Department of Cell Biology, Albert Einstein
College of Medicine, Bronx, New York 10461
Received 29 January 1999/Returned for modification 14 April
1999/Accepted 11 May 1999
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INTRODUCTION |
The Saccharomyces
cerevisiae cell invests enormous resources in the synthesis of
ribosomes. In a rapidly growing cell, the 100 or more rRNA genes are
responsible for at least 60% of total transcription (59).
The 137 ribosomal protein (RP) genes are among the most active in the
genome (57). As a result, the cell has evolved mechanisms
that tightly control the synthesis of the components of the ribosome.
The 78 RPs are synthesized in nearly equimolar amounts that are matched
to the synthesis of rRNA. Furthermore, the synthesis of these
components is coordinately responsive to many changes within or outside
the cell, including heat shock, carbon source, and nutrient
availability (reviewed in references 44 and
59).
The primary level of regulation is at transcription (7, 16,
24). Most RP genes have similar promoter motifs, consisting of
two Rap1p binding sites in tandem arrangement 250 to 400 bp upstream of
the transcription initiation site recently compiled by Lascaris et al.
(26), followed by one or two T-rich regions, and then a
region of about 180 bp that includes the putative TATA element. Serial
promoter deletions of RP genes, including RPS14A (CRY1), RPL25, and RPL28
(CYH2), have shown that the Rap1p sites are responsible for
about 90% of promoter activity, with the rest due to the T-rich
regions (41, 45, 47). Deletions downstream of the T-rich
elements have little effect on the rate of transcription but can affect
the initiation site (47). For a small minority of RP genes,
including RPL3, RPL4A, RPL4B, and
RPS28A, a single Abf1p site replaces the two Rap1p sites
(8, 13). Nevertheless, such genes appear to be regulated
coordinately with the others. (In this paper, we have used the new
nomenclature for RPs and their genes that was recently adopted
(32, 64a). Thus, L30 was previously known as L32, L3 was
previously known as Tcm1p, and L8 was previously known as L4.)
Rap1p is an essential, abundant DNA-binding protein that has several
functions in the cell. It is a context-dependent transcription regulator (50), responsible for activating the transcription of many of the most highly transcribed genes, encoding not only the RPs
and other translation factors but also the very abundant glycolytic
enzymes (reviewed in reference 49). Rap1p also acts as a transcriptional silencer in at least two contexts. At the silent
mating type loci, Rap1p cooperates with Abf1p and the origin of
replication complex (ORC) to repress transcription (1). At
the telomeres, Rap1p binds to the [C1-3A]n
repeats (63), leading to the silencing of genes adjacent to
telomeres. In both cases Rap1p recruits multiple copies of Sir3p and
Sir4p that participate in the silencing, perhaps by interacting with
the tails of histones H3 and H4 (38). A mutant allele,
rap1s, relieves the repression at silent mating
type loci. The overexpression of Sir3p or Sir4p can restore the
repression (53).
When yeast cells growing at 23°C are shifted to 37°C, both growth
and protein synthesis continue unabated; indeed, depending on strain
background, they sometimes increase (11). Furthermore, mRNA
synthesis, measured by the incorporation of [3H]uracil
into poly(A)+ RNA, is unaffected (24). Yet,
during the first 20 min after such a shift the level of RP mRNAs
declines by about 80% and then recovers to the original level by about
60 min (11, 15). This down-regulation of RP mRNA has been
attributed to a temporary transcriptional silencing of RP genes
(24) and to an increased turnover of RP mRNAs
(15). In neither case have the elements responsible been
identified (15, 45).
Recently, we have shown that a defect at any of several points in the
secretory pathway down-regulates the synthesis of ribosomes, through
the repression of transcription of both rRNA and RP genes (28,
36). Inhibitors that disrupt the secretory pathway, such as
brefeldin A and tunicamycin, also repress ribosome synthesis. A similar
effect on rRNA transcription was observed in a sec23 mutant
(29). These results suggest that there exists an
intracellular signal transduction pathway between the secretory
apparatus and the master control of ribosome biosynthesis.
