From the Department of Pathology, Brigham and
Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115, the ¶ Department of Genetics,
Massachusetts General Hospital, Boston, Massachusetts 02114, the
** Department of Pediatric Oncology, The Dana-Farber Cancer Institute,
Harvard Medical School, and the
Department
of Medicine, Brigham and Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, December 20, 2000
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ABSTRACT |
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Eukaryotic Rvb1p and Rvb2p are two highly
conserved proteins related to the helicase subset of the AAA+ family of
ATPases. Conditional mutants in both genes show rapid changes in the
transcription of over 5% of yeast genes, with a similar number of
genes being repressed and activated. Both Rvb1p and Rvb2p are required
for maintaining the induced state of many inducible promoters. ATP binding and hydrolysis by Rvb1p and Rvb2p is individually essential in vivo, and the two proteins are associated with each
other in a high molecular weight complex that shows
ATP-dependent chromatin remodeling activity in
vitro. Our findings show that Rvb1p and Rvb2p are essential
components of a chromatin remodeling complex and determine genes
regulated by the complex.
Differential gene expression is an important means by which
unicellular organisms respond to their environment and lies at the
heart of cell specialization in multicellular organisms. Recent discoveries have emphasized the complexity of the apparatus that governs transcription in eukaryotic cells (reviewed in Refs. 1 and 2).
The eukaryotic RNA polymerase II holoenzyme, responsible for the
transcription of protein-encoding genes, is a megadalton complex
containing over 50 components. This large complex must access the
template genes on chromosomes packed into chromatin, which restricts
access to the DNA. Packaging of genes into chromatin represses basal
transcription allowing several multisubunit complexes to regulate gene
expression by modulating the topology of the constituent nucleosomes in
a number of ways. Histone tails can be modified by either
phosphorylation or acetylation (3), and these modifications are
correlated with increased transcriptional activity. Another mechanism
involves ATP-driven molecular machines that destabilize or remodel
nucleosomes directly. These chromatin remodeling factors are all large
complexes and all include at least one helicase-like subunit with
DNA-dependent ATPase activity (4). The prototype chromatin
remodeling complex is the yeast Swi/Snf complex (5, 6), but genetic and
biochemical studies have revealed a number of other chromatin
remodeling complexes. Inspection of the yeast genome reveals at least
17 ORFs1 with homology to the
Swi/Snf helicase-like ATPase subunit (Snf2p) suggesting that
chromatin remodeling complexes are numerous and may each be involved in
specific cellular pathways (2, 7). Although Snf2p and its
homologs, such as Sth1p, a component of the RSC chromatin remodeling
complex (8), and the recently described Ino80p (Ari1p) transcription
factor (9, 10) all belong to the DEAD/H class of proteins and contain
sequence motifs typical of helicases, none of them has been shown to
possess intrinsic helicase activity when purified.
The bacterial RuvB protein belongs to the class of hexameric helicases.
Hexameric helicases are members of a larger family of proteins known as
the AAA+ class Chaperone-like ATPases (11) that all contain a number of
sequence motifs, including Walker A (P-loop) and Walker B boxes that
represent active sites for ATP (or dNTP) binding and hydrolysis,
respectively. The functional unit of most of these enzymes is a
circular hexamer. Recent work in structural biology has shed light on
how proteins of this class can translate allosteric changes produced
upon ATP hydrolysis into circular movement and vice versa (see Refs. 12
and 13). We first identified the human RVB1 ortholog in a
two-hybrid interaction screen with the 14-kDa subunit of the DNA
replication and repair factor RPA, and we subsequently identified yeast
RVB1 in data base searches (14). The protein was named
RuvBL1 (RuvB-like) because of its similarity to bacterial RuvB.
RVB2 (RuvBL2) was subsequently identified as a closely
related family member present in the yeast genome, with conserved
orthologs in human, fly, and worm. The high degree of sequence
conservation in the Rvb1p and Rvb2p proteins immediately suggested that
they have important functions in vivo, and subsequent
genetic studies in yeast showed that both genes are essential for
mitotic growth (14, 15). RVB1 and RVB2 have been
independently discovered a number of times, first in rat as interactors
of TATA-binding protein (named TIP49a and TIP49b) (15, 16) and later in
human cells as components of a large nuclear protein complex (named
ECP-51 and ECP-54) (17) and as essential interactors of At the outset of this study, little was known about the function of the
Rvb proteins, but in light of the findings that they interacted with
TATA-binding protein (15, 16) and were components of the large RNA pol
II holoenzyme complex (14), a role in transcription was plausible.
However, a different role was implied by the activities of the closest
homolog with a known function, eubacterial RuvB, which is involved in
the movement of Holliday junctions (branched four-way structures that
form upon homologous recombination of DNA). The association with RP-A,
a protein involved in DNA replication and repair, implied yet a third
possible function for Rvb1p and Rvb2p (14).
Recent publications add to the list of activities attributed to Rvbp.
Human Rvb1p and Rvb2p were reported to act independently as helicases
of opposite polarity (15, 21), although this result has been disputed
(22). More recently a study (23) of a temperature-sensitive mutant
strain showed the involvement of Rvb2p in the transcriptional
regulation of CLN2 and genes encoding four ribosomal
subunits, and a separate study (10) showed that yeast Rvb1p and Rvb2p
copurify with the Ino80p ATPase as a complex that has the ability to
remodel chromatin in vitro. The mammalian Rvb orthologs have
been implicated in cell transformation by c-Myc (20), and the human Rvb
proteins were also found to associate with the TIP60 histone acetylase
complex and to play a role in repair of DNA damage (22). Finally, a
recent study found that the respective Drosophila homologs
act antagonistically in the control of Wingless signaling through
In this paper, we report a global analysis of yeast genes affected by
removal of Rvb1p or Rvb2p. We used targeted degradation of the Rvb
proteins in combination with whole genome high density oligonucleotide
arrays (24) to characterize the in vivo functions of these
proteins. We find that Rvb1p and Rvb2p cooperate, directly or
indirectly, in transcriptional regulation of over 5% of yeast genes.
In addition, we demonstrate that ATP binding and hydrolysis by the Rvb
proteins is essential for their function in vivo and that
both proteins are components of an ATP-dependent chromatin remodeling complex in vitro.
