(Received for publication, May 22, 1995; and in revised form, August 30, 1995)
From the
We report in vitro studies showing that tRNA gene
transcription in yeast is down-regulated during the transition from
logarithmic to stationary phase growth. Transcription in a postdiauxic
(early stationary) phase extract of a wild-type strain decreased 3-fold
relative to a log phase extract. This growth stage-related difference
in transcription was amplified to 20-fold in extracts of a strain
containing a mutation (pcf1-4) in the 131-kDa subunit of
TFIIIC. The reduction in transcription activity in both wild-type and
mutant postdiauxic phase extracts was correlated with a decrease in the
amount of TFIIIB, the limiting factor in these extracts.
However, the 3.7 ± 0.5-fold decrease in amount of TFIIIB
in mutant extracts does not, by itself, account for the 20-fold
decrease in transcription. Accordingly, transcription in the mutant
postdiauxic phase extract could be reconstituted to a level equal to
the mutant log phase extract by the addition of two components,
TFIIIB
and TFIIIC. Addition of TFIIIB
increased transcription 10-fold, while a 2-fold effect of TFIIIC
was seen at saturating levels of TFIIIB
. The data suggest
that both TFIIIB
and TFIIIC play a role in coordinating
the level of polymerase III transcription with cell growth rate.
The expression of genes transcribed by each of the three nuclear
RNA polymerases (pols) is down-regulated during the
transition from logarithmic to stationary phase (G
)
growth(1, 2, 3) . The regulatory events that
mediate this general decrease in transcription are complex and not well
understood. In the yeast Saccharomyces cerevisiae, the
transition from logarithmic growth to stationary phase is accompanied
by a 50% decrease in total RNA and a decline in the number of ribosomes
to less than 25% of the maximum amount(4) . One of the earliest
events in this process is the decrease in pol I-dependent transcription
of rRNA. For cells growing in rich medium, this occurs well before the
end of the log phase and is followed closely by a decrease in pol
II-dependent transcription of ribosomal protein genes. The early
shut-off in the synthesis of ribosomal components, specifically the
large rRNAs, demonstrates that yeast cells respond to increasing cell
density prior to changing their growth rate. At the present time, the
nature of the signal and the manner in which it is transduced to affect
transcription is unknown. However, in higher eukaryotes and in Acanthamoeba, growth regulation of pol I transcription appears
to involve posttranslational modification of the polymerase or a
tightly associated initiation factor (see (1) and (5) , and references therein).
At the end of logarithmic
growth, yeast cells undergo a diauxic shift in which the nutrients
required for growth by fermentation become exhausted and the cells
switch to respiratory metabolism(2) . Many pol II-transcribed
genes that are expressed in abundance during logarithmic growth are
down-regulated to barely detectable levels following the diauxic shift.
In contrast, the expression of other genes remains relatively constant
or even increases in some cases. Nonetheless, by the time the cells
reach stationary phase, the level of total poly(A) RNA
has decreased 2-fold(6) . Since not all pol II-transcribed
genes are regulated coordinately during the growth cycle, decreased
transcription of specific genes is brought about by controlling the
activity of activator and/or repressor proteins. These, in turn, affect
the assembly or function of the basal transcription factors on
promoters under their control (reviewed in (7) ).
The RNAs transcribed by pol III are not limiting for cell growth under normal conditions but are available at all times for numerous cellular processes including protein synthesis, protein secretion, and RNA processing(3, 8) . Despite the apparent surplus of pol III transcripts for cell growth, the expression of pol III genes is not unregulated. Indeed, the coordination of pol III gene transcription with cell growth rate is well documented in higher eukaryotic systems (reviewed in (3) ). Pol III gene transcription in both human and mouse cells increases when growth is stimulated by serum(9, 10) . Conversely, down-regulation of pol III transcription is observed when mouse cells are treated with cycloheximide or grown to confluence(11) . In vitro systems that mimic these in vivo treatments show effects on transcription of 3-8-fold and have allowed identification of the regulated factors. Growth-related differences in transcription result almost entirely from changes in one or both of the multisubunit transcription factors (TFs) IIIC and IIIB; the activity of pol III changes very little with cell growth rate(9, 11) .