To identify the elements of an RP gene that makes it subject to
repression by a temperature shock and by the failure of the secretory
pathway, we have carried out experiments which demonstrate (i) that the
rapid decline in mRNA levels is due entirely to the rapid repression of
transcription, coupled to a normal, rapid turnover of the mRNA (this
repression does not depend on the presence of Sir2p, Sir3p, or Sir4p)
and (ii) that the repression of transcription is mediated by two
cis elements, the Rap1p binding sites and a 180-bp sequence
just upstream of the transcription initiation site. Either of these
elements alone can mediate at least 75% of repression.
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MATERIALS AND METHODS |
Strains.
The strains used in this study are listed in Table
1. Unless otherwise stated, cultures were
grown on 2% yeast extract-1% Bacto Peptone-2% glucose (YPD) at
23°C. Cultures were shifted from 23 to 37°C by pouring them into a
large, prewarmed flask shaking in a water bath. Cultures were shifted
from YPGal to YPD by filtration onto a Millipore filter which was
immediately transferred to the new medium. The whole operation took
less than 20 s.
Yeast total RNA isolation.
Generally, 10-ml volumes of
cultures in log phase were harvested by pouring them on crushed ice
(46). Cells were collected by centrifugation, resuspended in
400 µl of AE buffer (50 mM NaAc [pH 5.3], 1 mM EDTA), and
transferred to microcentrifuge tubes. Forty microliters of 10% sodium
dodecyl sulfate was added, and the mixture was vortexed. An equal
volume of fresh, AE buffer-saturated phenol was added, and the mixture
was vortexed for 2 min. The samples were then incubated at 65°C for 4 min and chilled rapidly in dry ice-ethanol until phenol crystals
appeared. The samples were centrifuged at maximum speed for 10 min, and
the aqueous layer was transferred to another tube, extracted with
phenol-chloroform at room temperature for 5 min, and then centrifuged
for 5 min. Total RNA was precipitated with ethanol, washed with 70%
ethanol, dried, and resuspended in 50 µl of sterile water.
RNA gel and Northern analysis.
Ten micrograms of total RNA
was fractionated on 1.5% agarose gels containing 6% formaldehyde. The
RNA was transferred to Nytran (Schleicher & Schuell) nylon membranes,
cross-linked with UV light, and then baked in a vacuum for 1 h at
80°C. Northern blot analysis was performed as described previously
(10). 32P-labelled antisense RNA probes and
oligodeoxynucleotides were used to detect mRNAs (36). rRNA
was labelled with [C3H3]methionine
(60) and analyzed by fluorography (36).
Construction of fused genes with site-directed mutagenesis.
For the ACT1 gene constructs, the A of ATG in the coding
region is at position +1. For RPL30, the transcription
initiation site is considered position +1 and is 58 nucleotides
upstream of ATG. To fuse the RPL30 promoter to the
ACT1 transcription unit, the URA3 gene was first
cloned into pMDJ8, which contains a 1.8-kb fragment with the complete
RPL30 sequence, from 
443 to +1520. The new plasmid,
containing divergently expressed URA3 and RPL30 genes, each from its own promoter, was then used as a template for PCR.
The left-hand primer contained ACT1 sequences from 410 to
364 and the reverse complement of URA3 sequences from +915 to +890. (The URA3 open reading frame [ORF] is 803 bp).
The right-hand primer contained ACT1 sequences from 96 to
141 and RPL30 sequences from 1 to 26. PCR amplified a
fragment in which the entire URA3 gene and RPL30
promoter sequences (to the transcription start site at +1) were
bracketed by ACT1 sequences. The PCR fragment was integrated
into the chromosomal ACT1 locus of strain 169ts or JW142 by
homologous recombination, selecting for URA3. The integration reconstituted the ACT1 transcription unit, under
the control of the RPL30 promoter (Fig. 4B). The correct
integration was verified by PCR. The correct initiation of
transcription was verified by primer extension. The resulting strain
was designated JW1201 (Table 1).
Constructs A to D (Fig. 4C) were made starting with a pUC19-based
plasmid carrying a 2.2-kb EcoRI fragment from
RPL30. The promoter region was replaced with a fragment
containing the promoter of ACT1 (construct A; Fig. 4B). From
this construct, the upstream activating sequence (UAS) of
ACT1 was replaced with portions of the UAS of
RPL30 (constructs B, C, and D; Fig. 4C). For use in yeast,
the constructs were subcloned into the CEN/URA3 plasmid, pRS316 (51). Primer extension revealed that about 50% of
the transcripts initiated at the normal site for RPL32, and 50%
initiated nine nucleotides downstream.