Constructs for Generating Temperature-degradable
Alleles--
Plasmids used to generate the rvb1-td and
rvb2-td alleles were constructed by replacing the CDC28
fragment in pPW66R (25) with polymerase chain reaction-generated
fragments from the N termini of RVB1 or RVB2. The
plasmid pKL54 (26) used to place the UBR1 gene under the
control of a GAL1 promoter was a gift from John F. X. Diffley.
Yeast Strains Genetic Methods and Media--
Yeast strains used
in this study are isogenic with W303-1a (27), except YRVB1D, YRVB2D,
YKG3, and YRA2, which were derived from with YSB455 (14) (Table
I). Mating, sporulation, and tetrad analyses were performed by standard methods (28). Rich (YPD), synthetic
complete (SD), 5'-fluoro-orotic acid (5-FOA), and sporulation media
were prepared as described (28). Media and plates for maintenance of
rvb-td strains contained 0.1 mM
CuSO4. For experiments involving galactose induction,
glucose in YPD or SD was substituted with 2% raffinose. Subsequent
induction was achieved by switching to medium containing 2% raffinose
and 2% galactose.
FACS Analysis--
Samples of ~107 cells were
harvested and fixed in 70% ethanol. Propidium iodide staining and
analysis was performed as described in Ref. 29.
Northern Analysis of Gene Expression--
Samples of 4 × 107 cells were harvested for each time point and
flash-frozen on dry ice. Total RNA was isolated by phenol extraction and precipitation with lithium acetate. Yields were quantified by
absorbance at 280 nm, and the samples were subsequently diluted to
equal concentration. 5 µg of total RNA were loaded onto each lane on
formaldehyde gels following standard protocol (30). Probes were
generated by random 32P labeling using ORFs obtained from
Research Genetics (Huntsville, AL) as template.
Microarray Analysis of Gene Expression--
Transcript profiling
with the Affymetrix GeneChips was performed using whole genome high
density oligonucleotide arrays (24) according to Affymetrix protocol.
Briefly, 8 µg of total RNA were reverse-transcribed using a primer
consisting of oligo(dT) coupled to a T7 RNA polymerase-binding site.
The cDNA was made double-stranded and biotinylated cRNA synthesized
using T7 polymerase. Unincorporated nucleotides were removed, and cRNA
was quantitated by UV absorbance. For each sample 25 µl of cRNA were
randomly sheared to an approximate length of 50 nucleotides and
hybridized (16 h) to the Affymetrix GeneChips. External standards were
included in each hybridization to control for hybridization efficiency,
to test for sensitivity, and to assist in the comparisons between data
sets from different experiments. These external standards were cRNA
transcribed from cloned bacterial genes (bio b, bio c, bio
d, and cre). The first hybridization was against a Test
II Chip, to determine the quality of the cRNA mixture. Hybridized
biotinylated cRNA was detected by incubation with
phycoerythrin-streptavidin and was quantitated by scanning using the
Hewlett-Packard GeneArray laser scanner. Following positive analysis of
the Test Chip, the same hybridization mixture was added to the
expression chip, and the chips were hybridized, reacted with
phycoerythrin-streptavidin, washed, and then incubated with a
polyclonal anti-streptavidin antibody coupled to phycoerythrin as an
amplification step to aid in the detection of lower abundance transcripts. Following further washing, the expression chips were scanned as above. Analysis of the scanned data was performed using GeneChip software (version 3.3), Microsoft Access and Microsoft Excel.
Analysis of Microarray Data--
Affymetrix GeneChip software
was used to calculate average difference (AvgDiff) and fold change (FC)
values. For the analysis reported in
Table II and in Fig. 4, the 0-h time
point for each strain was used as base for the 4-h time point
calculations. Analysis was limited to chromosomal ORFs, and non- or
marginally expressed genes were discarded based on absent/present calls
and a cut-off AvgDiff value of 200 across all six chips. Exceptions
were made when a gene was present with an AvgDiff value of >1000 for
at least one data set. For genes where negative AvgDiff values
remained, a factor increasing the lowest number to 5 was added across
the arrays. To select genes that are deregulated in the
rvb-td strains relative to the wild type strain, ratios of
the values for the 0- and 4-h time points for each gene were calculated
for the three strains and transformed to linearity by applying a base 2 logarithm, and then the log2 ratios for the wild type
strain were subtracted from the corresponding log2 ratios
for the two rvb-td strains. A difference in log2
ratios greater than 1 corresponds to a more than 2-fold difference in
response between rvb-td and wild type and was set as cut-off
for further analysis. Finally we removed ORFs where confidence in the
level of expression at base line was low because of excessive variation
in AvgDiff values in the three yeast strains at 23 °C as well as
non-annotated ORFs and Ty elements. 326 ORFs passed these criteria and
were included in the analysis presented in Fig. 4.
The "corrected fold change difference" values reported in Table II
were calculated based on GeneChip FC values that incorporate corrections for background noise in the experiments. To calculate differences between a pair of FC values we first reversed scaling applied by the GeneChip software to obtain raw expression ratios according to the formula (|FC|
