by gel filtration over a bed (l ml) of
Sephadex G-25 (Amersham Pharmacia Biotech) equilibrated in TE Buffer
containing 100 mM NaCl. The amount of labeled probe was
quantitated by both scintillation counting and a dot-blot assay using
ethidium bromide staining and a standard curve prepared with samples of
DNA of known concentration (51). The concentration of the probe was
~0.5 µg/ml, which corresponded to ~7.5 fmol of probe/µl with a
specific activity of ~1.6 × 104 cpm/fmol. Proteins
were mixed with l µl of probe-containing solution in DNA-Binding
Buffer (40 mM Tris-HCl, pH 8.0, 60 mM KCl, 5 mM MgCl2, 4% glycerol, 0.1% Brij 58, 1 mM DTT, 100 ng of poly(dI-dG) (Roche Molecular
Biochemicals) and 100 µg/ml BSA) in a total volume of 20 µl. After
a 30-min incubation at room temperature, either 0.25 mM ATP
(pH 8.0) or an equivalent volume of pH 8.0 water was added, and
incubation was continued for an additional 5 min at room temperature.
The reaction mixtures were immediately loaded onto a native 6%
polyacrylamide gel and separated by electrophoresis, essentially as
described in detail elsewhere (25). Gels were fixed in 10% methanol,
10% acetic acid, dried under vacuum, and analyzed by autoradiography
using x-ray film (Kodak Biomax-MR) or quantitated using a
PhosphorImagerTM (Molecular Dynamics).
Preparation of Psoralen Cross-linked DNA--
A sample (4 nmol)
of a "short" oligonucleotide (CM6, 5'-ACC GGG TGT TCC TGA AGG
TAG GCT-3') and a sample (4 nmol) of a complementary
"long" oligonucleotide (CM5, 5'-CGC GCC CCC ACC CCC TTT TAT AGC
CTA CCT TCA GGA ACA CCC GGT-3') were annealed and
cross-linked (between the nucleotides indicated in boldface) using the
reagent, 4'-hydroxymethyl-4,5',8-trimethylpsoralen ("psoralen"), essentially as described in detail elsewhere (52). The cross-linked oligonucleotides were 5'-end-labeled using [-32P]ATP
(Amersham Pharmacia Biotech) and T4 polynucleotide kinase (New England
Biolabs), as recommended by the suppliers. Reaction was terminated by
denaturation of the enzyme by incubation at 70 °C for 10 min. The
short oligonucleotide was then extended by addition of excess Klenow
fragment of E. coli DNA polymerase I and incubation in the
presence of 50 µM of each of the four dNTPs. After
extension, the resulting duplex sequence corresponds to the AdMLP,
except for the 2-bp change made to direct the psoralen cross-link to
that specific site (in boldface above). The full-length and
cross-linked product was separated from all of the other material by
boiling and quick-cooling on ice (the cross-linked product rapidly
renatures) followed by resolution by electrophoresis on an 8%
polyacrylamide gel containing 8 M urea. The cross-linked duplexes had a distinctly slower mobility than the separate, fully denatured constituent oligonucleotides. The band corresponding to the
radiolabeled, full-length, and cross-linked probe was eluted from the
gel in TE and used for gel retardation experiments, as indicated.
| |
RESULTS |
Purification of Mot1-(His)6--
Full-length Mot1 was
tagged at its C terminus with a tract of six His residues to facilitate
purification of the enzyme by use of Ni2+-chelate affinity
chromatography. When expressed in yeast from the MOT1
promoter on a low copy number (CEN) plasmid, the tagged protein, Mot1-(His)6, was indistinguishable from wild-type
Mot1 in its ability to rescue the viability of an otherwise lethal chromosomal mot1
mutation (data not shown). To simplify
further purification, MOT1-(His)6 was
expressed under the transcriptional control of the strong inducible
GAL1 promoter in S. cerevisiae strain SC295 (40),
which carries the reg1-501 mutation that prevents glucose
repression of GAL-dependent gene expression
(53). As described in detail under "Experimental Procedures,"
Mot1-(His)6 was purified to apparent homogeneity by
ammonium sulfate fractionation, Ni2+-chelate affinity
chromatography (Fig. 1A), and
gel filtration chromatography (Fig. 1B). Identity of
Mot1-(His)6 was confirmed by immunoblotting of samples
using both anti-Mot1 antiserum (45) and a commercial anti-poly(His)
antibody (data not shown). The yield was over 1.5 mg of purified
Mot1-(His)6 per 100 g of wet cell paste (Table
I). As observed previously for Mot1
prepared by in vitro translation (26),
Mot1-(His)6 migrated with an apparent molecular mass of
175-180 kDa upon SDS-PAGE, somewhat smaller than its calculated
molecular mass (210 kDa) (27). Mot1 contains several highly net
negatively and positively charged regions, and anomalous migration of
proteins with such highly charged regions has been observed often (see,
for example, Ref. 54). The purified enzyme should contain native
post-translational modifications (if any) because it was isolated from
yeast cells. Mot1-(His)6 (hereafter Mot1) of 95% purity
(fraction IV) was used in all of the experiments presented here, unless
otherwise specified. In particular, there was no detectable TBP in the
purified Mot1 preparations. This result was expected because, as we
have noted (45) and will describe in greater detail
elsewhere,2 Mot1-TBP
association is disrupted by high ionic strength, and for this reason,
high salt (500 mM NaCl) was included in all buffers at
every stage of the purification.