Constructs E to H (Fig. 9A) were made, starting with the vector pRS316
carrying RPL30. A large portion of the ORF downstream of the
intron was replaced by the ORF encoding the green fluorescent protein
(GFP), generating construct E (see text for details). From construct E,
different portions of the RPL30 promoter were replaced with
a 500-bp fragment containing the four Gal4p binding sites from
GAL1 (nucleotides 829 to 324; with the start codon numbered +1), generating constructs F to H.
Deletion mutations were constructed by PCR (18). A circular
plasmid carrying the gene of interest was used as a template for PCR
catalyzed by Tli DNA polymerase (Promega), which has proofreading ability. PCR products were purified from an agarose gel with a DNA
extraction kit (Qiagen). The purified DNA was used directly for
self-ligation with the standard protocol, except that 10 U of
polynucleotide kinase was included in the ligation reaction. The
deletion constructs were sequenced.
mRNA half-life measurement.
The strains to be tested were
grown overnight at 23°C to log phase. Cell cultures were concentrated
fivefold and then shifted to 37°C (43). At the indicated
time points 2-ml aliquots of the cultures were quickly collected and
frozen in dry ice-ethanol. Total RNA was isolated and analyzed as
described above. RNA levels were quantified relative to the U3
internal loading standard by using PhosphorImager (Molecular
Dynamics) analysis.
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RESULTS |
Repression of RP gene expression.
The mRNAs encoding RPs
appear to be particularly sensitive to environmental changes. As shown
in Fig. 1, a temperature shift from 23 to
37°C leads to a rapid decline in RP mRNA levels that is temporary in
wild-type (wt) cells (lanes 1 to 6) but permanent in cells with a
temperature-sensitive (ts) mutation in the secretory pathway,
exemplified by ypt6-1 (lanes 7 to 13). This is not true of
most non-RP genes, e.g., ACT1, or of the stable small
nucleolar RNA (snoRNA) U3. These results could be explained by the
hypothesis that the transcription of RP genes is extinguished after the
temperature shift, temporarily in wt cells and permanently in cells
with a defect in the secretory pathway. However, it has also been
suggested that the loss of RP mRNA after a heat shock is due instead to an accelerated turnover of RP mRNA (15).
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FIG. 1.
The level of RP mRNAs is down-regulated in W303 (wild
type), 169ts (ypt6-1), and Y260 (rpb1-1) strains.
The indicated strains (Table 1) were grown to log phase in YPD at
23°C. An aliquot was harvested, the rest of the culture was shifted
to 37°C, and aliquots were harvested as indicated. Total RNA was
isolated, and 10 µg of RNA was analyzed by Northern blotting.
Individual RNAs were detected by using either antisense RNA probes, for
RPL30 and ACT1, or oligonucleotide probes, for
RPL3, RPL8, and U3 snoRNA, as described in
Materials and Methods.
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In an attempt to distinguish between these alternatives we have
determined the half-life (t1/2) of an RP mRNA
when its transcription is halted in two distinct ways. In one case we
employed strain Y260, which carries rpb1-1, a ts allele of
the largest subunit of RNA polymerase II (Pol II); in such cells, RNA
Pol II-dependent transcription is reduced to less than 10% within 2 min after a shift to 37°C (42, 43). When Y260 is
transferred to the nonpermissive temperature, the mRNA derived from
most RP genes declines with a t1/2 of about 20 min (Fig. 1, lanes 14 to 20; Fig. 2;
Table 2). The contrast between the
effects of a secretory mutant (Fig. 1, lanes 7 to 13) and a polymerase
mutant (lanes 14 to 20) (Fig. 2) supports the hypothesis that the
turnover of RP mRNAs is accelerated due to heat shock in wt or
sec cells.
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FIG. 2.