Recently, an analysis of mutant yeast strains selected for their
ability to increase pol III transcription identified a number of
isolates in which this phenotype is recessive(12) . A genetic
characterization of one of these strains mapped the mutation to the
gene encoding the 131 kDa subunit of TFIIIC (PCF1/TFC4). Subsequently, a mutation in this subunit (pcf1-3) was shown to increase transcription of a
variety of pol III genes in vitro. One implication of these
results, namely that a recessive (presumed loss of function) mutation
increases pol III transcription, is that the wild-type TFIIIC protein may negatively regulate this process. We therefore
examined (i) whether pol III transcription in yeast is negatively
regulated during the transition from logarithmic to stationary phase
growth and (ii) whether a recessive mutation in TFIIIC
perturbs this regulation. The results provide in vitro evidence that pol III transcription is down-regulated at the end
of the logarithmic growth phase and show that the magnitude of this
regulation is amplified in extracts of a mutant strain. The
growth-related differences in transcription activity are attributed to
effects on TFIIIB
and TFIIIC.
Figure 6:
Detection of TFIIIB in cell
extracts. A, recognition of TFIIIB
in nuclear
extracts. Nuclear extracts prepared from a wild-type (WT)
strain or a strain containing the PCF4
gene
on a multicopy, 2-µm plasmid (PCF4-MC) were analyzed by
Western blotting. Blots were probed with a 1:5000 dilution of either
preimmune or immune serum. The arrow indicates the mobility of
recombinant TFIIIB
which was run in an adjacent lane. B and C, the level of TFIIIB
is reduced
in postdiauxic extracts. Quantitative Western analysis was performed to
determine the relative levels of TFIIIB
in log and
postdiauxic phase whole cell extracts (see ``Experimental
Procedures''). A representative Western blot is shown in panel
B, with the position of a recombinant TFIIIB
standard
shown by an arrow. A lighter exposure of the blot was
quantified by densitometry, and the data are presented in panel
C.
, pcf1-4 log phase;
, wild-type log
phase;
, pcf1-4 postdiauxic phase;
,
wild-type postdiauxic phase. D, a representative Western blot
analysis of TFIIIC
in pcf1-4 whole cell
extracts. The pcf1-4 extracts used in panel B were analyzed in a separate experiment with a polyclonal antibody
to TFIIIC
. The data were quantified as before. The
prominent band marked by the arrow shows the position of
TFIIIC
.
All transcription factors were
wild-type unless otherwise noted. Recombinant yeast TBP was a gift from
Dr. Michael Brenowitz. TFIIIB was synthesized in a rabbit
reticulocyte lysate as described below. TFIIIB
was
prepared by urea extraction of chromatin pellets and chromatography on
Bio-Rex 70(17) . TFIIIB
activity was then further
purified on DEAE-Sephadex A25, heparin-agarose, and Cibacron
blue-agarose following procedures used previously for
TFIIIB(18, 19, 20) . The preparation of
heparin-agarose TFIIIB and TFIIIC and Sephacryl S300 TFIIIC (from a PCF1-1 strain) have been described
previously(20) . The in vitro transcription activity
of TFIIIC purified from the PCF1-1 strain is
indistinguishable from wild type(20) . The pol III fraction
used in this work was a 500 mM step fraction from
DEAE-Sephadex(15) . The transcription activities of TFIIIB,
TFIIIB
, and in vitro synthesized TFIIIB
were determined in reconstitution experiments under single round
initiation conditions ( (15) and (18) ; see below).
Titrations of each component were performed to ensure that the activity
being assayed was limiting under the conditions employed. TFIIIC
activity represents specific DNA binding activity determined by gel
mobility shift assays(20) .
Figure 1:
Comparison of
pol III transcription in wild-type and pcf1-4 log phase
and postdiauxic phase cell extracts. Log phase and postdiauxic phase
extracts were prepared by glass bead disruption from cells grown to
densities of 5 10
and 2
10
cells/ml, respectively. A shows an extract titration
experiment performed under multiple round initiation conditions at 15
°C for 60 min.
, pcf1-4 log phase;
,
wild-type log phase;
, pcf1-4 postdiauxic phase;
, wild-type postdiauxic phase. B compares pcf1-4 log and postdiauxic phase extracts in a modified
single round reaction. Standard (lanes 1 and 2) and
modified (lanes 3-5) single round assays are compared
using a BR
fraction (see ``Experimental Procedures'').