For comparison of the effects of rvb1-td with
rvb2-td and to the effects of swi1/snf2
deletion (Fig. 5), a less stringent approach was used so as to compare
our results with those of Sudarsanam et al. (31). Only the
values obtained at the non-permissive temperature were used. AvgDiff
values were calculated using the wild type strain as base. Non- or low
expressing genes were removed based on absent calls and AvgDiff values
as above, but with a cut-off at 400. Only genes present in both our
data set and the swi1/snf2 data set were included, thus leaving
3991 ORFs for comparison.
Immunoprecipitation and Mass Spectrometry--
For
immunoprecipitation experiments, 2-liter cultures of the strains YRA2
(RVB1-3Myc) and YKG3 were grown in YPD at 30 °C to an
A600 of 1.0. Cells were harvested by
centrifugation, washed once with 500 ml of cold distilled
H2O and once with 500 ml of buffer A (25 mM
HEPES-KOH, pH 7.6, 2 mM MgCl2, 1 mM
sodium bisulfite, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 0.5 mM EGTA, 0.1 mM EDTA, 0.02% v/v Nonidet P-40, 20% v/v glycerol) with 0.3 M KCl (buffer A0.3). Pellets were resuspended at 2 ml/g
in buffer A0.3 with protease inhibitors (2 µg/ml pepstatin A, 2 µg/ml leupeptin, and 5 µg/ml aprotinin) and extracted on dry ice
with a coffee grinder. 20,000 units of DNase I were added to 25 ml of
lysate followed by incubation for 15 min on ice, 15 min at room
temperature, and 1 h of gentle rocking at 4 °C. After
ultracentrifugation for 90 min at 36,000 rpm in a Sorval SW41-Ti rotor,
the extracts were pre-cleared with 50 µl of protein A-Sepharose beads
(Amersham Pharmacia Biotech) at 4 °C with gentle mixing, incubated
with 75 µl of ATPase Assay--
Ten-µl reactions were incubated for 30 min
at 30 °C in 10 mM HEPES-KOH, pH 7.5, 5 mM
MgCl2, 1 mM dithiothreitol, 100 µg/ml bovine
serum albumin, and 20 µM [ Chromatin Remodeling Assay--
End-labeled nucleosomal array
5S-G5E4 (32) (gift from J. L. Workman) was formed as described
previously (33, 34). Remodeling was carried out at 30 °C in 60 mM KCl, 7% glycerol, 4 mM MgCl2 ± 2 mM ATP·Mg, 0.02% Nonidet P-40, 10 mM
HEPES, pH 7.9, and 10 mM Tris, pH 7.5. Reactions were
initiated by addition of 4 µl of end-labeled array to a final volume
of 20 µl. The final concentration of array was ~1 nM in
mononucleosomes with ~1.5 ng/µl of Rvb complex. At various times,
3-µl aliquots were removed and quenched in 4.5 µl of 2% SDS and
100 mM EDTA. The aliquots were deproteinized using 1 mg/ml
proteinase K. Cut and uncut arrays were separated on 1% 1xTBE agarose
gel and quantified by phosphorimaging.
Removal of Rvb1p or Rvb2p Leads to Growth Arrest at Various Stages
in the Cell Cycle--
To determine the functions of Rvb1p and Rbv2p
in vivo, a modification of the N-degron system for
temperature-dependent protein degradation was used (25,
26). Briefly, the chromosomal copies of the RVB genes were
replaced by a fusion consisting of ubiquitin (Ub), the N terminus of
mutant DHFR (DHFR-ts), an HA epitope, and finally
the respective RVB gene (Fig.
1A). Proteolytic removal of Ub
results in a fusion protein with an arginine N terminus. At 23 °C
the N-terminal arginine of DHFR-ts in not recognized by the
polyubiquitination/proteasome pathway (25), but upon shift to the
non-permissive temperature (38 °C), the N terminus is exposed, and
the protein is targeted for processive degradation. To increase the
efficiency of degradation, the UBR1 gene is placed under
control of a GAL1 promoter and tagged with a Myc epitope to
facilitate detection (26). After preinduction of Ubr1 with galactose,
cultures were shifted to 38 °C, and the degradation of Rvb proteins
was monitored by immunoblotting (Fig. 1C). The results show
that degradation of Rvb1p and Rvb2p is virtually complete within 2 h of temperature shift. Concurrently, growth of the yeast slows down
and eventually stops (Fig. 1B).
Because of the interaction with RP-A, and the essential nature of the
yeast RVB genes, we considered that these proteins might have a unique role in S-phase. The morphology of the cells, however, indicated that loss of Rvb1p or Rvb2p resulted in cells ceasing growth
in all stages of the cell cycle (not shown). This was confirmed by FACS
analysis of propidium iodide-stained cells (Fig. 1D). Thus
Rvb1p and Rvb2p do not uniquely affect a metabolic process specific for
S-phase. The stage in which the majority of cells arrested was highly
dependent on conditions, such as the composition of the growth medium,
indicating that growth arrest is not dependent on a single check point.
For example, in rich medium the cells accumulated predominantly with a
1C DNA content (corresponding to G1), whereas in minimal
medium the cells accumulated mostly with 2C (corresponding to
G2). Such differential effects of Rvb1p and Rvb2p on the
cell cycle, depending on whether growth was in rich or minimal media,
were reminiscent of the differential effects on transcription caused by
the Swi/Snf chromatin remodeling complex (31).
To determine whether the cell proliferation block was reversible and to
monitor the viability of the cells after temporary removal of Rvbp,
samples were taken at 2-h intervals and plated at 23 °C on YPD
containing 0.1 mM CuSO4. The block is
reversible with less than 2-fold loss of cell viability after 8 h
following the temperature shift. Although viability was only slightly
affected, the rbv-td strains showed a considerable lag
before growth resumed, resulting in a 24-h delay in the appearance of
colonies compared with the RVB+ strain, and a
great deal of variation in colony size (data not shown).
While this work was in progress Lim and co-workers (23) published a
study of a temperature-sensitive rvb2 allele
(tih2-160). When grown on rich medium the strain showed a
rapid but reversible accumulation of G1 cells upon
temperature shift, which the authors attributed to a G1
arrest. These results are in agreement with our observations, but our
additional results on minimal media suggest that Rvb1p and Rvb2p are
not uniquely required in G1.
Rvb1p/Rvb2p Are Required for Transcription of Galactose-induced
Promoters--
The relative difference in the levels of Myc-Ubr1p
following induction in galactose in strains with rvb1-td and
rvb2-td first led us to consider that the RVB1
and -2 genes had important roles in transcriptional
regulation. Whereas Myc-Ubr1p was highly induced by galactose in the
control strain (YUBR1), the loss of Rvb1p and Rvb2p in the
td strains (YR1UB1 or YR2UB1) was accompanied by a
progressive decrease in the amount of Myc-Ubr1 protein (Fig. 1C) which was concomitant with the disappearance of
UBR1 mRNA (not shown), consistent with RVB1
and RVB2 being required for induction of the GAL1
promoter that drives Myc-UBR1. To test whether this was true
for other galactose-responsive promoters, Northern blotting was
performed with samples taken at various time points following a
temperature shift in the presence of galactose (Fig. 2). The transcripts from several
galactose-inducible promoters (GAL2, GAL7, and
GAL10) were significantly decreased upon the removal of
Rvb1p and Rvb2p from yeast in vivo. It is interesting to
note that even after activation of the galactose-induced promoter (0-h lane), the induced expression is not maintained once
Rvb1p or Rvb2p is degraded. This could indicate that the proteins are needed to maintain transcription even after the establishment of a
transcriptionally active initiation complex.