A portion of the data used to generate Table 2. Cultures
were handled as described in the text. The values represent the levels
of the indicated RNA measured by PhosphorImager analysis, normalized to
the amount of U3 RNA in that lane of the gel, and further normalized to
100% at the start of the experiment. Note that the repression of the
GAL1-L30 transcripts by glucose is indistinguishable from
the repression of the RPL30 transcripts by the secretory
defect. The transcripts shown are as follows, with the method of
repression in parentheses: ACT1-L30 (rpb1-1) ( ), RPL30
(rpb1-1) ( ), RPL30 (ypt6-1) ( ), GAL1-L30
(glucose) ( ), ACT1 (rpb1-1) ( ), RPL30-ACT1
(rpb1-1) ( ), and RPL30-ACT1 (ypt6-1) ( ).
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To halt transcription in a way that does not depend on a temperature
shift we employed modified RPL30 in which the UAS was replaced with that of GAL1. When this gene is used as the
sole source of L30, cells will grow only in the presence of galactose. In that case substantial RPL30 mRNA is present (Fig.
3A). Once the galactose is replaced with
glucose, transcription is immediately repressed (20, 30);
the mRNA derived both from GAL1-RPL30 (Fig. 2) and from the
GAL1 and GAL10 genes themselves declines rapidly
(Fig. 3A), with a t1/2 of 5 to 7 min for the
transcript encoding L30 and 3 to 5 min for the GAL genes
(Fig. 3A and Table 2), as observed previously (3).
ACT1 mRNA and U3 RNA are unaffected. This result suggests
that the t1/2 for the RPL30 mRNA is
artificially extended in the rpb1-1 strain. Indeed, when the
rpb1-1 strain is grown on galactose and shifted to the
nonpermissive temperature, the t1/2 of the
GAL1 and GAL10 mRNAs is increased greatly,
independent of whether the galactose has been replaced with glucose
(Fig. 3B). Thus, the influence of rpb1-1 on the measured
t1/2 of mRNAs is not limited to the transcripts
of the RP genes.
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FIG. 3.
(A) Direct measurement of the
t1/2 of the mRNA encoding L30. Strain JV7-2a
(rpl30 ::HIS3 [pYE: CEN, URA3
GAL1-RPL30]) was grown at 37°C in YPGal medium and shifted to
YPD (4% dextrose) as described in Materials and Methods. Cells were
harvested just before and at intervals after the shift, and RNA was
prepared and analyzed by Northern blotting as described previously.
After PhosphorImager analysis, the t1/2 of the
GAL1, GAL10, and GAL-RPL30 mRNAs was
determined by using the stable U3 RNA as a loading control (Table 2).
Note that the same experiment was carried out at 23°C, in which case
the t1/2 of the mRNAs was about twice as long.
(B) Extended t1/2 of the GAL1 and
GAL10 mRNAs in an rpb1-1 strain. Strain Y260
(rpb1-1) was grown in YPGal medium at 23°C. At zero time,
one sample was taken; half the remaining culture was shifted to 37°C,
and the other half was filtered and resuspended in YPD (4% dextrose),
prewarmed, and maintained at 37°C. Samples were taken at the
indicated times, and RNA was prepared and analyzed as for panel A.
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Integrating the data of Fig. 1, 2, and 3 and Table 2 suggests the
following scenario. A temperature shift from 23 to 37°C leads to an
immediate, but temporary, repression of the transcription of RP genes.
In wt cells, the transcription of RP genes resumes after about 20 min.
In a sec mutant, however, the transcription of RP genes does
not resume, and RP mRNA is reduced to a very low level. These are
likely to be two separate phenomena because either the ablation of the
protein kinase C pathway (40) or mutation of the silencing
domain of Rap1p (35) relieves the repression of
transcription due to a secretory defect without affecting the
repression due to heat shock. The immediate kinetics of the repression
due to a defect in the secretory pathway are obscured by the cell's
response to heat shock. Nevertheless, we have found that the inhibition
of the endoplasmic reticulum-Golgi communication with brefeldin A
appears to repress the transcription of RP genes within 15 min
(36), while direct stress on the plasma membrane, with the
intercalating drug chlorpromazine, leads to repression almost
immediately (40).
