From the
Following protein synthesis inhibition in cycloheximide
growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis
decrease with apparent half-times of about 20 and 10 min, respectively.
This effect is mimicked by extracts of treated cells, and the
impairment of tRNA gene transcription activity that is observed in
vitro parallels the in vivo inactivation of RNA
polymerase III transcription. As revealed by experiments in which
partially purified class III transcription factors were singly added to
extracts of treated cells, only the activity of the multiprotein
transcription factor TFIIIB is severely impaired after 3 h of
cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by
a combination of the TATA box-binding protein (TBP), the 70-kDa
component TFIIIB70, plus a partially purified fraction known as B" have
shown that the latter two components are both necessary and sufficient
to restore control levels of transcription. Their activity, but not TBP
activity, is considerably reduced in extracts of treated cells.
TFIIIB70 and a component of fraction B" thus appear to be the selective
targets of the down-regulation of polymerase III transcription that is
brought about by cycloheximide. A substantial depletion of the TFIIIB70
polypeptide was detected by Western immunoblot analysis of extracts
derived from cycloheximide growth-arrested cells, indicating that the
inactivation of this TFIIIB component results primarily from its
enhanced destabilization under conditions of protein synthesis
inhibition.
The intracellular levels of the RNA components of the eukaryotic
translational apparatus are regulated in response to growth conditions,
and nuclear run-on analyses have shown that this regulation occurs in
large part at the transcriptional level (1, 2 and references therein).
Both in the yeast Saccharomyces cerevisiae and in higher
eukaryotes, rRNA synthesis by RNA polymerase (pol)
5 S RNA and
tRNA synthesis by RNA pol III is similarly reduced following a
cycloheximide-induced growth arrest, under conditions in which RNA pol
II transcription is only slightly affected
(1) . A negative
biosynthetic response to cycloheximide administration has been observed
both in mammalian cells
(1) and, albeit to variable extents,
also in yeast
(3, 4, 6) . Soluble extracts
derived from mouse cells, either treated with cycloheximide or grown
into stationary phase, mimic the in vivo response and
down-regulation of RNA pol III transcription in these cells has been
shown to result mainly from a reduced activity of the general class III
transcription factor TFIIIB (7). The same transcription factor has been
found to be involved in the down-regulation of class III gene
transcription that occurs in differentiating mouse F9 cells and in frog
oocytes undergoing mitosis (8, 9). These findings thus raise the
possibility that TFIIIB may represent a general target for the negative
modulation of RNA pol III transcription in eukaryotic cells.
In view
of the recent discovery that TFIIIB from various organisms is a
multiprotein transcription factor (reviewed in Ref. 10), a further
question of interest concerns the identification of the TFIIIB
component(s) that is either modified or destabilized under protein
synthesis inhibitory conditions. The highest resolution of the TFIIIB
complex has been achieved, so far, for the transcription factor from
yeast. In this system, two distinct polypeptides, TFIIIB70 (also known
as BRF, TDS4, or PCF4) and the TATA box-binding protein (TBP), plus a
fraction (B") that is enriched in a 90-kDa polypeptide have been shown
to be necessary and sufficient to reconstitute TFIIIB activity
(11-17). A fourth component, TFIIIE, which is also required to
reconstitute in vitro class III transcription in the presence
of a partially purified TFIIIB fraction, has recently been resolved in
our laboratory and found to co-fractionate with highly purified B"
fractions
(18) . Notwithstanding the availability of cloned or
highly resolved class III transcription components, tRNA and 5 S RNA
synthesis have not yet been analyzed in crude extracts of
cycloheximide-treated yeast cells, and nothing is known about the
molecular events underlying negative modulation of RNA pol III
transcription under conditions of protein synthesis inhibition.
We
are interested in defining the targets and the mechanisms of
inactivation of yeast pol III transcription in response to
cycloheximide-induced protein synthesis inhibition. We were also
intrigued by the possibility that TFIIIE might be involved in such
response. The effect of cycloheximide upon tRNA and 5 S RNA synthesis
has thus been initially re-examined under in vivo pulse
labeling conditions. We have found that the rates of tRNA and 5 S RNA
synthesis decrease with apparent half-times of 20 and 10 min,
respectively, in cycloheximide-treated cells and that in vitro transcription of a tRNA gene by extracts of treated cells
faithfully mimics the in vivo response. Only TFIIIB activity
is severely impaired in these extracts, and a selective loss of
TFIIIB70 and B" activities, but not TBP activity, fully accounts for
the observed inactivation.