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Fig. 1.
Purification of Mot1-(His)6 from
budding yeast. S. cerevisiae strain SC295 harboring
plasmid pCM112 was grown, induced, harvested, lysed, and clarified by
centrifugation as described under "Experimental Procedures." The
resulting whole-cell extract (W) was precipitated with
ammonium sulfate; the fraction (25-50% pellet) containing
Mot1-(His)6 was resuspended, dialyzed, and loaded
(L) onto a bed of Ni2+-saturated
iminodiacetate-agarose in a small column. The flow-through was passed
over the column again and then collected (FT). The column
was washed and eluted stepwise with buffers containing the indicated
concentrations (5, 20, 40, and 500 mM) of imidazole.
Samples of each fraction were resolved by SDS-PAGE and visualized by
staining with Coomassie Blue dye (A). Molecular weight
markers (MW) are indicated on the left, and the
band (Mot1) corresponding to Mot1-(His)6 is indicated by an
arrow on the right. Four fractions of the eluate
(40-mM wash, lanes 1-3, and 500-mM
wash) from metal-chelate affinity chromatography were pooled, dialyzed,
concentrated, and subjected to size-exclusion chromatography.
B, samples of the resulting eluate (fractions
22-30) were resolved by SDS-PAGE and visualized by staining with
silver nitrate, and the indicated fractions were pooled.
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Native State of Mot1--
During the gel filtration step of
purification, Mot1 eluted from the Sephacryl S-300 column earlier than
expected for its calculated molecular mass, suggesting that Mot1 might
be larger than a monomer. Therefore, size-exclusion chromatography was
used analytically to obtain a more rigorous estimate of the apparent size of Mot1 in solution. Samples of purified Mot1 (fraction III) were
subjected to gel permeation chromatography on a Sephacryl S-300-HR
column that had been calibrated immediately beforehand. The observed
elution volume (Ve) yielded a
Kav for Mot1 of 0.183, corresponding to a
molecular mass of ~380 kDa, based on the Kav
values obtained for the four protein standards used for calibration
(Fig. 2A). This result
suggested that Mot1 either has a non-globular rod-like shape or
undergoes a rapid monomer-dimer equilibrium in solution.
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Fig. 2.
Determination of native molecular mass of
Mot1. Purified Mot1-(His)6 (fraction III, Table I) was
subjected to size-exclusion chromatography (A) using an FPLC
apparatus (Waters/Millipore, model 650E) on a bed of Sephacryl S-300-HR
under non-denaturing conditions, as described under "Experimental
Procedures." For this column, vo = 38 ml and
vt = 120 ml. Molecular mass standards (and the
corresponding observed Kav values, where
Kav = (ve vo)/(vt vo)) were aldolase, 158 kDa (0.280); catalase, 260 kDa (0.268); ferritin, 440 kDa (0.171); and, thyroglobulin, 669 kDa
(0.067). Arrow indicates relative elution position of Mot1
(Kav = 0.183), corresponding to an apparent
molecular mass of ~380 kDa. B, samples (10 µg) of
purified Mot1 (fraction III) were mixed with globular
protein standards (100 µg), layered onto a 5-20% (w/v) linear
sucrose gradient, and subjected to centrifugation. Fractions were
collected, and the distribution of each protein was measured as
described under "Experimental Procedures." Globular protein
standards were hemoglobin, 64 kDa (squares); aldolase, 158 kDa (shaded diamonds); and, catalase, 260 kDa
(circles). The peak Mot1-containing fraction is indicated by
the arrow. Inset, samples (15 µl) of the
indicated fractions were analyzed by SDS-PAGE and immunoblotting with
rabbit polyclonal anti-Mot1 antibodies.
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To distinguish between these possibilities, the apparent size of Mot1
in solution was also estimated by rate zonal sedimentation, using the
method of Martin and Ames (55). The Stokes radius of a molecule has an
opposite effect on its separation (and thus its estimated size) as
judged by this method as compared with size-exclusion chromatography.
Samples of purified Mot1 (fraction III) were mixed with globular
protein standards of known molecular mass and subjected to
centrifugation through a linear 5-20% (w/v) sucrose gradient (Fig.
2B). As assessed by immunoblotting, only one peak of Mot1
was observed (and no Mot1 was detected in any of the other fractions).
Compared with the protein standards used for calibration, the
sedimentation rate of Mot1 yielded an apparent molecular mass of ~160
kDa, consistent with the conclusion that Mot1 is an elongated monomeric
protein (rather than a dimer). Moreover, as judged by either gel
filtration or sedimentation, the hydrodynamic behavior of Mot1 was
unchanged in the presence of 1 mM ATP and 4 mM
MgCl2 (data not shown).