These data suggest that there is no need to invoke accelerated turnover
(15) to explain the response of RP mRNA to a temperature shift. The t1/2 of RP mRNAs observed in response
to a temperature shift in either a wt or a sec cell is the
same as that observed at 37°C when transcription is halted due to
glucose repression of the GAL1 promoter (Fig. 3A). However,
the extended t1/2 of both the RP and the GAL
mRNAs when transcription is extinguished by the inactivation of RNA
polymerase II suggests that this experimental approach may be having
broader physiological effects on the RNA metabolism of the cell.
RP promoter mediates repression.
The experiments whose results
are shown in Fig. 1 and 2 demonstrate an almost instantaneous and
complete repression of RP gene transcription. What sequence elements of
the RP genes are responsible for this repression? The promoter of
RPL30 (Fig. 4A) resembles the
promoters of most RP genes, with two Rap1p binding sites as the major
UAS, and one or two T-rich regions which have some promoter activity
(41, 45, 47), followed by a less well defined region that
contains the putative TATA box. Previous work has implicated the Rap1p
binding sites as the elements mediating the regulation of
transcription, in the response to a carbon source shift (7,
16), to amino acid starvation (37), and to cyclic AMP
(cAMP) (25, 39), but not in the response to a temperature shift (41, 45).
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FIG. 4.
(A) The promoter of RPL30. The start codon is
boxed. The two Rap1p binding sites are indicated by a heavy underline,
and the two T-rich regions are indicated by a light underline. The
initiation of transcription, termed +1 and marked with an arrow, is 58 nucleotides upstream of ATG. (B) The RPL30-ACT1 fusion gene.
See Materials and Methods for details. (C) Constructing the fused gene
with the ACT1 promoter driving the RPL30
transcript (constructs A to D); see Materials and Methods. The stippled
area represents sequences from ACT1. Nucleotides from the
RPL30 promoter were fused to the ACT1 TATA region
to form constructs B, C, and D. The hatched boxes are ACT1
sequences; the open boxes are RPL30 sequences; the black
boxes represent the RPL30 Rap1p binding sites. The line
represents the L30 transcript. The nucleotide boundaries of the
RPL30 sequences are shown above the constructs, and those of
the ACT1 sequences are shown below the constructs. Because
the ACT1 gene has multiple sites of transcription
initiation, the numbering is in reference to ATG of the coding
region.
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To determine which of the elements in the promoter of RP genes mediate
the repression of transcription in response to a defect in the
secretory pathway we made two promoter swap constructs, in which the
RPL30 promoter drives the ACT1 transcript (Fig.
4B) and the ACT1 promoter drives the RPL30
transcript (Fig. 4C). Primer extension demonstrated that the resulting
ACT1 transcripts were initiated at the same site as that for
the endogenous ACT1; about half of the RPL30
transcripts were initiated at the correct site, with the rest being
initiated nine nucleotides downstream (data not shown). The
ACT1-RPL30 construct is not responsive to the ypt6-1 mutation (Fig. 5, lanes
1 to 5). The RPL30-ACT1 construct is responsive to the
ypt6-1 mutation, with the level of its transcript decreasing
monotonically from the time of the temperature shift (Fig. 5, lanes 18 to 23, and Fig. 2). The rate of decline in RPL30-ACT1 mRNA
is lower than that of RPL30, with a
t1/2 of about 16 min, presumably because the
intrinsic stability of the ACT1 transcript is greater. Once
again, the t1/2 measured here is substantially shorter than that measured with the rpb1-1 mutation (Fig. 2
and Table 2), reinforcing the suggestion that rpb1-1 has a
broad effect on mRNA stability.
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FIG. 5.
Rap1p binding sites mediate the repression of RP mRNAs.
ypt6-1 rpl30::HIS3 strains
containing constructs A to D (strains BL174 to BL177; Table 1) and
strain JW1201, in which RPL30-ACT1 is the only source of
actin sequences, were grown to log phase at 23°C and then shifted to
37°C. Aliquots were harvested at the indicated times, and RNA was
prepared and analyzed as described previously.
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Rap1p binding sites mediate RP gene repression.