While confirming the results of the add back experiment as
to the primary involvement of TFIIIB in the transcriptional response to
cycloheximide, the latter data show that the reduced activity of this
component parallels the decrease of total transcription capacity
measured in unfractionated extracts. tRNA and 5 S rRNA transcription by
control extracts is limited by different activities, either TFIIIB or
TFIIIA, yet no transcriptional component other than TFIIIB has been
found to vary between control extracts and extracts of treated cells.
This selectivity of inactivation also indicates that the effect of
cycloheximide does not merely result from a generalized alteration of
the nuclear extractability of class III transcription components.
Indeed, practically identical results as to the impaired
transcriptional capacity of extracts derived from treated cells were
obtained when varying the ammonium sulfate concentration used for
nuclei extraction from 0.5 to 0.9 M (data not shown).
The observed
inactivation of TFIIIB70 and B" may result from their selective
destabilization under conditions of protein synthesis inhibition.
Alternatively, it may derive from a cycloheximide-induced modification
of these components, leading to a substantial impairment of their
transcription factor activity. To begin to distinguish between these
two possibilities, we used anti-TFIIIB70 antibodies for a Western
immunoblot analysis of control and cycloheximide extracts. TBP was used
as an internal reference for this analysis and, in agreement with the
results of previous titration experiments, it was found not to vary
between the two types of extract (Fig. 6A). In contrast,
under identical experimental conditions, a substantial depletion of the
TFIIIB70 polypeptide was detected in extracts derived from
cycloheximide-treated cells (Fig. 6B).
We have found that yeast pol III transcription, though
appreciably less sensitive to cycloheximide-induced inactivation than
pol I transcription, is similarly down-regulated under conditions of
protein synthesis inhibition. The results of our in vivo pulse
labeling analysis are in keeping with the previously reported
differential sensitivity of these two transcription systems to
cycloheximide
(1, 3, 4, 6) . In
particular, tRNA transcription declines under in vivo conditions with an apparent half-time about 4-fold longer than the
corresponding half-time for pol I transcription. In turn, pol III
transcription has been found to decline 4.5-9-fold more rapidly
than pol II transcription, indicating that the effect of cycloheximide
is not due to a generalized destabilization of the RNA biosynthetic
machinery. Furthermore, the steady state levels of tRNAs and 5 S RNA do
not change following cycloheximide addition, as if their turnover rates
were considerably slowed-down under growth-arrested conditions. A
similar divergence between the rate of synthesis and the steady state
level of various class III transcripts has been observed in other
systems, and the regulation of tRNA half-life has previously been
reported (10, 30, and references therein). By enabling cells to
maintain a level of essential pol III products that is sufficient to
support basal cellular functions, this mechanism would explain the
complete reversibility of the cycloheximide-induced growth arrest as
observed under our experimental conditions.
Extracts of
cycloheximide-treated cells programmed with a cloned tRNA gene mimic
the in vivo response to protein synthesis inhibition. The same
extracts did not faithfully reproduce the earlier decline of 5 S RNA
synthesis that was observed under in vivo labeling conditions.
Since TFIIIA has the ability to bind both the 5 S gene and the 5 S RNA
transcript
(10) , this discordance of behavior may result, for
example, from an in vivo shortage of transcriptionally
competent TFIIIA, due to a reduced availability of the ribosomal 5 S
RNA binding protein YL3 in cycloheximide growth-arrested
cells
(20, 31) . Alternatively, it may reflect a
predominant chromatin effect under in vivo conditions, arising
from the peculiar chromosomal location of yeast 5 S rRNA genes within
the ``spacer'' regions interposed between individual rDNA
transcription units
(32) . Our present in vitro data do
not allow distinguishing between these two possibilities, and the only
transcription component that has been found to be affected by
cycloheximide treatment is TFIIIB. However, the observation that the
level of TFIIIA activity is initially lower than the level of TFIIIB
(), at least accounts for the delayed involvement of TFIIIB
in the in vitro detected inactivation of 5 S RNA
transcription.