Northern Analysis Reveals that the Transcriptional Regulation of a
Large Number of Promoters Depends on Rvb1p and Rvb2p--
The strong
effect of Rvb1p and Rvb2p on the galactose-induced promoters prompted
us to examine the effects of rvb-td on the levels of several
other transcripts after temperature shift. Expression of two
G1 cyclins, CLN1 and CLN2 (Fig. 2),
was strongly decreased upon loss of Rvb1p or Rvb2p, but the persistence
of another G1 cyclin, CLN3, is sufficient to
allow cells to progress through G1. In addition, induction
of some heat shock-induced genes (e.g. HSP26) was
lost upon the removal of Rvb1p or -2p (Fig. 2). However, the expression
of CLB5, SPT15, MATa1, and
ACT1 was not significantly affected by temperature shift in
the rvb1-td and rvb2-td
strains (Fig. 2). Thus Rvb1p or Rvb2p are required to regulate the
expression of some but not all RNA pol II-transcribed genes.
The rapid changes in gene expression closely follow the degradation of
Rvb1p and Rvb2p proteins (Fig. 1C) and precede noticeable changes in cell proliferation (Fig. 1B). For the most
rapidly responsive genes (e.g. GAL2 and
GAL10) the transcripts are more than 50% reduced within
1 h of heat shock and almost completely absent after 2 h in
the rvb2-td strain (Fig. 2 and data not shown). The absence
of a lag time is similar to the rapid effect on transcripts seen upon
inactivation of RNA pol II core in rpb1-1 mutants (35), strongly suggesting that the decrease in GAL transcripts is
a primary effect of Rvb inactivation and not due to general effects of
cellular toxicity or growth arrest. Interestingly, several transcripts
were up-regulated to different degrees in the absence of either Rvb1p
or Rvb2p (AGA1, DAL5, and GDH3), which
contrasts with what is seen in temperature-sensitive mutants of RNA pol II or TFIID components.
rvb-td Mutants Escape from
Since the induction and maintenance of a G1 block by
pheromone depends on a strict transcriptional program, we reasoned that this program was deregulated by the loss of either Rvb1p or Rvb2p. The
secreted Bar1p protease is responsible for degradation of
In contrast to the increase in CLN1 transcript upon
temperature shift of rvb1-td or rvb2-td in the
presence of pheromone, similar temperature shift in the absence of
pheromone resulted in down-regulation of CLN1 transcript
(Fig. 2). Such opposite effects on the same promoter strongly suggest
Rvb1p and Rvb2p are not conventional positive or negative transcription
factors but have a more complex function.
Genome Wide Microarray Analysis Reveals That Transcription of Over
5% of Yeast Genes Is Affected in the Absence of Rvb1p or
Rvb2p--
To address how widespread the requirement of Rvb1p or Rvb2p
in yeast transcription is, a genome-wide analysis of gene expression was undertaken by hybridization of cRNA from control and test strains
of yeast to high density oligonucleotide arrays representing the whole
yeast genome (Affymetrix). Samples taken at the 0-h time point, before
temperature shift, were compared with samples taken 4 h after
shift to the non-permissive temperature. The 4-h time point was chosen
based on the response time seen by Northern blotting with
Gal-responsive genes (Fig. 2) to maximize the chance of detecting
changes in gene expression while avoiding potential secondary effects
due to the growth arrest. As seen in Fig. 1B neither
td strain had ceased growth at this time point. Fig.
4 presents the 326 genes whose expression
levels were most significantly altered 4 h after rvb
inactivation. In addition transcripts of 113 Ty elements or Ty long
terminal repeats were at least 2-fold up-regulated upon temperature
shift in either rvb1-td or rvb2-td strains. Thus
the transcription of over 5% of active yeast genes is responsive to
changes in Rvb1p or Rvb2p levels. The affected genes did not belong to
any particular class or cellular pathway as reflected in Table II which
shows genes whose transcription was changed more than 5-fold in the
rvb-td strains compared with wild type. Results from
Northern blotting (Fig. 2) agreed well with those obtained by
microarray hybridization.
The effects of inactivation are generally similar in the two
rvb-td strains. This is best demonstrated by the analysis in Fig. 5A where the
log2 ratios of the expression level of a gene in the
presence or absence of Rvb2p (y axis) is plotted against the
ratio in the presence or absence of Rvb1p (x axis). The
strong positive correlation of the two ratios suggests that the two
proteins predominantly work together. The correlation coefficient is
similar to that reported when comparing the effect of Swi1p with that of Snf2p, which act together in a complex (31).
As seen in both Northern blots (Fig. 2) and the microarray data (Fig. 4
and 5A), removal of Rvb2p has, for the most part, a stronger
negative effect on transcription than removal of Rvb1p. This raises the
possibility that Rvb1p and Rvb2p may also have functions independent of
each other. Similarly in the analysis of global gene transcription in
Fig. 5A, there are a few genes that are in the upper left of
lower right quadrants, demonstrating that at least for some promoters
Rvb1p and Rvb2p may act in opposite directions. This could be either
due to the proteins acting independently of each other or because of
differences in the kinetics of protein degradation in the two
rvb-td strains leading to different kinetics of
transcriptional changes in the two strains.
The ATP-binding Sites of Rvb1p/Rvb2p Are Essential for
Viability--
Lysine to aspartic acid mutations in the Walker A motif
of an ATP-binding protein are known to disrupt its ability to bind ATP,
whereas aspartic acid to glycine mutations of the Walker B motif do not
affect its ability to bind ATP but prevent its hydrolysis (36). These
mutations were introduced into the Walker A or B motifs of the yeast
RVB1 or RVB2 genes (Fig.