If the
RPL30-ACT1 construct is repressed while the
ACT1-RPL30 transcript is not, the influence of the secretory
pathway on RPL30 must depend solely on sequences upstream of
the transcription initiation site. In an attempt to identify which
sequences are involved, the UAS of the ACT1 promoter of
construct A was replaced with fragments of the RPL30
promoter (constructs B, C, and D shown in Fig. 4C). Plasmids carrying
these genes were used to transform BL17, a diploid strain with the
genotype RPL30/rpl30::HIS3
YPT6/ypt6-1. Following sporulation and dissection of tetrads,
strains carrying ypt6-1 and with constructs A to D as the
only source of L30 were identified (strains BL174 to BL177 in Table 1).
At the permissive temperature, the level of mRNA derived from each of
the constructs was about the same (Fig. 5, zero time), suggesting that
the two Rap1p sites (construct D) are sufficient to substitute for the
UAS of ACT1. Upon a shift to the nonpermissive temperature,
there is a rapid decline in the levels of mRNA derived from
constructs B, C, and D. Clearly the presence of Rap1p binding sites,
either with (construct B) or without (construct D) T-rich regions,
makes the test gene responsive to the secretion defect. These results
suggest that in this context the 40-bp sequence containing the two
Rap1p binding sites is a sufficient cis element to effect
repression in response to a defect in the secretory pathway.
It has been reported that Rap1p is degraded in cells depleted of cAMP
(39), resulting in the reduced transcription of RP genes. To
determine if limiting Rap1p lies behind the repression of RP
transcription in response to a defect in the secretory pathway, we
overexpressed Rap1p in both W303 and 169ts, by transforming each strain
with a 2µm plasmid carrying wt RAP1. The shift of the
transformants from 23 to 37°C led to a loss of RP mRNA that was
indistinguishable from that shown in Fig. 1 and 5 (data not shown),
suggesting that the repression of RP gene transcription is not due to
limiting Rap1p.
The SIR complex is not involved in the repression induced by
secretion defects.
While Rap1p is a major transcriptional
activator, it is also a major transcriptional silencer, at the silent
mating type loci and at telomeric regions (reviewed in reference
49). In both cases, Rap1p recruits Sir3p and Sir4p
to form a complex that inhibits the transcription of the adjacent genes
(38). Since our results implicate Rap1p in mediating the
repression of RP gene transcription, we asked if Sir3p and Sir4p are
involved in the repression. The effects of tunicamycin on the mRNA
levels of RP genes and others were determined in strains lacking
components of the SIR complex (Fig. 6).
It is apparent that the presence of tunicamycin leads to a substantial
repression of the transcripts of both RPS6 and RPL30, with little effect on ACT1 or
PYK1. The deletion of SIR2, SIR3, or
SIR4 does not alter the repression of the RP genes. The induction of KAR2 is an expression of the "unfolded
protein response" demonstrating that the tunicamycin is active on
these cells (4).
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FIG. 6.
The SIR complex does not mediate the repression of RP
genes in response to a defect in the secretory pathway. Cultures of
strains YDS2 (wt), YDS714 (sir2), YDS430
(sir3), and YDV122 (sir4) were grown in YPD
at 30°C. One half of each culture was treated with 1 µg of
tunicamycin per ml for 2 h. All eight cultures were harvested on
crushed ice, and RNA was prepared and subjected to Northern analysis,
by using several probes.
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As a more direct test, strains containing null alleles of either
SIR3 (YDS430) or SIR4 (YDV122) were crossed with
169ts, the strain carrying ypt6-1. From each diploid, we
selected a tetrad that provided the four combinations of the
YPT6 and SIR3 alleles (strains BL180 to BL183;
Table 1) or the SIR4 allele (strains BL185 to BL188; Table
1). If either Sir3p or Sir4p were essential for the repression, its
absence would eliminate the repression of RP mRNAs in cells carrying
the ypt6-1 allele. As shown in Fig. 7, the repression of the transcription of
RP genes, induced either in YPT6 cells by heat shock or in
ypt6-1 cells by a failure in the secretory pathway, depends
neither on Sir3p nor on Sir4p.
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FIG. 7.
Neither Sir3p (A) nor Sir4p (B) is involved in the
repression of RP gene transcription in a sec mutant or
during heat shock. Strains of the indicated genotypes (Table 1) were
grown to log phase in YPD at 23°C. An aliquot was harvested, the
cultures were shifted to 37°C, and aliquots were harvested at the
indicated times. RNA was prepared and analyzed as described previously,
except that all RNAs were detected with 32P-labelled
oligonucleotide probes.