Similar to what has previously been found in the case
of growth-restricted mouse cells
(7) , TFIIIB is the only class
III transcription component whose activity is severely impaired in
extracts of cycloheximide-treated yeast cells. The observation that an
identical class III component mediates a negative transcriptional
response in so diverse organisms supports the view that TFIIIB may
indeed be a general target for eukaryotic pol III down-regulation. The
selective impairment of TFIIIB activity under protein synthesis
inhibitory conditions is consistent with the key role that this
component is known to play in transcription initiation
(33) . In
various mammalian systems, TFIIIC has previously been shown to be
implicated in both positive and negative pol III regulatory events
(reviewed in Ref. 10). At variance with TFIIIC, however, TFIIIB
inactivation has the potential ability not only to prevent
transcription complex formation, but also to abolish initiation from
preassembled transcription complexes. Particularly interesting, in this
regard, is the involvement of TFIIIB in controlling the differential
expression of constitutive and silk gland-specific tRNA
TFIIIB has recently been
shown also to mediate an up-regulation event that is induced by phorbol
esters in Drosophila Schneider S2 cells
(35) . In this
case, stimulation of pol III transcription correlates with an increased
level of TBP, but no other TFIIIB component has been examined.
Based
on the high resolution that has recently been achieved for the yeast
TFIIIB complex (reviewed in Ref. 29), our studies represent the first
analysis of the negative modulation of individual components of a
multiprotein class III transcription complex, under conditions that
determine an in vivo detectable inactivation of transcription.
Two TFIIIB components, TFIIIB70 and B", are the specific targets of the
pol III transcriptional response to protein synthesis inhibition. We do
not yet know whether a qualitative or a quantitative variation is
responsible for the observed inactivation of B", but the latter
possibility appears to hold in the case of TFIIIB70. In fact, Western
immunoblot analysis has revealed a substantial depletion of the
TFIIIB70 polypeptide in extracts of cycloheximide growth-arrested
cells. In the same extracts, TBP remains essentially constant, and it
is capable of interacting with exogenously added TFIIIB70 and B" to
reconstitute control levels of in vitro transcription
activity. Since TBP is known to direct transcription by all three
eukaryotic RNA
polymerases
(10, 14, 15, 24, 36) ,
its marked stability under protein synthesis inhibitory conditions is
by itself quite remarkable. It is consistent both with the differential
cycloheximide sensitivity of the three transcriptional machineries
evidenced by our in vivo pulse labeling analysis, and with the
fact that a component other than TBP has been found to be involved in
the down-regulation of mouse RNA pol I transcription under protein
synthesis inhibitory conditions
(5) .
The inactivation of
TFIIIB70 appears to be even more relevant if one considers that this
particular TFIIIB component has previously been shown to limit pol III
transcription in intact yeast cells
(13) . In addition, both
genetic and biochemical data have demonstrated the existence of a
fairly stable interaction between TFIIIB70 and
TBP
(11, 12, 37, 38) . Interestingly, a
computer sequence analysis of these two TFIIIB components detects the
presence of a degradation promoting signal (``PEST,'' 39) in
TFIIIB70, but not in TBP.
We are grateful to Gian Luigi Rossi for support and
encouragement. We thank George Kassavetis for detailed instructions on
the preparation of fraction B"; Howard Riezman and Steve Buratowski for
yeast strains; Tony Weil, Jean Marc Egly, and Steve Hahn for plasmids;
and Diane Hawley and Frank Pugh for antibodies. The assistance of
Silvia Pizzi and Andrea Ballabeni in the preparation of rTBP and
fraction B" is also gratefully acknowledged.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
I is known to be down-regulated by a number of treatments,
including growth arrest induced by protein synthesis inhibitors,
starvation for an essential nutrient, and growth into stationary phase
(1-4 and references therein). Interestingly, extracts prepared
from growth-restricted mammalian cells have an impaired capacity for
RNA pol I transcription due to depletion or modification of an
essential transcription factor
(1, 5) .