Ternary complexes assembled in the presence of ATP, UTP and GTP were
not (NC) or were (C) chased with CTP and heparin. For
reactions performed under the modified conditions, the time of
incubation (in seconds) after addition of NTPs to assembled complexes
and before addition of heparin is shown. Lanes 6-8 and lanes 9-11 compare the activity of the postdiauxic and
log phase pcf1-4 whole cell extracts (WCEs),
respectively.
Log and postdiauxic phase whole cell
extracts were prepared in parallel by glass bead disruption and assayed
for transcription activity. The pcf1-4 log phase extract
increased pol III-specific transcription activity 9-fold over the
wild-type log phase extract (Fig. 1A and (12) ). This effect of pcf1-4 on transcription
is consistent with the strong in vivo phenotype of this mutant
and is appropriate (in magnitude) relative to the transcriptional
activities and in vivo phenotypes of the pcf1-3 and PCF1-1 alleles(12) . Transcription in
wild-type extracts prepared from postdiauxic phase cells was reduced by
70% compared to the log phase control. A quantitatively similar low
level of transcription was also observed in the mutant postdiauxic
phase extract. Consequently, transcription activity in the wild-type
extracts decreased 3-fold between the log and postdiauxic phase, while
transcription activity in the pcf1-4 extracts decreased
by 20-fold. To examine whether the differential activity in pcf1-4 extracts resulted from a difference in the number
of transcriptionally competent complexes, a modified single round
initiation assay was used to determine the number of these complexes
(see ``Experimental Procedures''). Briefly, preinitiation
complexes assembled in the absence of NTPs were allowed to transcribe
for 30-90 s following NTP addition. Using this assay and a
BR fraction, the 30-, 60-, and 90-s time points were found to
yield 1.8, 1.3, and 1.0 transcripts/active complex/30 s, respectively (Fig. 1B, compare lanes 3-5 with lane 2). A comparison of transcription in pcf1-4 log and postdiauxic phase extracts under these conditions yielded
a 25 ± 1-fold difference in the number of active complexes (Fig. 1B, lanes 6-11). This is similar
to the difference seen in multiple round assays. Thus, a difference in
the number of active complexes can account for the transcriptional
differential between the pcf1-4 log and postdiauxic
phase extracts.
The preceding experiments suggest that pol III
transcription in yeast is subject to down-regulation as the cells
approach stationary phase. This conclusion is in concordance with in vitro and in vivo studies on growth regulation of
pol III transcription conducted in mammalian systems(3) .
Interestingly, despite the high level of transcription seen in the pcf1-4 log phase extract, the pcf1-4 mutation does not impair the down-regulation of transcription in
extracts of postdiauxic cells. Therefore, the mutation at amino acid
728 in TFIIIC does not render the protein defective in
postdiauxic phase regulation. Nonetheless, the ability of the pcf1-4 mutation to amplify the transcriptional
difference between log and postdiauxic phase cell extracts provides a
useful system to investigate the factors and the molecular mechanisms
regulating pol III gene transcription during the growth cycle.
Figure 2: Transcription in mixed pcf1-4 extracts. Multiple round transcription reactions were carried out with pcf1-4 log and postdiauxic phase extracts (100 µg of each extract) assayed separately or in combination. The bar graph shows the average -fold increase in transcription over the postdiauxic phase extract and the standard error from three experiments.
Transcription of a tRNA gene
in yeast requires the initiation factor TFIIIB (which comprises TBP,
TFIIIB, and TFIIIB
), TFIIIC (a multisubunit
complex of six polypeptides), and the 16-subunit RNA polymerase
III(3, 8, 17, 24) . Additionally, a
requirement for a distinct new factor, TFIIIE, has been demonstrated
using a different purification scheme than that used by most
laboratories(25) . The nature of the activities that are
limiting for pol III transcription in wild-type log phase and mutant
postdiauxic phase cell extracts was examined by adding various factors
(or fractions) individually or in combination to these extracts. The
TFIIIB components used in this experiment included recombinant yeast
TBP, TFIIIB
synthesized in a rabbit reticulocyte lysate,
and a purified TFIIIB
fraction from urea-extracted
chromatin pellets (see (17) and ``Experimental
Procedures''). Previous studies have demonstrated the ability of
two of these fractions (recombinant TBP and urea-extracted
TFIIIB
) to function in pol III transcription (17, 26) . The activity of the third component, in
vitro synthesized TFIIIB
, is demonstrated in Fig. 3A. Addition of a rabbit reticulocyte lysate
programmed with RNA encoding TFIIIB
, but not a negative
lysate control, to a wild-type log phase cell extract produced a
significant (5-fold in Fig. 3A) increase in
transcription. This result is consistent with our previous finding that
TFIIIB
is limiting for pol III transcription in vivo(27) and extends the related observation, that TFIIIB
activity is limiting in whole cell extracts (18) by identifying
the limiting component of this factor in vitro. Other
experiments have demonstrated that in vitro synthesized
TFIIIB
, but not lysate alone, is active in reconstituting
transcription in the presence of TBP, urea-extracted
TFIIIB
, purified TFIIIC and pol III (see
``Experimental Procedures'').