Absence of appreciable homo-oligomerization of Mot1 in vivo
was confirmed by co-expression of either Mot1 or
Mot1-(His)6 and a fully functional Mot1 derivative tagged
at its C terminus with c-Myc epitope (Mot1-myc), followed by
Ni2+-chelate affinity chromatography of cell extracts. Both
Mot1-myc and co-expressed native Mot1 were absent in the 500 mM eluate (Fig. 3, left
side), demonstrating that, as expected, neither protein was
retained on the Ni2+-chelate column. In contrast, in the
extract containing Mot1-myc and co-expressed Mot1-(His)6,
Mot1-(His)6 was found in the 500 mM eluate
(Fig. 3, right side), indicating that a significant fraction
of the Mot1-(His)6 bound tightly to the affinity matrix, as
anticipated. However, no detectable Mot1-myc was present in this
Mot1-(His)6-containing eluate (Fig. 3, right
side). The same result was obtained with extracts prepared at salt
concentrations varying from 150 to 500 mM NaCl (data not
shown), indicating that Mot1 monomers do not stably interact in cell
extracts. Thus, this experiment provides independent and complementary
evidence that, like highly purified Mot1, native Mot1 does not
self-associate. Hence, large Mot1-containing species observed by others
in cell extracts, for example by sedimentation in a glycerol gradient (56), most likely represent a single molecule of Mot1 complexed with
other proteins.
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Fig. 3.
Mot1 does not self-associate in cell
extracts. A protease-deficient yeast strain (BJ2168), carrying a
multicopy plasmid expressing from the GAL1 promoter
Myc-epitope-tagged Mot1 (Mot1-myc), was co-transformed with another
multicopy plasmid expressing from the GAL1 promoter either
unmodified Mot1 (left panel) or Mot1-(His)6
(right panel). Cells were grown in glucose (2%)-containing
medium selective for both plasmids and then shifted to galactose
(2%)-containing medium for 2 h to induce protein expression.
Lysates were prepared by cavitation with glass beads and loaded
(L), in parallel, onto two identical,
Ni2+-saturated iminodiacetate-agarose columns. The
flow-through fractions (FT) were collected, and the columns
was washed sequentially in buffers containing increasing concentrations
(in mM) of imidazole as follows: 20 (W-20), 40 (W-40), and 60 (W-60). The columns were then
eluted (EL) with buffer containing 500 mM
imidazole and stripped (S) with an EDTA-containing buffer.
Top, the indicated fractions were resolved by SDS-PAGE in an
8% gel, and all Mot1-related species were detected by immunoblotting
with rabbit polyclonal anti-Mot1 antibodies. Bottom, the
same fractions were separated on a second gel, which was analyzed by
immunoblotting with mouse anti-Myc monoclonal antibody 9E10 to
specifically detect only Mot1-myc.
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Catalytic Properties of Purified Mot1--
We previously expressed
the C-terminal third (residues 1254-1867) of Mot1 in E. coli and purified this fragment of the enzyme (which was dubbed
Mot1C) from this source (26). This segment of Mot1 contains the
conserved sequence motifs shared by ATPases of the Snf2 family
(27) and, as expected, was a highly active ATPase in vitro
(26). To determine the optimal conditions for ATP hydrolysis by this
catalytic domain, reactions were performed under a wide range of values
of both pH and ionic strength. Mot1C exhibited maximal activity at pH
6.7 (Fig. 4A) and in the
presence of 300 mM NaCl (Fig. 4B). When measured
under these optimal conditions, the specific activity of Mot1C was
~330 nmol of ADP formed per min/mg.
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Fig. 4.
Effect of pH and salt concentration on the
rate of ATP hydrolysis catalyzed by Mot1C and full-length Mot1.
Samples of purified, bacterially expressed Mot1C (700 ng) or
Mot1-(His)6 purified from yeast (765 ng, fraction
IV) were incubated with [-32P]ATP under the
conditions indicated, and the rate of ADP formation was measured, as
described under "Experimental Procedures." Values shown are the
average of at least two independent determinations, each performed in
duplicate, and are given as percent of the maximal activity observed.
Error bars represent the range of observed values.
A, Mot1C (at 300 mM NaCl); B, Mot1C
(at pH 6.7); C, Mot1-(His)6 (at 800 mM NaCl); and D, Mot1-(His)6 (at pH
8.0). The scales on the ordinates of B and D are
different.
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When measured using the same assay and under the same conditions, the
specific activity of purified full-length Mot1 was reproducibly quite
low (~15-20 nmol/min/mg). Therefore, reactions were conducted over a
broad range of pH and ionic strength values to determine the optimal
conditions. Full-length Mot1 exhibited maximal activity at pH 8.5 (Fig.
4C) and in the presence of 0.8-1.2 M NaCl (Fig. 4D). A similar pH activity profile was observed at 200 mM NaCl, but the maximal activity achieved was only ~10%
of that seen at 800 mM NaCl (data not shown). Under optimal
reaction conditions, the specific activity of Mot1 was in excess of 150 nmol/min/mg. Under the same conditions, the Km value
of full-length Mot1 for ATP was ~100 µM (Fig.