Cell Growth and Pulse Labeling Analysis
The
diploid, protease-deficient S. cerevisiae strain RH804C775D
( lys2 leu2 pep4-3/a leu2 trp1::URA3 gal2 pep 4-3, a
gift of H. Riezman, University of Basel, Switzerland) was used
throughout this work. Cells were grown at 30 °C in
phosphate-depleted YEPD medium (19). Cycloheximide (Fluka, 10
µg/ml) was added to yeast cultures grown to a density of 10
cells/ml; mock-treated aliquots of the same cultures were used as
controls. For pulse labeling experiments
(20) , aliquots of
either cycloheximide-treated or control cultures (a total of 10
cells each) were collected by centrifugation at various times
after cycloheximide addition and resuspended in 0.5 ml of the
corresponding growth medium. Cells were then incubated for 10 min in
the presence of radioactive [
P]orthophosphate
(20-30 µCi, 8500 Ci/mmol), washed twice with ice-cold growth
medium, and frozen in liquid nitrogen in preparation for RNA
extraction. To determine the time course of radioactive phosphate
accumulation by either control or cycloheximide-treated cells, samples
were taken at 10-min intervals from a scaled-up incubation mixture,
washed twice with ice-cold growth medium, and counted.
RNA Isolation and Analysis
Total RNA was extracted
with the hot phenol procedure
(21) . After the addition of
ammonium acetate (3.75 M, final concentration) and ethanol
(2.5 volumes), samples were kept on dry ice for 20 min, and RNA was
collected by centrifuging at 12,000 g for 30 min. The
pellets were then washed once with 70% ethanol, dissolved in diethyl
pyrocarbonate-treated water, and used for either spectrophotometric,
electrophoretic, or chromatographic analyses. RNA samples (15 µg of
total RNA) were fractionated on 6% polyacrylamide, 7 M urea
gels. The gels were first stained with ethidium bromide to monitor
total amounts of the different RNA species; they were subsequently
exposed overnight for autoradiography, followed by the quantitation of
individual RNA bands by Cerenkov counting. Poly(A
) RNA
was resolved by chromatography on oligo(dT)-cellulose (Boehringer)
following the manufacturer's instructions.
Plasmid DNAs
Plasmids pUCLeu3, containing a yeast
tRNA gene, pPM16, containing a yeast tRNA
gene, and pUC9-5S, containing the yeast 5 S rRNA gene (from
P. A. Weil, Vanderbilt University School of Medicine, Nashville, TN),
were used as templates for in vitro transcription reactions.
Plasmids pET3b(rTFIIDY)
(22) and pSH360
(12) were used to
express yeast TBP and TFIIIB70 in Escherichia coli and were
kindly provided by J. M. Egly (INSERM, Strasbourg, France) and S. Hahn
(Fred Hutchinson Cancer Research Center, Seattle, WA). Plasmid pETIIIA
(23) was used for the expression of yeast TFIIIA.
Extract and Transcription Factor
Preparations
Nuclear extracts were prepared from either control
or cycloheximide-treated cells grown to a final density of 1-1.5
10
cells/ml (18). Ammonium sulfate concentrations
ranging from 0.5 to 0.9 M were tested for their nuclear
extraction ability. The resulting extracts were found to exhibit
essentially identical in vitro transcriptional activities (see
below), and a final salt concentration of 0.65 M was routinely
used for nuclear extract preparation. Transcription factor fractions
(TFIIIB, TFIIIC, and TFIIIE) and polymerase were partially purified
from control extracts as described previously
(18) . Fraction B"
was extracted from chromatin pellets and partially purified on BioRex
70 (Bio-Rad) according to Kassavetis et al.(17) .
Recombinant yeast TFIIIA was expressed in E. coli and purified
to near homogeneity as described previously
(23) . Recombinant
yeast TBP and TFIIIB70 were purified from E. coli-soluble
lysates using modifications of previously described
procedures
(12, 22) . After an initial fractionation on
DEAE-Sephacel and heparin-Ultrogel
(22) , rTBP was purified to
near homogeneity by gel filtration on Ultrogel AcA-54 (IBF, 2
150 cm column) equilibrated, and eluted with 25 mM Tris-HCl
(pH 8.0), 0.2 mM EDTA and 0.5 M KCl at a flow rate of
0.35 ml/min. Histidine-tagged rTFIIIB70 was purified to about 50%
homogeneity by chromatography on Ni
-NTA agarose
(Quiagen) under native conditions, using 250 mM imidazole
dissolved in 50 mM sodium phosphate (pH 8.0), 0.3 M
NaCl, 1.5 mM
-mercaptoethanol, plus protease inhibitors
(1 µM each of leupeptin and pepstatin, 0.1 mM
phenylmethylsulfonyl fluoride, and 0.5 mM benzamidine) as
eluent. The in vitro complementation of extracts derived from
TBP mutant yeast cells (YSB67 strain, 24) and the transcriptional
stimulation of extracts prepared from wild type haploid cells
(12) were initially used to assay for rTBP and rTFIIIB70,
respectively. Protein concentrations were determined by the method of
Bradford
(25) , using bovine serum albumin as standard.