6A), and the mutant alleles
were shown to be incapable of supporting growth. Since both
RVB1 and RVB2 are essential, dissection of
tetrads with rvb1 Rvb1p and Rvb2p Associate in a Chromatin Remodeling
Complex--
The similar effects on gene expression by Rvb1p and Rvb2p
(Fig. 5A) led us to ask whether the proteins were associated
with each other. To this end a yeast strain with a chromosomal deletion of Rvb1p, complemented by a plasmid carrying Rvb1p with three C-terminal Myc tags, was constructed. Immunoprecipitation of Rvb1p-3Myc co-precipitated another yeast protein whose size corresponded to Rvb2p
(Fig. 7A). MALDI-TOF mass
spectrometry was used to identify unambiguously the band as Rvb2p. When
compared with mock immunoprecipitations from an isogenic strain
harboring a plasmid with untagged Rvb1p, the Rvb1p-3Myc
immunoprecipitates reveal a number of bands that are present in
substoichiometric ratios to the predominant Rvb1p-Rvb2p protein
complex. By analogy to the hexameric helicases, Rvb1p and Rvb2p could
form a hetero-hexamer or a hetero-dodecamer, and the presence of lesser
amounts of interacting proteins could therefore indicate that they form
the core of a larger complex. We attempted to identify these
interacting proteins by excising them from silver-stained gels and
performing MALDI-TOF mass spectrometry on their tryptic digests. Only
two bands could be unambiguously identified as Actin (Act1p) and the
actin-related protein Arp4 (Fig. 7A). Interestingly,
The immunoprecipitated Rvb1p-Rvb2p complex was tested for biochemical
activities consistent with the suggested role in chromatin remodeling.
The complex revealed weak ATPase activity that was slightly stimulated
by double- or single-stranded DNA and strongly stimulated by
chromatinized DNA (Fig. 7B). Helicase assays, utilizing labeled oligonucleotides hybridized to circular M13 DNA failed to
detect helicase activity in the complex (data not shown). Finally we
assayed the ability of the Rvp1p-Rvb2p complex to remodel chromatin using a linear array of 12 nucleosomes as template. The two central nucleosomes contained 5 Gal4-binding sites, the E4 promoter, and several unique sites recognized by restriction enzymes. These are
flanked on either side by 5 nucleosomes positioned via the 5 S
ribosomal gene nucleosome positioning sequence. For the remodeling assay, cleavage at the HhaI site within one of the central
nucleosomes was monitored. As shown in Fig. 7, C and
D, the Rvb complex strongly stimulates cleavage by
HhaI in the presence of ATP but not in its absence or in the
presence of the non-hydrolyzable analog AMP-PNP. No chromatin
remodeling activity was seen with equal amounts of the mock
immunoprecipitated extract. The level of stimulation by the Rvb complex
is similar to that seen with the Swi2/Snf1 complex in vitro
(data not shown). Taken together these results show that Rvb1p and
Rvb2p are key components of a complex that has highly specific
chromatin remodeling activity in vitro. As discussed below,
these results are in agreement with the recently published results of
Shen et al. (10).
Rvb1p/Rvb2p and Swi/Snf Act on Different Sets of
Promoters--
Given the general similarities between the effects that
Rvb proteins and the Swi/Snf chromatin remodeling complex have on transcription, we asked whether the two complexes act on the same or
different sets of promoters (Fig. 5B). We combined our data with data obtained from Sudarsanam et al. (31), which used a similar experimental design to study the effects of deletions of
swi1 or snf2. The ratio of transcription
levels in the presence or absence of Rvb2p (y axis) was
plotted against the corresponding ratio in the presence or absence of
Snf2p. The lack of correlation between the two ratios is in
marked contrast to what was seen with Rvb1p and Rvb2p (Fig.
5A) or with Swi1 and Snf2 (31). This analysis
suggests that the two chromatin remodeling complexes act independent of
each other.
Transcription in many Ty transposon elements was activated upon
temperature shift in the rvb1-td and rvb2-td
strains, indicating that Rvb1p and Rvb2p are required for repressing
these promoters. Most interesting, the opposite effect, a decrease in
Ty element transcription, is seen by disruption of components of the
SAGA acetyltransferase complex (38). Further comparison of our
data with the data obtained in that study for TAFII145 (a
component of TFIID), SPT3, SPT20, or GCN5 (components of SAGA)
did not reveal further correlation with the genes affected by
rvb1-td or rvb2-td (not shown). A notable distinction is the
predominantly negative effects on transcription caused by mutations in
TFIID or SAGA components compared with the relatively equal up-
and down-regulation seen in swi Rvb1p and Rvb2p have widespread and complex effects on gene
expression in yeast as shown by the transcriptional up- or
down-regulation of hundreds of genes in the rvb1-td and
rvb2-td mutants. As a consequence several essential cellular
pathways are interrupted which explains why mitotic growth
predominantly ceases in different stages of the cell cycle, depending
on conditions. Another example of the general effects caused by Rvb1p
and Rvb2p removal is a loss of response to mating factor The selective list of promoters that are affected by the loss of Rvb1p
or Rvb2p, together with the demonstrated in vitro chromatin remodeling activity, suggest that Rvb1p and Rvb2p affect gene expression in vivo as components of an ATP-driven chromatin
remodeling complex. The fact that many genes are up-regulated in the
absence of Rvb1p or Rvb2p makes it unlikely that these are structural elements in the general transcription machinery. A function of Rvbp as
a chromatin remodeling factor is consistent with previous reports that
a portion of mammalian Rvb1p (RuvBL1) is part of the RNA pol II
holoenzyme complex (14) or is associated with TATA-binding protein
(16). This result is also consistent with the suggestion that the
portion of the myc oncogene that interacts with Rvbp
is also involved in transcriptional repression, cell transformation,
and interaction with TRAAP (also known as PAF400), a 400-kDa protein
believed to act in a complex with chromatin-modifying factors (20).
The decreased levels of GAL2, GAL7, and
GAL10 mRNAs indicate that even after these genes have
been induced with galactose, removal of Rvb1p and Rvb2p results in a
rapid decrease in transcription. This suggests that Rvb1p and Rvb2p are
required after the establishment of a transcriptionally active complex
at these promoters. Similarly, the HSP26 gene was induced
following the temperature shift, but an induced state was not
maintained when Rvb2p was removed. These results imply that the
Rvb1p/Rvb2p-containing complex is required to maintain a state of
active transcription. It is interesting that the Swi/Snf complex has
also been reported to be required for continuous transcription from
some promoters (39), further highlighting the similarities between the
two chromatin remodeling complexes.