6A), in the same range as the Km value
for Mot1C (~200 µM) determined under its optimal
conditions (45).
Effect of TBP and DNA on Mot1 Activity--
Because Mot1 in
extracts is found in soluble complexes that contain TBP and other
polypeptides (56-58), and because we have shown that TBP can interact
with Mot1 in the absence of any other yeast proteins (45), the effect
of TBP on the ATPase activity of purified Mot1 was tested. Under salt
conditions optimal for ATPase activity (800 mM NaCl),
addition of TBP, even in large excess, had no effect (data not shown).
This result is consistent with our finding that Mot1 cannot bind TBP at
ionic strengths above 300 mM NaCl (45).2
However, at lower salt concentrations (100-125 mM NaCl),
where the ATPase activity of Mot1 alone was barely detectable, addition of TBP had a dramatic stimulatory effect on Mot1-catalyzed ATP hydrolysis (Fig. 5B). The
apparent dissociation constant for the Mot1-TBP interaction averaged
from five independent experiments was 100 nM. This value
measured on the basis of stimulation of reaction rate is in good
agreement with the Kd value for TBP binding to Mot1
(50 nM) determined by an independent physical method (45),
as will be described elsewhere.2 At a molar ratio of TBP to
Mot1 of 10:1, the specific activity of Mot1 was 150 nmol/min/mg,
identical to that achieved by Mot1 under high salt conditions in the
absence of TBP. As expected, addition of TBP had no effect on the
activity of Mot1C under any condition tested (data not shown) because
this fragment of Mot1 lacks the region required for TBP binding (45,
59).
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Fig. 5.
Response of Mot1-catalyzed ATPase activity to
increasing ATP and TBP concentration. A, the ATP
hydrolysis activity of purified Mot1-(His)6 (765 ng;
fraction IV) was measured at pH 8 in 800 mM NaCl
at the indicated ATP concentrations. To ensure nucleotide was always
present as Mg2+·ATP complex, the MgCl2
concentration in each reaction mixture was in 1 mM excess
of the ATP concentration (81). B, the ATPase activity of
Mot1-(His)6 (765 ng; fraction IV) was measured
at pH 8 in 100 mM NaCl at the indicated concentrations of
purified, bacterially expressed Spt15 (full-length yeast TBP). Values
shown are the average of at least three independent determinations,
each performed in duplicate. Error bars represent the range
of values observed.
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Addition of either a 28-nucleotide single-stranded DNA (ssDNA) or a
28-bp duplex DNA (dsDNA), which contain the AdMLP TATA sequence, up to
a concentration of 7 µM (100:1 molar ratio of DNA to
Mot1), had no effect on the ATPase activity of purified Mot1 (Fig.
6). Moreover, within experimental error,
incubation of Mot1 with either form of DNA in the presence of TBP
failed to stimulate the ATP hydrolysis activity of Mot1 above the
levels seen with TBP alone. This lack of effect of DNA is consistent with the lack of DNA binding affinity observed for Mot1 (25) (see
below).
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Fig. 6.
Effect of DNA on Mot1-catalyzed ATPase
activity. The ATP hydrolysis activity of Mot1-(His)6
(765 ng; fraction IV), corresponding to an enzyme
concentration of ~70 nM, was measured at pH 8 and 100 mM NaCl in either the absence or presence of a 2-fold
excess of purified yeast TBP with or without either a single-stranded
or a double-stranded 28-base oligonucleotide encoding the AdMLP
TATA-sequence (~7 µM), as indicated.
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Formation and ATP-dependent Dissociation of
Mot1·TBP·DNA Complexes--
The ability of highly purified Mot1
(fraction IV) to interact with purified TBP and a radiolabeled DNA
probe spanning the AdMLP TATA-sequence was examined using a gel
retardation assay (60) (Fig. 7). As
judged by an electrophoretic mobility shift, addition of TBP formed a
readily detectable complex with the probe in either the presence or
absence of ATP (lanes 2 and 3). Identity of this
species as a TBP·DNA complex was confirmed by adding anti-TBP antiserum, which resulted in a marked supershift of the mobility of the
complex (lane 4), whereas addition of anti-TBP antiserum to
the probe alone had no detectable effect (data not shown). In contrast
to TBP, addition of Mot1 alone produced no detectable complex with DNA
in either the presence or absence of ATP (lanes 5 and
6). However, in the absence of ATP, the simultaneous
presence of Mot1 and TBP generated a substantial amount of a
Mot1·TBP·DNA ternary complex. Because the samples analyzed in
lanes 2 and 7 contained identical amounts of TBP,
yet the total amount of the probe shifted was far greater when Mot1 was
present, it can be inferred that, in the absence of ATP, the
Mot1·TBP·DNA ternary complex is more stable than a TBP·DNA binary
complex. Identity of this species as the Mot1·TBP·DNA ternary
complex was confirmed by addition of anti-Mot1 antiserum, which
prevented formation of the complex (data not shown). As expected on the
basis of prior work (25, 26), in the presence of ATP, the species
corresponding to both the Mot1·TBP·DNA ternary complexes and the
residual TBP·DNA complexes were almost completely abolished,
confirming that Mot1 acts catalytically to dissociate TBP·DNA
complexes and requires the hydrolysis of ATP to do so. The fact that
Mot1, purified to apparent homogeneity, efficiently generated ternary
complexes with TBP-bound DNA and dissociated those complexes in an
ATP-dependent manner indicates that Mot1 requires no other
yeast protein or small molecule cofactor for these activities.