Transcription Assays
Standard transcription assay
mixtures contained 80 fmol of template DNA, 15 mM Tris-HCl (pH
7.9), 120 mM KCl, 6 mM MgCl, 10% (v/v)
glycerol, 600 µM each of ATP, GTP, and CTP, 25
µM UTP, and 5-10 µCi of
[
-
P]UTP (800 Ci/mmol). Transcription assays
were carried out in a final volume of 20 or 37 µl, in the case of
either crude extracts or variously reconstituted transcription systems.
The amounts of extracts, fractions, and purified components employed
for each experiment are indicated in the figure legends. Multiple round
transcription reactions were incubated for 20-30 min at room
temperature. The concentration of transcriptionally active components
in either partially purified transcription factor fractions and in
recombinant transcription factor preparations was determined by single
round transcription assays as described
previously
(18, 26) . Reactions were terminated by the
addition of 10 µl of a solution containing 4% (v/v) SDS and 5 mg/ml
of carrier RNA (Sigma, type R-6625 T6). After phenol-chloroform
extraction and ethanol precipitation, RNA products were analyzed by
electrophoresis on 10% polyacrylamide, 8 M urea gels as
described previously
(18) . Transcription products were
visualized by autoradiography of wet gels and were quantitated by
measuring Cerenkov radiation in gel slices.
Western Blot Analysis
Equal amounts of total
protein from either extracts of cycloheximide-treated cells or from the
corresponding control extracts were electrophoresed, along with
molecular weight markers, on polyacrylamide-SDS gels
(27) .
Electrotransfer to nitrocellulose membranes (Schleicher & Schuell,
BA-85) and immunoblotting were carried out as described by Harlow and
Lane
(28) . TFIIIB70 was detected with an antiserum raised
against the partially purified recombinant yeast protein (kindly
provided by F. Pugh, Pennsylvania State University, University Park,
PA). Polyclonal antibodies against yeast TBP were a gift of D. Hawley,
University of Oregon, Eugene, OR. Horseradish peroxidase-conjugated
anti-rabbit Ig antibodies and enhanced chemiluminescence reagents
(Amersham) were used according to the manufacturer's instructions
to detect antigene-bound primary antibodies.
Effect of Cycloheximide upon tRNA and 5 S RNA Synthesis
in Yeast Cells
Before analyzing RNA pol III transcription in
cell-free extracts of cycloheximide-treated cells, we re-examined the
effect of this protein synthesis inhibitor on tRNA and 5 S RNA
synthesis under in vivo pulse-labeling conditions. To this
end, exponentially growing yeast cells were treated for various periods
of time with cycloheximide, at a concentration (10 µg/ml) which
reversibly blocked both protein synthesis and cell growth within a few
minutes from its addition. Following pulse labeling of control and
cycloheximide-treated cells with
[P]orthophosphate (10 min), radioactively
labeled RNA species were resolved by either polyacrylamide gel
electrophoresis or oligo(dT)-cellulose fractionation. As shown in
Fig. 1
(panels A and B), radioactive phosphate
incorporation into 5 S RNA and tRNAs decreased with apparent half-times
of about 10 and 20 min upon cycloheximide administration, and it
leveled off at 10 and 15% of the corresponding control values after
about 2 h. Under identical experimental conditions, labeling of total
poly(A
) and poly(A
) RNA, as
estimated from the incorporation of radioactive phosphate into RNA
species that either bound to or flowed through oligo(dT)-cellulose,
diminished with apparent half-times of 90 and 5 min, respectively (data
not shown). Consistent with the fast decline of
poly(A
) RNA synthesis, labeling of the processed 5.8
S rRNA species was also rapidly inhibited in growth-arrested cells
(Fig. 1A). The observation that the in vivo pulse labeling of distinct classes of RNAs decreases in
growth-arrested cells with considerably different kinetics, together
with the fact that radioactive phosphate accumulation proceeded
linearly for at least 30 min after cycloheximide addition (data not
shown), indicates that the effect of cycloheximide does not merely
result from a reduced specific activity of the intracellular nucleotide
pool. Moreover, no decrease in the steady state amounts of either 5 S
RNA or tRNAs, as estimated from the intensity of ethidium bromide
staining of the same gel used for pulse labeling analysis, was detected
after 3 h of cycloheximide treatment (Fig. 1C). The
effect of cycloheximide on 5 S RNA and tRNA accumulation thus occurs
primarily at the level of transcription.