We also present a comparison of the activities of two chromatin
remodeling factors on a genome wide level. Although the Swi/Snf complex
and the Rvb1p/Rvb2p-containing complex regulate independent subsets of
promoters, transcription of some genes appears to be regulated by both
complexes (e.g. HO and GAL1). This
could signify that more than one chromatin remodeling factor could act
at a given promoter, either simultaneously or in succession.
Our results agree with the recently published finding that the
Snf2p homolog Ino80p can be isolated in a complex with Rvb1p and
Rvb2p and a number of other proteins, including Act1p and Arp4p (10).
The immunoprecipitated Ino80 complex increased the accessibility of a
chromatinized DNA template to restriction endonuclease. The authors
compared the Ino80p complex with an immunoprecipitate of FLAG-tagged
Rvb2p which yielded a complex with a composition that appears nearly
identical to our Rvb1p-3Myc complex, although we cannot unambiguously
identify the ~175-kDa Ino80 subunit in the Rvb1-3Myc
immunoprecipitated complex. Other differences between the Ino80 complex
used in their study and our Rvbp complex are reflected in the relative
abundance of the various bands and the presence of a few bands that are
specific to the Rvbp complex. The low amounts of Ino80 in the Rvbp
complex could explain the relatively weak ATPase activity and apparent
lack of helicase activity in our preparations compared with what is
reported for the Ino80 complex. However, the chromatin remodeling
activity in the Rvbp complex is remarkably strong, suggesting that
Rvb1p/Rvb2p may contain the active chromatin remodeling activity or
that some of the unidentified subunits of the complex may substitute
for Ino80p. Accordingly, ino80 null mutants are viable but
show decreased expression of a number of genes such as PHO5,
GAL1, CYC1, and ICL1 (9), whereas
rvb null mutations are lethal.
Another recent study of the 14-subunit TIP60 histone acetylase complex
from human cells revealed the presence of both Rvb orthologs in the
complex along with The precise function of the Rvb proteins in transcription in yeast and
human cells awaits further study, but the results presented here show
that both proteins are transcriptional regulators that form a complex
with chromatin remodeling activities. Further genetic and biochemical
analyses of Rvb1 and Rvb2 will help us understand the mechanisms by
which they act, as well as their roles in controlling transcription
in vivo and their possible contribution to the pathogenesis of human malignancies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin
(named pontin52 and reptin52) (18, 19) or c-Myc (20).
-catenin-mediated transactivation (19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
Genes most affected by removal of Rvb1p or Rvb2p
1)×(FC/|FC|). The corrected
fold change difference (FCdiff) was then calculated for a given gene by
subtracting the raw expression ratio of the wild type strain from that
of the rvb-td strain.
-Myc (9E10) antibody-coupled protein A-Sepharose
beads for 90 min at 4 °C, washed 1× with buffer A with 0.1 M KCl (A0.1), and incubated in 500 µl of A0.1 with
protease inhibitors and 1000 units of DNase I for 45 min at 22 °C.
Beads were then washed 3× with buffer A0.3, 2× with A0.5, once in
A0.1, and eluted by two sequential 30-min incubations at room
temperature in 100 µl of A0.1 with 5 mg/ml Myc peptide (EQKLISEEDL).
Proteins were subjected to SDS-polyacrylamide gel electrophoresis and
silver staining. Bands were excised, destained in-gel, digested with
sequencing grade trypsin (Promega), and analyzed by MALDI-TOF mass spectrometry.
-32P]ATP (33 Ci/mmol) without or with 250 ng of M13mp18 single-stranded DNA,
double-stranded plasmid DNA, or chromatinized linear double-stranded DNA. Rvb1p/Rvb2p complex (~50 ng) or equal volumes of mock
immunoprecipitate or buffer were added as indicated. Reactions were
quenched by adding 1 µl of 0.5 M EDTA on ice. 2.5 µl of
each reaction were separated by TLC on PEI-cellulose in 1 M
formic acid with 0.5 M LiCl, and visualized by
phosphorimaging. Chromatinized DNA (gift from J. Parvin) was prepared
with histones purified from chromatin pellet of BJABS cells by gradient dialysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conditional mutants generated by N-degron
fusions to Rvb1p and Rvb2p arrest growth under non-permissive
conditions. A, a schematic representation of the
integrated N-degron constructs and the galactose-inducible
Myc-UBR1. B, cultures of the strains, YUBR1 ( )
(W303-1a,
ubr
1::GAL-Myc-UBR1),
YR1UB1 (
) (YUBR1,
rvb1
::DHFR-HA-rvb1-td),
YR2UB1 (
) (YUBR1,
rvb2
::DHFR-HA-rvb2-td),
and the parent strain W303-1a (
) were grown to early log phase in YP
raffinose at 23 °C. Expression of UBR1 from the
GAL1 promoter was induced by adding galactose to 2%.
One-hour post-induction the cultures were spun down and transferred to
pre-warmed YP containing 2% each, galactose and raffinose. Growth was
monitored by measuring A600. Overexpression of
Ubr1p does not significantly affect the growth rate, but degradation of
either Rvb1p or Rvb2p leads to a gradual cessation of growth.
C, samples were taken before Gal induction and temperature
shift, and at 2-h intervals thereafter. Total protein was extracted as
described under "Experimental Procedures," and equal amounts were
loaded onto SDS-polyacrylamide gel electrophoresis for Western
blotting. Monoclonal
-Myc (9E10) was used to visualize the induction
of Myc-Ubr1p, and 12CA5
-HA antibody was used to monitor the
degradation of the HA tagged rvb-td proteins. Both Rvb-td
proteins are effectively removed within 2 h. D,
rvb-td mutants do not arrest at a single point in the cell
cycle, but cease growth gradually depending on growth conditions. Rapid
degradation of Rvb1p or Rvb2p with concurrent Ubr1p overexpression in
rich medium leads to accumulation in G1, whereas strains
with endogenous Ubr1 levels grown on minimal medium accumulate with 2C
DNA content.