Figure 3:
TFIIIB is the limiting
component in wild-type log phase and pcf1-4 postdiauxic
phase extracts. Transcription was assayed under multiple round
initiation conditions as in Fig. 1. A, stimulation of
transcription by in vitro synthesized TFIIIB
. A
wild-type log phase extract (65 µg of protein) was supplemented
with a negative control rabbit reticulocyte lysate (1.5 and 3 µl, lanes 2 and 3) or a lysate that had been programmed
with RNA encoding TFIIIB
(1.5 and 3 µl, lanes 4 and 5). No lysate was added to the reaction in lane
1. B, a wild-type log phase extract (100 µg of
protein) was supplemented with exogenous factors as follows: lanes
1-3, 20, 40, and 80 fmol of recombinant yeast TBP; lanes
4-6, 0.7, 1.4, and 2.1 fmol of in vitro synthesized
TFIIIB
; lanes 7-9, 0.85, 1.7, and 3.4 fmol
of TFIIIB
; lane 10, 80 fmol of TBP + 2.1
fmol of TFIIIB
; lane 11, 80 fmol of TBP +
3.4 fmol of TFIIIB
; lane 12, 2.1 fmol of
TFIIIB
+ 3.4 fmol of TFIIIB
; lane
13, 80 fmol of TBP + 2.1 fmol of TFIIIB
+
3.4 fmol of TFIIIB
; lane 14, 32 fmol of TFIIIC; lane 15, 100 ng of pol III. C, a pcf1-4 postdiauxic phase extract was supplemented with exogenous factors
as in panel B.
The ability of TFIIIB and other pol III factors/fractions to increase transcription
when added to wild-type log phase and pcf1-4 postdiauxic
phase extracts is examined in Fig. 3(B and C,
respectively). The results for both extracts are similar and show a
significant increase in transcription for TFIIIB
(up to
6-fold, lanes 4-6), no effect of TBP (lanes
1-3), and a slight decrease in transcription for
TFIIIB
(at higher concentrations, lanes
7-9). Moreover, additions of TBP and/or TFIIIB
in combination with TFIIIB
did not stimulate
transcription above the level obtained with TFIIIB
alone
(compare lanes 10-13 to lane 6). Addition of
pol III had no effect on transcription in either extract (lane
15). In contrast, purified TFIIIC had a small stimulatory effect (lane 14, 1.2-1.7-fold, see also Fig. 5). These
data show that pol III transcription activity in both wild-type log
phase and pcf1-4 postdiauxic phase extracts is limited
primarily by the amount of TFIIIB
(see also Fig. 4A).
Figure 5:
Reconstitution of pcf1-4 log phase-type transcription in postdiauxic phase extracts and in
the wild-type log phase extract. Multiple round reactions were
conducted as in Fig. 4using 75 µg of the various whole cell
extracts. A, in lanes 1-8, the postdiauxic
phase extract from the pcf1-4 strain was supplemented
with heparin-agarose-purified TFIIIB (1.0 fmol except for lane
3, which contained 1.2 fmol) and various other fractions as
follows: lane 3, TFIIIB (2.5 fmol); lane
4, TBP (80 fmol); lane 5, pol III (175 ng); lane
6, TFIIIC (6.4 fmol); lane 7, TFIIIC (19.2 fmol); lane 8, TFIIIC (10 fmol) and pol III (90 ng). Transcription in
the pcf1-4 log phase extract is shown in lane
9. B, the bar graph shows the quantitation of the data in panel A. Bar designations correspond to the lanes in panel
A. The presence (+) or absence(-) of supplementary
components in each reaction is indicated above the graph. The 20-fold
differential between pcf1-4 log and postdiauxic phase
extracts is shown as a dotted line. C, the wild-type
postdiauxic phase extract was supplemented with TFIIIB
as
follows: lane 2, 0.6 fmol; lane 3, 1.2 fmol; lane
4, 1.8 fmol; lane 5, 2.4 fmol; lanes 6 and 9-11, 3.0 fmol. In addition, lanes 10 and 11 contained 0.45 and 0.9 µg of heparin-agarose TFIIIC. Lanes 1 and 8 contained no exogenous factors. D, the wild-type log phase extract (lane 1) was
supplemented with increasing amounts (0.4-1.2 fmol) of
heparin-agarose-purified TFIIIB (lanes
2-5).