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Fig. 7.
Binding and ATP-dependent
dissociation of TBP·DNA complexes by purified Mot1. A
radiolabeled DNA probe (7.5 fmol) containing the AdMLP TATA sequence
was incubated, as indicated, in the absence of any added protein or
with purified TBP (0.5 pmol), either alone or with anti-TBP antiserum
(1 µl), or with purified Mot1-(His)6 (0.125 pmol;
fraction IV), or with both TBP and Mot1. After incubation
for 30 min at room temperature, 0.25 mM ATP was either
added or omitted, as indicated, and incubation was continued for an
additional 10 min. Reactions were terminated by cooling on ice, and
reaction products were analyzed by subjecting each mixture to
electrophoresis on a non-denaturing polyacrylamide gel at 4 °C and
visualizing by autoradiography. Migration positions of the resulting
complexes and the free probe are indicated by arrows.
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Mechanism of Mot1 Action--
Sequence alignments first revealed
that motifs also found in certain DNA helicases are present in the Mot1
ATPase domain (27). Given that TBP binding to a TATA sequence radically
alters the conformation of B-form DNA by causing a severe bend and a
positive writhe (61), ATP-driven strand unwinding of promoter DNA is a
plausible mechanism by which Mot1 could catalyze the displacement of
TBP from DNA. To test this possibility, both purified full-length Mot1
and purified Mot1C were tested for helicase activity using a standard
strand displacement assay (Fig. 8). For
this purpose, three different substrates were constructed in which
oligonucleotides corresponding to the 28-bp sequence that contains the
AdMLP TATA box were bound to circular single-stranded M13 DNA
containing an insert with a complementary sequence. This 28-bp sequence
is identical to that in the probe used for the gel mobility shift assay
(Fig. 7), in which Mot1 was able to displace TBP from the DNA in an
ATP-dependent manner. In one substrate, the oligonucleotide was perfectly complementary to the insert (Fig. 8A); for the
other two substrates, oligonucleotides with additional,
non-complementary 10-nucleotide 3'- or 5'-overhangs were used,
generating branched forms (Fig. 8B).
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Fig. 8.
Purified Mot1-(His)6 lacks
detectable helicase activity. Purified Mot1-(His)6
(750 ng, fraction IV), or purified SV40 T antigen (500 ng)
as a positive control, or purified Mot1C (700 ng) was mixed with DNA
substrates, prepared and labeled as described under "Experimental
Procedures," containing either a perfect 28-base pair duplex
corresponding to the AdMLP TATA sequence (A) or an otherwise
identical substrate with either a 10-base non-complementary
5'-extension (B, left side) or a 10-base
non-complementary 3'-extension (B, right side),
and incubated for 1 h at 30 °C. Reaction products were resolved
by electrophoresis of each reaction mixture on a non-denaturing
polyacrylamide gel and visualized by autoradiography. As a measure of
the complete dissociation of labeled oligonucleotide from the
complementary single-stranded circle, a sample of each substrate was
heated to 100 °C ("boiled").
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As a positive control, an equivalent concentration of a known DNA
helicase, SV40 large T antigen (T-Ag), was assayed under the same
conditions. In reactions with all three substrates, SV40 T-Ag displayed
readily detectable strand displacement activity (Fig. 8A, lane
5; Fig. 8B, lanes 5 and 11), even in the
absence of a ssDNA-binding protein to capture the displaced
oligonucleotide. Addition of TBP inhibited the helicase activity of
SV40 T-Ag (Fig. 8A, lane 6; Fig. 8B, lanes 6 and
12), suggesting that TBP binds to and stabilizes the dsDNA
substrate under these conditions, preventing strand displacement.
Likewise, it has been reported previously that binding of Lac repressor
to its operator can block the helicase activity of SV40 T-Ag (62). In
contrast to SV40 T-Ag, neither full-length Mot1 nor Mot1C displayed any
detectable strand displacement, in either the absence or presence of
TBP, even upon prolonged incubation. The apparent lack of helicase activity cannot be attributed to a lack of all catalytic activity in
these reactions because, in parallel assays conducted under otherwise
identical conditions (but containing radioactive ATP), both Mot1 and
Mot1C hydrolyzed ATP and exhibited significantly greater ATPase
activity than SV40 T-Ag (data not shown). Thus, if Mot1 possesses any
helicase activity at all, it is decidedly less robust than that of SV40
large T antigen.