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Fig. 2.
RVB1 and RVB2 regulate the expression of a
number of genes from different cellular pathways. Yeast were grown
as described in Fig. 1, and samples were taken before addition of
galactose ( 1 h) and before and after temperature shift. Total RNA was
isolated and mRNA levels probed by Northern analysis. The mRNA
levels of several genes are strongly affected as discussed in the
text.
-Factor-induced Cell Cycle Block Due
to Defects in Transcriptional Regulation--
Experiments designed to
examine whether the Rvb1p and Rvb2p were required in S-phase provided
further evidence that the proteins were involved in transcriptional
regulation. The
-factor mating pheromone was used to arrest cells in
G1 with the aim of releasing the cells into S-phase after
inactivation of the Rvbp by temperature shift (Fig.
3). Ubr1p was concurrently induced by
addition of galactose. At 23 °C all three strains remained arrested
in G1 by
-factor, but in contrast to the YUBR1 strain
(RVB1 and RVB2), a significant proportion of
cells in both rvb1-td or rvb2-td strains escaped
from
-factor block when shifted to 38 °C (Fig. 3A).
Subsequent removal of
-factor did not cause further cell cycle
progression in the rvb1-td or rvb2-td strains at
the non-permissive temperature (data not shown) indicating that the
promotion of cell cycle progression was transient. Loss of either Rvb1p
or Rvb2p eventually inhibited entry into S-phase and completion
of mitosis. The loss of
-factor arrest did not adversely affect
mating, since both strains mated as well as wild type, at both 23 and
37 °C in plate mating assays.
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Fig. 3.
rvb-td mutants
escape prematurely from -factor block.
A, strains were grown at 23 °C in minimal medium
containing 2% raffinose, to an A600 of 0.1.
-Factor was added to a final concentration of 6 µM and
incubated for 1 h; galactose was then added to 2% and incubation
continued at 23 °C for 1 h. Additional 6 µM of
-factor was added and incubation continued at room temperature for
1 h by which time shmoos exceeded 80%. The cultures were
then split and incubated at either 23 or 38 °C for an additional
hour. Samples of the cultures were fixed in 80% ethanol, stained with
propidium iodide, and analyzed by FACS. B, escape from
-factor block is reflected by changes in the expression of
-factor-regulated genes. After deletion of BAR1 strains
were grown as above. 3 h prior to temperature shift galactose was
added to 2%. 1 h later samples were taken (
2 h) for RNA
isolation and FACS analysis, and
-factor was added to a final
concentration of 0.1 µM. At the 0-h time point samples
were taken, the cultures were shifted to the non-permissive temperature
of 38 °C, and
-factor was replenished. Changes in expression were
followed for 2 h after temperature shift. The figure shows a
reduction in transcription of FAR1 and an increase in
CLN1 levels that reflects the transient release of
-factor block.
-factor.
Deletion of BAR1 in the rvb-td strains delayed
the escape from
-factor block and reduced the number of cells
entering S-phase, probably due to a higher effective concentration of
-factor leading to a stronger pheromone response, but did not
abolish the effect (Fig. 3B). Since
-factor-mediated
arrest of the cell cycle depends both on the induction of the Cdc28p
inhibitor FAR1 and repression of G1 cyclins
CLN1 and CLN2, we monitored the responsiveness of these promoters to loss of Rvb1p or Rvb2p. Northern analysis of the
-factor-treated bar1
, rvb1-td, or
rvb2-td strains revealed that FAR1 is
down-regulated by Rvb removal, whereas the G1 cyclin CLN1 is up-regulated (Fig. 3B). We conclude that
the escape from pheromone-induced block is due to additive changes in
the pheromone-specific transcription program, which are reflected by
the ratio of cell cycle inhibitor (Far1p) to cell cycle activator
(Cln1p) being effectively decreased upon loss of Rvb.
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Fig. 4.
Whole genome microarrays (Affymetrix)
were used to assess the genome-wide effects of Rvb inactivation. 8 µg of total RNA were used as substrate as described under
"Experimental Procedures." Samples of the three strains, YUBR1,
YR1UB1, and YR2UB1, taken before shift to the non-permissive
temperature were compared with samples taken 4 h post-shift. Only
a subset of ORFs that passed a series of stringent criteria (sufficient
abundance and low variation at the permissive temperature) were
analyzed as described under "Experimental Procedures."
Mitochondrial genes, tRNA genes, and Ty elements were excluded from the
analysis. Average Difference values calculated by the GeneChip software
(version 3.3) give an estimate of the relative abundance of each
transcript. A log2 ratio was calculated for each ORF for
25/38 °C. The figure shows the log2 ratios for the 326 genes that were deemed to have a significantly different response to
temperature shift in the wild type and mutant strains. The data were
clustered with the Cluster software (version 2.11) and visualized by
TreeView (version 1.50, Stanford University). Green
indicates a decrease, and red indicates an increase in
mRNA abundance. The changes for each gene have been normalized, so
the colors indicate relative changes in gene expression in the three
strains but are not absolute values.
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Fig. 5.
Rvb1p and Rvb2p are required for
transcription of the same genes in vivo but regulate a
set of genes different from Swi/Snf. A, the scatter
plot portrays the difference in expression of genes in YR1UB1
(rvb1-td) and YR2UB1 (rvb2-td) relative to the
wild type (YUBR1) strain. The transcription ratios in mutant
versus wild type, after 4 h at the non-permissive
temperature, were transformed to linearity by applying a base 2 logarithm such that a 2-fold increase in gene expression in a mutant
would equal 1, and 1 corresponded to a 2-fold decrease. A strong
correlation (R2 = 0.52) similar to what has been
reported for swi1 and snf2
(R2 = 0.54) (31) exists between changes in the
two strains. B, the changes caused by degradation of Rvb2p
versus the changes in gene expression in the absence of
Snf2 show a lack of correlation between the genes affected. For
details see under "Experimental Procedures." Note that points can
be found far off center in all four quadrants of the graph, reflecting
that transcription can be strongly affected in both the same and
opposite directions by the two complexes.