Figure 4:
Titration of TFIIIB and
TFIIIB in pcf1-4 extracts. A shows the effect
of adding increasing amounts of in vitro synthesized
TFIIIB
on transcription in a pcf1-4 log
phase extract (circles and lanes 1-4) and a pcf1-4 postdiauxic phase extract (squares and lanes 5-9). Multiple round reactions were employed using
75 µg of each extract. Lane 1 contained no exogenous
TFIIIB
. The amount of added TFIIIB
was
determined by single round transcription (see ``Experimental
Procedures''). B shows the effect of adding increasing
amounts of heparin-agarose-purified TFIIIB on transcription in a pcf1-4 log phase extract (circles and lanes
1-6) and a pcf1-4 postdiauxic phase extract (squares and lanes 7-12). The same reaction
conditions were used as in panel
A.
At the highest concentration of
TFIIIB used in Fig. 3, transcription had not
reached a plateau and was therefore still limited by the amount of this
factor. To assess the extent to which TFIIIB
might
contribute to the differential activities of pcf1-4 log
and postdiauxic phase extracts, transcription was examined over a wider
range of exogenous TFIIIB
concentrations (Fig. 4A). Addition of saturating amounts of
TFIIIB
to the mutant postdiauxic phase extract resulted in
a 10-fold increase in transcription. This level of transcription is
within a factor of 2 of that exhibited by the unsupplemented pcf1-4 log phase extract. Even the high transcription
activity of the mutant log phase extract can be increased by adding
TFIIIB
. In this case, however, only a modest 1.7-fold
increase could be achieved over the unsupplemented extract at
saturating levels of TFIIIB
. From these data we conclude
that TFIIIB
is primarily responsible for the low level of
transcription in the postdiauxic phase extract.
The data in Fig. 4B suggest that
either TFIIIC or pol III becomes limiting in the postdiauxic phase
extract at saturating levels of TFIIIB. An additional
add-back experiment was performed to resolve these possibilities.
Saturating levels of TFIIIB (i.e. TFIIIB
) were
determined from Fig. 4B and were confirmed by comparing
transcription at two different concentrations of this factor (Fig. 5, A, lanes 1 and 2, and B). Addition of a pol III fraction to the TFIIIB-supplemented
extract showed no stimulation of transcription. However, two different
concentrations of TFIIIC increased transcription 2.4- and 2-fold,
respectively, over the TFIIIB-supplemented control. The addition of
TFIIIC together with pol III also showed a 2-fold effect. Importantly,
the transcription observed in these reactions is comparable to that
seen in the log phase extract of the mutant strain (Fig. 5, lane 9). Thus, the transcription activity of the mutant
postdiauxic phase extract can be increased to the level obtained in the
corresponding log phase extract by adding both TFIIIB
and
TFIIIC. A similar high level of transcription can also be achieved by
adding both factors to the wild-type postdiauxic phase extract (Fig. 5C). Addition of saturating amounts of
TFIIIB
yields a 12-14-fold increase in transcription
over the unsupplemented wild-type extract. Transcription is further
stimulated upon addition of TFIIIC for an overall increase of
26-28-fold. We note that the ability to increase transcription in
the wild-type postdiauxic phase extract is slightly higher than for the
corresponding mutant extract. This correlates with the slightly lower
activity of unsupplemented wild-type extract (Fig. 1A).