It remained possible, however, that Mot1 could cause a local unwinding
of the DNA, sufficient to displace the bound TBP but insufficient to
observe wholesale strand displacement, even of a relatively short
oligonucleotide. If so, such local strand separation activity might go
undetected in the assay we used. To address this issue directly, a
substrate was constructed that contained a covalent interstrand
cross-link introduced using psoralen photochemistry. Based on the known
base specificity of psoralen-induced DNA cross-links (63, 64), and the
method used to prepare the substrate (see "Experimental
Procedures"), the cross-link was introduced at a 5'-TpA site (Fig.
9A) that falls between the
TBP-binding site and the nucleotides that lie in close proximity to
Mot1, as judged both by their protection in footprint analysis of
Mot1·TBP·DNA ternary complexes (25) and by the ability of
photoactivable nucleotide derivatives at these positions to undergo
UV-induced cross-linking to Mot1 (26). Thymines in 5'-TpA sites of
B-form duplex DNA are the preferential targets of psoralens (63, 64). As judged by NMR analysis, interstrand psoralen cross-links distort the
double helix of B-form DNA only slightly (65). Consistent with this
conclusion, we observed that TBP was able to bind to the cross-linked
substrate we prepared as efficiently as to an identical, but
non-cross-linked, probe (Fig. 9B, lanes 2 and 6), although the resulting complex displayed a slightly slower mobility, perhaps indicating that the cross-linked DNA did not bend as
dramatically as non-cross-linked DNA in response to TBP binding. In
addition, the complexes of cross-linked DNA and TBP were able to form
ternary complexes with Mot1 with nearly the same efficiency as the
non-cross-linked control DNA·TBP complexes (Fig. 9B, lanes
3 and 7). Finally, in the presence of ATP, the
Mot1-TBP·DNA complexes were dissociated, as observed before (Fig. 7),
regardless of whether the DNA was cross-linked or not (Fig. 9B,
lanes 4 and 8). Thus, the presence of a psoralen
cross-link, which should prevent localized strand separation
(especially that initiated 5' to the TATA box), did not inhibit the
ability of Mot1 to dissociate TBP·DNA complexes. These data provide
additional evidence against the hypothesis that ATP-driven strand
separation is the mechanism by which Mot1 displaces bound TBP from the
DNA.
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Fig. 9.
A psoralen-induced interstrand cross-link
does prevent Mot1-catalyzed dissociation of TBP·DNA complexes.
A, DNA sequence of the probe. Short (CM6) and
long (CM5) oligonucleotides were designed so that the only
5'-TpA dinucleotide (white-on-black letters) in the
resulting hybrid was situated between the regions contacted by TBP and
Mot1 (brackets). After cross-linking, the full duplex was
generated by extension (underlined) of the shorter
oligonucleotide from its 3'-OH end using the Klenow fragment of
E. coli DNA polymerase I in the presence of all four dNTPs.
The nucleotides directly contacted by TBP, as assessed from the x-ray
structures of TBP-DNA co-crystals (60, 74), are indicated
(boxed). Sites that, when occupied by photoactivable
nucleotide derivatives, can form either strong (bold
asterisks) or weak (plain asterisks) photo-induced
cross-links with Mot1 in Mot1·TBP·DNA ternary complexes are also
shown. B, either the cross-linked (lanes 1-4) or
non-cross-linked (lanes 5-8) DNA probes were incubated, as
described in the legend to Fig. 7, with TBP alone or with TBP and an
equal molar ratio of Mot1 in the presence or absence of ATP, as
indicated. TBP·DNA complexes (1) and Mot1·TBP·DNA
ternary complexes (2) are indicated by the
arrows.
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DISCUSSION |
Mot1, an essential protein in S. cerevisiae, regulates
Pol II transcription via a novel, as yet undefined, mechanism that requires ATP hydrolysis. To investigate its catalytic properties and
mode of action, we purified active full-length Mot1 from yeast to
apparent homogeneity in good yield (Fig. 1), and we examined its
biochemical properties in vitro.
Hydrodynamic measurements (Fig. 2) indicated that, in either the
presence or absence of ATP-Mg2+, purified Mot1 is a
monomeric protein with an apparent Stokes radius larger than expected
from its mass, suggesting that it has an elongated non-globular shape.
Indeed, when viewed by negative staining in the electron microscope,
Mot1 is a particle with a distinct "kidney bean"
appearance.3 Native Mot1 in
cell extracts does not self-associate, as indicated by the lack of
interaction between two functional, but differentially tagged, forms of
Mot1 co-expressed in vivo (Fig. 3). These results are
relevant to the mechanism of Mot1 action because most (although not
all) DNA helicases operate as homo-oligomers (66, 67), typically dimers
or hexamers, and display cooperativity between subunits as they unwind
the nucleic acid strands (68). Some helicases, like E. coli
Rep, are monomers in solution but dimerize upon binding to DNA (69).
However, purified Mot1 did not display detectable DNA binding activity
(in the absence of TBP) (Fig. 7). Cruder Mot1 preparations also lack
DNA binding ability (25, 26, 59, 70).