/RVB2 or
rvb2
/RVB2 results in two viable spores
(RVB+) and two non-viable spores
(rvb
). Transformation of the diploid yeast with a vector
expressing the respective wild type Rvb protein but not with empty
vector rescued the lethality of rvb
(Fig. 6B).
Plasmids expressing the respective rvb with mutations in the
Walker A or B motifs also failed to rescue the lethality of rvb
. To eliminate the possibility that the ATP-binding
motifs were required for a function of Rvb proteins unique to meiosis or sporulation, haploid yeast were constructed with chromosomal deletions of either rvb1 or rvb2, supported by
the wild type allele on an episome that also carried the
URA3 gene. Neither strain grew on 5-FOA that selects against
URA3. Transformation with a second plasmid carrying the
respective wild type RVB, however, allowed growth on 5-FOA
(Fig. 6C). In contrast, transformation with an empty vector
or a vector expressing the respective rvb with mutations in
the Walker A or Walker B motifs did not support growth on 5-FOA.
Therefore, ATP binding and hydrolysis by Rvb1p and Rvb2p are required
for their essential functions in normal mitotic cell growth and
division.
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Fig. 6.
ATP binding and hydrolysis by Rvb1p and Rvb2p
are independently essential. A, schematic
representation of the Walker A and B motifs in Rvb1p and -2p. The
mutations indicated were introduced into both motifs in both proteins
by site-directed mutagenesis. B, Walker motifs in Rvb1p and
2p are individually essential for viability. None of the mutant alleles
could substitute for wild type RVB1 or -2. The
figure shows tetrad dissection of diploid strains, heterozygous for
rvb1 ::HIS3 (YRVB1D) or
rvb2
::HIS3 (YRVB2D), transformed
with either an empty pRS315 or constructs expressing the respective
wild type RVB or rvb mutants from the endogenous
promoters. C, haploid strains, homozygous for the
rvb deletions complemented by the wild type alleles on
URA3 expressing plasmids, were used to verify the results.
The Walker A and B mutants did not support growth on medium containing
5-FOA.
-actin is found in the mammalian BAF chromatin remodeling complex (37), and both Act1p and Arp4p are components of the yeast NuA4 histone
acetyltransferase complex (22).
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Fig. 7.
Yeast Rvb1p and Rvb2p associate in a complex
with ATPase and chromatin remodeling activities. A,
immunoprecipitation of Rvb1p and Rvb2p. Indicated bands
(squares) were excised from the silver-stained gel,
digested, and analyzed by MALDI-TOF mass spectrometry. MS-Fit was used
to identify Rvb1p from 14 unique peptides (Mr = 1044.55, 1215.67, 1248.63, 1260.65, 1333.69, 1410.81, 1416.75, 1487.70, 1651.93, 1904.04, 1915.96, 1922.00, 1988.06, and 2234.15), Rvb2p from
10 unique peptides (Mr = 1035.53, 1167.63, 1301.66, 1331.72, 1356.57, 1489.75, 1724.87, 1918.03, 2046.12, and
2241.17), Act1p from 4 unique peptides (Mr = 975.44, 1117.50, 1197.70, and 1789.88), and Arp4p from 6 peptides
(Mr = 892.42, 914.41, 1108.50, 1114.50, 1139.63, and 1245.67). B, the Rvb complex has chromatin-stimulated
ATPase activity. Reactions were carried out in the absence or presence
of 250 ng of either chromatinized linear double-stranded DNA, circular
double-stranded DNA, or single-stranded DNA, followed by separation of
products by TLC. C, chromatin remodeling assay.
PhosphorImager scan of an assay monitoring the digestion of a
12-nucleosome DNA array by HhaI in the absence or presence
of ATP. The reactions contained end-labeled array template at a
concentration of ~1 nM in mononucleosomes and ~30 ng of
Rvb1p/Rvb2p complex. D, quantitation of PhosphorImager scans
shows strong ATP-dependent chromatin remodeling activity in
the Rvb complex. , Rvb complex + ATP;
, Rvb complex
ATP;
, Rvb complex + AMP-PNP; and
, mock IP + ATP. IP,
immunoprecipitation.
/snf
and
rvb1-td/rvb2-td.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which
causes cells to progress to S-phase, although they are unable to
complete the cell cycle.
-actin and the actin-related protein BAF53 (22).
These results suggest that the mammalian Rvb1p/Rvb2p orthologs may have
an alternative chromatin remodeling function through interaction with
histone acetylase. The Esa1p acetylase of yeast is a homolog of TIP60
and is associated with Tra1p, Act1p, and Arp4p in the NuA4 complex.
Tra1p is a homolog of PAF400, which is a component of the TIP60
complex. Similarly Act1 and Arp4p are homologs of
-actin and BAF53,
respectively, implying that NuA4 and TIP60 have similar functions. We
could not, however, detect the presence of HA-tagged Esa1p in Rvb1p immunoprecipitates,2 and
others have failed to detect Rvb subunits in yeast NuA4 (22). This
suggests differences in the structural composition of Rvb1p-Rvb2p complex in yeast and mammalian cells.
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ACKNOWLEDGEMENTS |
---|
We thank James Wohlschlegel for assistance with MALDI-TOF spectrometry; Neelima Mondal and Jeffrey Parvin for chromatinized plasmid DNA and critical discussions, and Nicole Francis for providing the assembled array template.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Swiss National Fund for Science (to Z. O. J.) and National Institutes of Health Grant CA60499 (to A. D.).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.
§ Supported by Postdoctoral Fellowship DAMD17-00-1-0166 from the United States Army Medical Research Acquisition Activity.
Supported by a predoctoral fellowship from Howard Hughes
Medical Institutes.
§§ To whom correspondence should be addressed: Dept. of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. E-mail: adutta@rics.bwh.harvard.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M011523200
2 Z. O. Jónsson, R. Auty, and A. Dutta, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
ORFs, open reading
frames;
pol, polymerase;
5-FOA, 5'-fluoro-orotic acid;
MALDI-TOF, matrix-assisted laser desorption ionization/time of flight;
FACS, fluorescence-activated cell sorter;
Ub, ubiquitin;
HA, hemagglutinin;
AMP-PNP, adenosine 5'-(,
-imino)triphosphate;
SAGA, Spt-Ada-Gen5-acetyltransferase.
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