In contrast to the postdiauxic phase extracts, TFIIIB alone is sufficient to increase transcription of the wild-type log phase extract to a level equivalent to (and slightly above) the unsupplemented mutant log phase extract. Amounts of heparin-agarose-purified TFIIIB that were sufficient to saturate the postdiauxic extracts (Fig. 4B and data not shown) show a linear response in the wild-type log phase extract (Fig. 5D). At 1.5 fmol of exogenous TFIIIB, transcription was 11-fold higher than the unsupplemented level (recall that the unsupplemented log phase extracts showed a 9-fold differential, Fig. 1A). This result suggests that TFIIIC is not limiting to the same extent in log phase extracts as it is in the postdiauxic phase extracts.
The relative amounts of
TFIIIB present in whole cell extracts of log and
postdiauxic phase wild-type and mutant strains were determined by
quantitative Western blot analysis. A representative experiment and the
resulting densitometric analysis is shown in Fig. 6(B and C). The data in Fig. 6C reveal a
2-fold difference in the amount of TFIIIB
between log
phase extracts of the mutant and wild-type strains (circles and squares). Over five experiments, a 2.1 ±
0.3-fold difference was determined. These extracts, however, differ in
transcription activity by a factor of 9 (Fig. 1A). A
similar disparity is seen when comparing log and postdiauxic phase
extracts of the mutant strain. Although these extracts differ in
transcription by 20-fold, the amount of TFIIIB
protein
shows only a 3.6-fold difference in Fig. 6C (circles and triangles) and a 3.7 ±
0.5-fold difference over four experiments. Because TFIIIB
is limiting in whole cell extracts and is stoichiometrically
required for initiation(27, 28) , these data suggest
that changes in the amount of TFIIIB
are not sufficient to
account entirely for the differences in transcription. In contrast, a
good correlation is observed between the amount of TFIIIB
and the transcriptional activity of wild-type extracts from the
log and postdiauxic phase. The TFIIIB
levels in these
extracts differ by an average of 2.9 ± 0.4-fold over four
experiments (in Fig. 6C, compare squares and inverted triangles), while transcription differs by 3-fold.
Quantitative Western analysis was also performed on the pcf1-4 cell extracts using an antibody to the 131-kDa
subunit of TFIIIC (Fig. 6D). The ratio of the slopes
obtained by linear regression of the densitometry data from two
experiments showed a 1.1-fold and a 1.2-fold difference in the amount
of TFIIIC in mutant log and postdiauxic phase extracts.
This result confirms the differences in the TFIIIB
levels
and the transcriptional capacities of the extracts.
In human and mouse systems, the level of pol III
transcription is known to vary according to the cell growth rate
(reviewed in (3) ). The present study indicates that growth
regulation of pol III transcription also exists in yeast. Specifically,
transcription of a tRNA gene was shown to be reduced in extracts of
cells approaching stationary phase (Fig. 1). Over two
generations of growth in which the cell doubling time increased from 90
min to greater than 270 min, tRNA gene transcription decreased 3-fold
in extracts of a wild-type strain and 20-fold in extracts of a pcf1-4 strain. The magnitude of the effect in the
wild-type extracts is similar to that observed in extracts of confluent
or growth-inhibited mouse cells(11) . In the mouse extracts,
decreased pol III transcription was attributed largely to a reduction
in the amount or activity of TFIIIB(11) . Interestingly, the
yeast factor most responsible for the difference in transcription
between pcf1-4 log and postdiauxic phase extracts is a
subunit of TFIIIB (TFIIIB, Fig. 4and Fig. 5). Thus, the mouse and yeast in vitro systems
appear to behave similarly in response to changes in cell growth rate.
This observation, together with the ability of the yeast in vitro system to reproduce in vivo phenomena (at least for
TFIIIB
, discussed below), suggests that the log and
postdiauxic phase extracts mimic the growth-related changes occurring
in the cell.
Previous studies in our laboratory have shown that the
general initiation factor TFIIIB is stoichiometrically
limiting for pol III transcription in vivo(27) . This
result together with studies showing that TFIIIB activity is limiting
in extracts of wild-type log phase cells (e.g. 18) suggested
that the amount of TFIIIB
might also be limiting for
transcription in vitro. The experiments presented here confirm
and extend this observation to include log phase extracts of a pcf1-4 mutant strain where pol III transcription is
elevated 9-fold over wild-type (Fig. 3, A and B, and 4A). Additionally, we have shown that
TFIIIB
is limiting in extracts of wild-type and pcf1-4 strains that are approaching stationary phase
(postdiauxic phase extracts, Fig. 3C and Fig. 5D). Thus, both in vivo and in
vitro, TFIIIB
is limiting for transcription.