Figure 1:
Analysis of newly synthesized and
steady state levels of tRNAs and 5 S RNA in cycloheximide
growth-arrested yeast cells. A, in vivo pulse
labeling analysis. Cycloheximide (Cy, 10 µg/ml) was added
to exponentially growing yeast cells at time 0. At the indicated times,
aliquots of the culture were removed and pulse labeled with
[P]orthophosphate (10 min). Total RNA was
extracted and fractionated on 6% polyacrylamide, 7 M urea gels
(see ``Experimental Procedures'' for details). The
autoradiographic result of a representative pulse labeling experiment
is shown; the migration positions of 5.8 S rRNA, 5 S rRNA, and tRNAs
are indicated. B, quantitation of radioactive, newly
synthesized tRNA (
) and 5 S RNA (
) transcripts at different
times after cycloheximide addition. Gel slices corresponding to tRNA, 5
S rRNA, and background areas were excised and, after background
deduction, radioactivity associated to each RNA species was determined;
data are the average (± S.D.) of three independent experiments.
C, ethidium bromide staining of the same gel shown in
panel A.
Extracts of Cycloheximide Growth-arrested Cells Have an
Impaired Capacity for RNA Polymerase III Transcription
To begin
to elucidate the molecular mechanisms responsible for the
down-regulation of pol III transcription in cycloheximide
growth-arrested cells, extracts were prepared from cell cultures that
had been exposed to the inhibitor for either 1.5 or 3 h under the above
described experimental conditions, as well as from parallel cultures of
mock-treated cells. Extracts were prepared from the same number of
cells and contained nearly identical amounts of total protein. Parallel
transcription assays were carried out with increasing amounts of either
extract, using the yeast tRNA gene and the 5 S rRNA gene
as templates. As shown in Fig. 2A, extracts of cells
harvested at different times from cycloheximide addition became
increasingly less competent for tRNA gene transcription with a 70%
inactivation after 1.5 h and almost no detectable activity after 3 h. A
different in vitro response was observed in the case of 5 S
RNA synthesis, with practically no variation of transcriptional
activity after 1.5 h of treatment and a nearly complete loss of
transcription after 3 h. Extracts derived from treated cells thus
appear to mimic cycloheximide-induced down-regulation of pol III
transcription. However, at variance with tRNA gene in vitro transcription, which faithfully reproduces the in vivo response (cf. Fig. 1B and 2A),
the time course of inactivation of in vitro 5 S RNA
transcription was found to be slower than that measured in intact cells
(cf. Fig. 1B and 2B).
Figure 2:
Comparison of the transcriptional
capacities of control extracts and extracts derived from
cycloheximide-treated cells. A, tRNA gene transcription.
Increasing amounts of extracts prepared from cells that had been
exposed to cycloheximide (Cy) for either 1.5 h (lanes
4-6) or 3 h (lanes 10-12), and the
corresponding control extracts (Co, lanes 1-3 and 7-9, respectively) were tested for their
ability to transcribe the yeast tRNA gene. The following
amounts of extract protein were added to individual reaction mixtures:
8 µg, lanes 1, 4, 7, and 10; 16
µg, lanes 2, 5, 8, and 11; 32
µg, lanes 3, 6, 9, and 12.
B, 5 S rRNA gene transcription. Lanes are numbered as in
panel A; the same experimental conditions were used. The
positions of transcripts (tRNA
, 5 S RNA) after
resolution on polyacrylamide gels are shown.