Full-length Mot1 was optimally active at higher values of both pH and
salt concentration than those required for maximum activity of Mot1C,
which lacks residues 1-1253 (Fig. 4). These observations suggest that
the N-terminal domain of Mot1 exerts an inhibitory effect on the
C-terminal catalytic domain and that electrostatic interactions may
contribute to this interaction. Three lines of evidence support this
proposal. First, partial unfolding of Mot1 by low concentrations of a
mild denaturant (urea) caused an ~50% stimulation of Mot1 ATPase
activity under otherwise sub-optimal conditions (46). Second, addition
of TBP, which binds to the N terminus of Mot1 (45),2
stimulates Mot1 ATPase activity to the same extent as high pH and high
salt (Fig. 5B), implying that TBP binding causes a
conformational change in Mot1. Third, as shown here, under optimal
conditions (high salt, high pH, or presence of TBP), ATPase activity of
full-length Mot1 approaches that of Mot1C, suggesting that, when the N
terminus is absent, the C-terminal catalytic domain is maximally active.
Taken together, these results suggest that under physiological
conditions (low ionic strength and neutral pH), the N terminus of Mot1
acts to inhibit the C-terminal ATPase domain via electrostatic interactions and that binding of TBP to the N terminus alleviates this
negative intramolecular constraint. Published observations suggest that
such an auto-inhibition model may apply to Snf2; its C-terminal
domain exhibited readily measurable and DNA-stimulated ATPase activity,
whereas purified full-length Snf2 had no detectable activity
(71). In the SWI·SNF complex, however, Snf2 displayed DNA-dependent ATPase activity (72, 73). Thus, the N
terminus of Snf2 may act as an auto-inhibitory domain, and its
interaction with other proteins in the SWI·SNF complex presumably
overcomes this inhibition. This behavior may be general for
Mot1-related proteins because all family members are large and share a
common organization, with the conserved catalytic (ATPase) domain at the C terminus and divergent N-terminal domains that presumably target
each enzyme to its substrate (26).
Under optimal conditions, the Km value of Mot1 for
ATP (Fig. 5A) is very close to that measured for Mot1C.
Hence, the stimulation of ATPase activity caused by TBP binding (Fig. 5B) cannot be explained by an increase in affinity of the
enzyme for its nucleotide substrate, consistent with the proposed
relief of intramolecular constraint model. Furthermore, addition of
single- or double-stranded DNA (with or without a TATA sequence) had no reproducible effect (either stimulatory or inhibitory) on the ATPase
activity of purified Mot1 (in either the presence or absence of TBP)
(Fig. 6). These results were somewhat surprising because Mot1 appears
to make contact with nucleotides in the DNA 5' of the TATA box, as
judged by both footprinting and cross-linking experiments, and requires
this 5'-flanking region in order to remove bound TBP from the DNA (25,
26). Moreover, it was reported that the human version of Mot1, hTAF172,
exhibits weak intrinsic ATPase activity that is not stimulated by
either TBP or DNA alone but is stimulated by TBP·DNA complexes (74).
This difference between Mot1 and hTAF172 is not understood.
Availability of a homogenous preparation of full-length Mot1 allowed us
to test its DNA-binding properties in the apparent absence of any other
yeast protein or small molecule cofactor. Mot1 did not stably interact
with DNA (in either the absence or presence of Mg2+-ATP);
however, in the absence of ATP, Mot1 readily formed ternary complexes
with TBP bound at the TATA sequence (Fig. 7). These results are
consistent with the ability of TBP, but not DNA, to stimulate the ATP
hydrolysis activity of Mot1 (Fig. 6). Although it is possible that the
strong (90°) kink in TATA DNA induced by TBP binding (61, 75) is
necessary to expose the contacts sites for DNA recognition by Mot1, it
is more likely that Mot1 is recruited to DNA-bound TBP via
protein-protein interactions. This conclusion is supported by several
observations. First, Mot1 forms stable complexes with TBP in the
absence of DNA both in cell extracts (56-58) and in vitro
(45, 59).2 Second, although nucleotides 5' to the TATA box
that appear to be contacted by Mot1 are required for the TBP
displacement reaction, this DNA is not required for formation of
Mot1·TBP·DNA ternary complexes (25). Third, as shown here,
TBP·DNA complexes do not stimulate Mot1 ATPase activity any more than
TBP alone (Fig. 6).