TFIIIB, as a limiting initiation factor, is a likely
target for global regulation of pol III transcription(27) .
Consistent with this expectation, we have shown (i) that the amount of
TFIIIB
decreases in both wild-type and pcf1-4 postdiauxic phase extracts relative to the corresponding log phase
extracts (Fig. 6) and (ii) that addition of TFIIIB
to the pcf1-4 postdiauxic phase extract increases
transcription (10-fold) to a level equal to half that of the log phase
extract ( Fig. 4and Fig. 5). These results indicate a
role for TFIIIB
in growth regulation of pol III
transcription and suggest that regulation may be achieved by affecting
the amount of this factor. Other data, however, suggest that growth
regulation of pol III transcription may be more complex. For example,
whereas the calculated 2.9 ± 0.4-fold decrease in the level of
TFIIIB
could potentially account for the 3-fold change in
transcription in wild-type extracts, a 3.7 ± 0.5-fold change in
the amount of this factor is unlikely to account for the entire 20-fold
decrease in transcription observed for the pcf1-4 extracts. Consistent with this view, saturating levels of
exogenous TFIIIB
do not reconstitute pcf1-4 log phase-type transcription in postdiauxic phase extracts of the
wild-type or the mutant strain ( Fig. 4and Fig. 5). The
participation of a factor other than TFIIIB
in growth
regulation is indicated by the ability of a TFIIIC fraction (but not
other fractions) to restore mutant log phase-type transcription to
TFIIIB
-supplemented postdiauxic phase extracts (Fig. 5, B, lanes 6-9, and C, lanes 8-10). Notably, TFIIIC is not required to achieve
this result in a wild-type log phase extract (Fig. 5D).
Thus, TFIIIC appears to be subject to growth regulation although the
effect is only modest (2-fold). Finally, while it is not yet known
whether the activity or the amount of TFIIIC is subject to change
during the growth cycle, the 131-kDa subunit of this factor is present
in the same amount in both log and postdiauxic phase extracts (Fig. 6D).
As noted above, the log to postdiauxic ratios of
TFIIIB are similar in extracts of both wild-type and
mutant strains (2.9 ± 0.4 and 3.7 ± 0.5, respectively)
even though the ratios of their transcriptional activities differ
significantly. Nonetheless, the reduced level of TFIIIB
in
the postdiauxic extracts and the limiting nature of this factor suggest
that changes in the amount of TFIIIB
may account, in part,
for the observed differences in transcription. However, an additional
(or alternative) explanation not excluded by our data is that
TFIIIB
activity may be regulated during the growth cycle.
Regulation of TFIIIB activity has been demonstrated during the cell
cycle in Xenopus where phosphorylation of a TFIIIB component
was shown to inhibit pol III transcription during mitosis(29) .
Mitotic repression of TFIIIB has also been reported in human cells
although the mechanism has not been determined(30) . Further
support for the regulation of TFIIIB activity during the growth cycle
in yeast is provided by in vivo footprinting studies of tRNA
genes in logarithmically growing and saturated cultures(31) .
Both growth stages exhibit identical patterns of protection in the
region between -40 and -10 relative to the SUP53 tRNA gene transcription start site. This protected region
corresponds precisely to that protected in TFIIIB-DNA complexes
assembled in vitro(28, 32) . Interestingly,
cell growth-related changes in the in vivo footprint on the SUP53 gene map immediately downstream of the TFIIIB complex to
a region (-10 to +15), where a number of pol III subunits
and the 131-kDa subunit of TFIIIC have been photo-cross-linked in
vitro (33, 34). These data indicate reduced occupancy of the DNA
by pol III and/or TFIIIC in the stationary phase and suggest that pol
III transcription may be regulated after TFIIIB has bound to the DNA.
Accordingly, modification of TFIIIB activity (on the DNA) could
potentially control the rate of initiation by pol III in non-dividing
cells. In this regard, one obvious future direction of our work is to
determine whether the specific activity of TFIIIB
is
different in log and postdiauxic phase extracts.