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Sir2p is not necessary for repression of either rRNA or RP
genes.
Sir2p is another participant in the repression of the
silent mating type loci, although its relationship to Rap1p is less clear than that of Sir3p and Sir4p. Surprisingly, Sir2p has recently been shown to participate in the silencing of a Pol II-transcribed gene
within the ribosomal DNA locus (52), although it has not been implicated directly in the control of rRNA transcription. To
determine whether Sir2p plays a role in the repression of ribosome synthesis, we deleted SIR2 or its close relative
HST1, which can partially substitute for SIR2 in
silencing (5), from a strain carrying a ts mutation in
sly1, an essential component of the secretory pathway
(36), yielding strains JW1210 and JW1211. The results of
shifting these two strains to the nonpermissive temperature are shown
in Fig. 8. In this case, the cells were grown in a medium lacking methionine and pulsed for 3 min with [C3H3]methionine as a measure of rRNA
transcription. RNA prepared from the cells was separated on an
acrylamide gel, the upper portion was subjected to fluorography, and
the lower portion was subjected to Northern analysis. From the latter
it is apparent that the mRNA for L30 disappears rapidly, just as in the
ypt6-1 strain shown in Fig. 1. C3H3
groups are incorporated predominantly into 35S rRNA, which is then
processed through the intermediate 27S and 20S species to the mature
25S and 18S rRNAs (56). In a 3-min pulse (Fig. 8, 0-min
lanes), most of the radioactivity has already passed into the 27S and
20S intermediates, and some is in mature 18S rRNA. We have previously
shown that the transfer to the nonpermissive temperature causes a rapid
repression of rRNA transcription in sly1-1 cells, with
little effect on wt cells (36). Similarly, the transfer of
the sly1-1 sir2 or the sly1-1
hst1 double mutant strains to the nonpermissive
temperature leads to a strong inhibition of the incorporation of
C3H3 into rRNA (Fig. 8, 20- and 60-min lanes),
just as for the cells carrying sly1-1 alone. What little 35S
RNA is formed is processed slowly if at all, presumably due to a lack
of RPs. We conclude that neither Sir2p nor Hst1p is involved in the
repression of ribosome synthesis in response to a defect in the
secretory pathway.
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FIG. 8.
Neither Sir2p nor Hst1p is involved in the repression of
ribosome synthesis in response to a defect in the secretory pathway.
Cultures of strains JW1210 and JW1211 were grown in methionine-free
dropout medium at the permissive temperature of 23°C. At zero time an
aliquot was labelled with 60 µCi
[C3H3]-methionine for three minutes and
poured onto crushed ice. The remainder of the culture was shifted to
37°C, and aliquots were similarly pulsed with
[C3H3]-methionine after 20 and 60 min. RNA
was prepared, and equal amounts were subjected to polyacrylamide gel
electrophoresis. The upper portion of the gel was impregnated with
En3Hance and subjected to fluorography to show the
incorporation of C3H3 groups into rRNA. The
lower portion was used for Northern analysis with probes against
RPL30 and against 5.8S rRNA as a loading control (5.8S rRNA
has no methyl groups).
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Non-Rap1p binding sites also mediate RP gene repression.
Although Rap1p binding sites contribute most of the transcriptional
activation of RP genes, some activity remains after the deletion of the
Rap1p binding site, due to the T-rich elements (41, 45, 47).
This residual activity of the RP genes still responds to heat shock,
implicating sequences other than the Rap1p sites in the regulation of
transcription of these genes. To identify such sequences and to
determine if they are also involved in the response to a secretion
defect, we developed a series of constructs based on the reporter gene
RPL30-GFP, in which the ORF of GFP replaced the ORF of L30,
starting with amino acid 4 in the second exon of RPL30 (Fig.