As verified
by experiments in which a control extract was preincubated with
cycloheximide prior to transcription, the impaired transcriptional
capacity of extracts prepared from treated cells is not due to a direct
effect of the inhibitor on the transcription machinery (data not
shown). Furthermore, extracts of cycloheximide-treated cells did not
inhibit control extracts when the two were mixed together prior to
in vitro transcription (Fig. 3). This result indicates
that the transcriptional impairment of extracts derived from treated
cells is not due to the presence of a diffusible inhibitor and that
these extracts do not trans-inactivate any transcriptional component of
the control extract. Rather, it argues that inactive extracts have
reduced amounts of a positive activity that is required for tRNA and 5
S RNA transcription.
Figure 3:
In vitro transcription in mixed
extracts. Transcription reaction mixtures, programmed with the yeast
tRNA gene, contained either 8 µg of control extract
(Co, lane 1), 8 µg of extract derived from cells
that had been exposed to cycloheximide for 3 h (Cy, lane
2), or 8 µg of each type of extract (lane 3). The
position of transcripts (tRNA
) after resolution on a
polyacrylamide gel is shown.
TFIIIB Activity Is Selectively Impaired in Extracts of
Cycloheximide-treated Cells
To identify the class III
component(s) which is responsible for the cycloheximide-induced
down-regulation of pol III transcription, we initially tried to restore
tRNA gene transcription by extracts of treated cells with the addition
of individual transcription factors partially purified from control
cells. As shown in Fig. 4, supplementation of either TFIIIC,
TFIIIE, or polymerase to extracts of treated cells had no effect on
transcription (cf.lane 2 with lanes
6-8). In contrast, the addition of partially purified TFIIIB
fully restored transcription (lanes 3-5). Similar
experiments with the 5 S rRNA gene showed that TFIIIB also restores 5 S
RNA transcription (data not shown). TFIIIB thus appears to be primarily
responsible for the impairment of tRNA synthesis that occurs in
cycloheximide-treated cells, and a reduced activity of this component
also accounts for the delayed decline of in vitro 5 S RNA
transcription.
Figure 4:
Complementation of extracts derived from
cycloheximide-treated cells by the TFIIIB fraction. The transcriptional
activity of an extract prepared from treated cells (Cy, 3 h of
exposure to cycloheximide, 16 µg of total protein) was measured in
the absence of additional factors (lane 2), or in the presence
of partially purified TFIIIB (B, 0.5, 1, and 2 µg of total
protein, lanes 3-5), TFIIIC (C, 0.6 µg,
lane 6), polymerase (p, 0.4 µg, lane 7),
or TFIIIE (E, 0.5 µg, lane 8). The
transcriptional output of the control extract (Co, 16 µg
of total protein) is shown for comparison in lane
1.
An intrinsic restraint of ``add back''
experiments is their inability to detect even significant variations of
components other than the one that is limiting for transcription. We
thus assayed the activity of individual class III transcription
components by supplying graded amounts of extracts from either control
or cycloheximide-treated cells to partially reconstituted transcription
systems providing an excess of all components except for the one being
tested. As shown in , TFIIIB activity is about 10-fold
reduced in extracts of treated cells, whereas nearly identical TFIIIA,
TFIIIC, TFIIIE,and polymerase activities are present in either type of
extract.
The TFIIIB70 and B" Components of TFIIIB Are Selectively
Inactivated Following Cycloheximide-induced Protein Synthesis
Inhibition
Yeast TFIIIB is a multiprotein complex constituted by
at least three distinct components (reviewed in Ref. 29). To determine
which of these components is negatively modulated in
cycloheximide-treated cells, we first set up a reconstituted
transcription system in which TFIIIB is replaced by recombinant
TFIIIB70 and TBP, plus a fraction, B", that was partially purified from
yeast nuclear pellets (Fig. 5A, lanes
3-9). The latter fraction was found to contain sizeable
amounts of cross-contaminating TFIIIE (data not shown). At variance
with its transcriptional requirement in the presence of the native
TFIIIB complex (Fig. 5A, lanes 1 and
2), TFIIIE thus turned out to be dispensable for transcription
in the presence of fraction B" (Fig. 5A, lanes 3 and 6). On the contrary, TFIIIB70, TBP, and B" were all
required to reconstitute TFIIIB activity, and the level of tRNA
transcription efficiency achieved with internally calibrated amounts of
these three components was comparable to that obtained in the presence
of native TFIIIB and TFIIIE (Fig. 5A).