There are four plausible models to explain how Mot1 might catalyze
release of TBP from DNA via an ATP-dependent mechanism (Fig. 10). First, Mot1 could act as a
DNA helicase to unwind DNA near the TATA sequence, thus disrupting
TBP-DNA contacts. However, as shown here (Fig. 8), Mot1 lacked
detectable strand displacement activity in either the presence or
absence of TBP on three different substrates (duplex, 3'-tail, and
5'-tail) under conditions where Mot1 binds TBP, is a highly active
ATPase, and dissociates TBP·DNA complexes. Cruder preparations of
Mot1 also reportedly lacked DNA helicase activity (25). Moreover, we
found that an interstrand psoralen cross-link (Fig. 9A)
specifically introduced between the DNA sites contacted by Mot1 and
TBP, respectively, does not block the ability of Mot1 to dissociate
TBP·DNA complexes (Fig. 9B). Because a cross-link should
prevent strand unwinding, yet did not interfere with Mot1-catalyzed
dissociation of bound TBP, a mechanism in which Mot1 unwinds the TATA
DNA sequence from its 5'-side seems unlikely. This experiment does not
rule out unwinding from the 3'-side, but this possibility also seems
unlikely because no contacts between Mot1 and DNA have been observed 3'
to the TATA sequence (25, 26). Furthermore, DNA-stimulated ATP
hydrolysis is a characteristic feature of known DNA helicases (66, 68), yet Mot1 ATPase activity was not stimulated by DNA, and DNA did not
enhance the stimulatory effect of TBP (Fig. 6). Taken together, the
monomeric nature of Mot1, the lack of stimulation of Mot1 ATPase
activity by DNA, the lack of strand displacement activity, and the
ability of TBP bound to cross-linked DNA to act as a substrate provide
evidence against a helicase mechanism for Mot1 action. To date, DNA
helicase activity has not been detected in any other Mot1-related
enzyme, including the Snf2 ATPase domain (71); full-length
Snf2 in the SWI·SNF complex (73); and the nucleotide-excision repair protein, Cockayne Syndrome gene B product (76), and its yeast
homolog, Rad26 (77).
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Fig. 10.
Models for the mechanism of action of
Mot1. In the absence of ATP, Mot1 forms a stable ternary complex
with TBP bound to TATA-containing DNA. In the presence of ATP,
Mot1-catalyzed nucleotide hydrolysis results in the complete
dissociation of TBP from DNA. Four possible models for the mechanism by
which the energy of ATP hydrolysis might be harnessed by Mot1 to
displace TBP from DNA are shown. 1, strand unwinding
(helicase activity); 2, DNA straightening; 3, DNA
tracking; and, 4, induced conformational change in TBP. See
text for details.
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A second model for Mot1 activity is based on the fact that TBP has a
higher affinity for a TATA sequence in a small, closed circular DNA
than in linearized DNA (78) and that TBP binding causes DNA to bend and
partially open (61, 75, 79). Mot1, by spanning the DNA and TBP (Fig.
10), might act as an ATP-driven molecular "lever" that straightens
the DNA, thereby weakening TBP-DNA contacts. Although Mot1 contacts
nucleotides 5' of the TATA box in the 28-base AdMLP (Fig.
9A), as assessed by footprinting and cross-linking
experiments (25, 26), this interaction appears to be very weak (Figs. 6
and 7). Thus, this model seems unlikely but has not been ruled out by
any direct test.
A third model proposes that ATP hydrolysis powers Mot1 to translocate
processively along the DNA, pushing TBP off, like a snow plow. Such a
"tracking" mechanism had been proposed for SWI·SNF and for the
Snf2 family in general (33). However, a direct test for DNA
tracking found no evidence for this mechanism (70).
In a fourth model, which we favor, ATP hydrolysis provides the energy
to drive a conformational change in Mot1, which is transmitted via
protein-protein interactions to TBP. The Mot1-induced changes in TBP
conformation would then be incompatible with the binding of TBP to its
TATA site. Thus, Mot1 would displace TBP in a "power stroke"
similar to that of cytoskeletal motor proteins (80). In support of this
model, we have found that Mot1 and TBP form a stable complex both in
solution and on the DNA, whether in the absence of nucleotide or in the
presence of ADP or two different non-hydrolyzable ATP analogs
(45).2 These findings suggest that ATP hydrolysis, not
nucleotide binding per se, drives TBP dissociation.
Consistent with this view, Mot1(D1408N), which displays no detectable
ATPase activity in vitro and cannot complement a
mot1 mutation in vivo (26), nonetheless binds TBP with normal affinity as judged by co-immunoprecipitation
experiments (45),2 and Mot1(K1303A), another
ATPase-defective mutant (26), forms ternary complexes with TBP-DNA with
wild-type affinity (59).
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ACKNOWLEDGEMENTS |
We thank Bob Lesch for assistance with the
fermenter, Stephen Johnston for strain SC295, Marjorie C. Brandriss for
assistance with optimization of the ammonium sulfate fractionation,
Grace Gill and Robert Tjian for anti-yeast TBP antiserum, Eric Fouts and Mike Botchan for purified SV40 T antigen and assistance with electron microscopy, John Hearst and Marie Fergusen for advice on
psoralen and assistance with preparation of the cross-linked probe, and
members of the Thorner laboratory for helpful discussions.
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FOOTNOTES |
*
This work was supported by a predoctoral fellowship from the
Howard Hughes Medical Institute (to J. I. A.), by Postdoctoral Fellowship LT-316/92 from the Human Frontier Science Program
Organization (to C. G. F. M.), by a university fellowship from the
graduate division of the University of California, Berkeley, by 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 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.
Current address: Institut Curie, INSERM U255, 26 Rue d'Ulm, Paris
75005, France.
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INTRODUCTION