Figure 5:
Restoration of tRNA gene transcription by
resolved TFIIIB subcomponents. A, reconstitution of TFIIIB
activity by TFIIIB70, TBP, and B". Transcription reactions were carried
out in the presence of TFIIIC (C, 0.6 µg of total
protein), TFIIIE (E, 0.5 µg), and polymerase
(pol, 0.4 µg), plus either the native TFIIIB fraction (0.5
µg, lane 1) or internally calibrated amounts of the three
TFIIIB subcomponents (rTFIIIB70 80 ng, rTBP 40 ng, fraction B" 0.5
µg, lane 3). As indicated above each lane, all of these
components were individually omitted from the latter reaction mixture
(lanes 4-9), whereas only TFIIIE was omitted from
reactions supported by the native TFIIIB fraction (lane 2).
All the lanes shown come from the same exposure of a single gel.
B, complementation of extracts derived from
cycloheximide-treated cells by TFIIIB70 plus B". Transcription reaction
mixtures contained 16 µg of an extract prepared from treated cells
(Cy, 3 h of treatment, lanes 2-12), either
alone (lane 2) or supplemented with various combinations of
TFIIIB subcomponents as indicated above each lane (lanes
3-11). The native TFIIIB fraction (nat. IIIB, 0.5
µg of total protein) was added to the reaction in lane 12.
A control extract prepared from mock-treated cells (Co, 16
µg of total protein) was used for the reaction in lane 1.
The amounts of TFIIIB subcomponents added to the cycloheximide extract
were as follows: rTBP, 40 ng; rTFIIIB70, 80 ng; B", 0.3, 0.7, 2 µg
in lanes 6-8, respectively, and 2 µg in lanes
9-11. The position of transcripts
(tRNA) after resolution on a polyacrylamide gel
is shown.
As shown in
Fig. 5B, neither TBP, B", nor a combination of these two
components could restore the activity of an extract prepared from
cycloheximide-treated cells. Only a slight stimulation of transcription
was observed with either TFIIIB70 or TFIIIB70 plus TBP (cf.
lane 2 with lanes 4 and 5), and essentially
identical results were obtained upon the additional supplementation of
TFIIIE (data not shown). In contrast, the same extract was restored to
about 90% of the control activity by the addition of TFIIIB70 and B"
(cf.lanes 1 and 9), with only a little
additional recovery upon the further supplementation of TBP (lane
11). TFIIIB70 and a component of fraction B" thus appear to be the
selective targets of the cycloheximide-induced inactivation of TFIIIB.
This conclusion was further supported by titration experiments, which
showed that largely reduced (7-10-fold) B" and TFIIIB70
activities, but essentially unchanged levels of TBP activity, are
present in extracts of treated cells (data not shown).
Figure 6:
Comparative analysis of the levels of TBP
and TFIIIB70 in extracts derived from cycloheximide and mock-treated
cells. A, TBP: 10 µg of either a control extract
(Co, lane 1) or an extract prepared from
cycloheximide-treated cells (Cy, 3 h of treatment, lane
2), and 30 ng of recombinant yeast TBP (rTBP, lane
3) were subjected to SDS-polyacrylamide gel electrophoresis and
Western immunoblot analysis as described under ``Experimental
Procedures.'' The migration position of the 27-kDa TBP polypeptide
is indicated. B, TFIIIB70: identical amounts of the same
extracts used for the experiment reported in panel A were
analyzed. 10 ng of recombinant yeast TFIIIB70
(rIIIB) were loaded in lane 3; the
migration position of the TFIIIB70 polypeptide (70 kDa) is
indicated.
genes in silkworms
(34) , and in the down-regulation of pol
III transcription during differentiation of mouse F9 cells
(8) .
TFIIIB is also involved in the repression of class III gene
transcription that occurs in Xenopus oocytes as they go
through mitosis
(9) . The latter event has been found to be
mediated by the phosphorylation of a TFIIIB subunit
(9) , but the
specific TFIIIB components that are involved in the above mentioned
negative modulations are not yet known.
(
)
The trans-recognition
and targeted proteolysis of specific components of multiprotein
complexes has recently been reported (reviewed in Ref. 40). It will
thus be important to establish whether similar mechanisms also pertain
to the selective inactivation of the TFIIIB70 and B" components of
yeast TFIIIB.
Table:
Transcription factor activities in extracts of
control and cycloheximide-treated cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.