9A, construct E). The spliced transcripts contain the 5' untranslated region of RPL30, seven
nucleotides from the RPL30 ORF, the GFP ORF, and the 3'
untranslated region of RPL30. Note that the repression of
RPL30 is independent of sequences downstream of the
transcription initiation site (ACT-RPL30; Fig. 5, lanes 1 to
5). Increasing portions of the RPL30 promoter were replaced
with four Gal4p binding sites derived from the GAL1 promoter, generating constructs F, G, and H (Fig. 9A). The reporter gene constructs, on CEN-based plasmids, were transformed
into cells carrying the ypt6-1 allele. The necessary L30
protein is supplied by the genomic RPL30. Since the GFP ORF
is 400 nucleotides longer, the RPL30 and
RPL30-GFP mRNAs can be compared directly on Northern blots
by using a probe complementary to the first exon of RPL30.
View larger version (42K):
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|
FIG. 9.
(A) Construction of fused genes between GAL1
and RPL30-GFP (constructs E to H). The stippled area
represents sequences from the GAL1 UAS. The nucleotide
boundaries of the RPL30 sequences are shown, and numbering
follows the conventions described in the legend to Fig. 3A. The black
boxes represent the RPL30 Rap1p binding sites. (B) The
180-bp cis element mediates the repression of RP gene
transcription in a sec mutant. ypt6-1 cells
carrying constructs E to H were grown to log phase in uracil-free
medium containing 2% glucose at 23°C. An aliquot was harvested, the
rest of the cultures were shifted to 37°C, and aliquots were
harvested at intervals. Total RNA was isolated and analyzed as
described previously. mRNAs for RPL30 and
RPL30-GFP were detected with an RNA probe that is
complementary to the first exon of RPL30. The snoRNA U3 was
detected with an oligonucleotide probe. (C) The same as panel B except
that cells were grown in 2% galactose to induce the GAL1
UAS. GAL1 mRNA was detected with an antisense RNA probe.
|
|
For the first set of experiments the cells were grown in glucose to
repress transcription from the GAL1 region, thereby
revealing the transcriptional contribution of the residual
RPL30 sequences (Fig. 9B). In a ypt6-1
background, following a temperature shift, construct E
(RPL30-GFP) is repressed as severely as endogenous RPL30, confirming that the promoter of the RP genes mediates
the repression caused by a failure in the secretory pathway. Deletion of the two Rap1p binding sites, leaving the T-rich domains (construct F), leads to the loss of 90% of the transcriptional activity. The 10%
of the activity remaining is repressed as severely as endogenous
RPL30. The presence of a single T-rich domain (construct G)
has only 5% of normal transcription, also repressible. Deletion of
both Rap1 sites and both T-rich domains (construct H) leads to a loss
of any detectable transcription. These results confirm that the T-rich
region can activate some transcription of RPL30, consistent
with the observations for the other RP genes RPL28, RPS14, and RPL25 (41, 45, 47).
Nevertheless, upon a temperature shift whatever residual transcription
that remains is repressed in the ypt6-1 mutant (Fig. 9B).
This result, in the absence of Rap1p sites, implicates the remaining
downstream elements of the RPL30 promoter in the repression
of transcription.
Growth of these strains in a medium containing galactose permits Gal4p
binding to the sites present in constructs F, G, and H, activating
transcription, at the permissive temperature, to slightly higher levels
than with the RPL30 promoter itself (constructs F, G, and H;
Fig. 9C). Nevertheless, when the cells are shifted from 23 to 37°C,
the transcription of constructs F, G, and H is strongly repressed. The
level of GAL1 transcripts is not dramatically affected, at
least at 20 and 40 min after the temperature shift (Fig. 9C). Since the
t1/2 of GAL1 transcripts is <5 min
(3) (Fig. 3A), Gal4p-promoted transcription must be
continuing. Comparison of construct E with constructs F, G, and H
suggests that a defect in the secretory pathway can largely, though not
completely, repress the transcription of an RP gene that is driven by a
novel activator, in this case Gal4p. As the results for constructs F
and H barely differ, we again conclude that the T-rich regions play
little role in the repression of transcription. Thus, the repression of
construct H suggests that the 180 bp that lie between the T-rich elements and the origin of transcription of RPL30 contain
sequence elements that are sufficient for a major proportion of the
sec-dependent repression of transcription of an RP gene.
Comparing the data from Fig. 5 and 9 leads us to conclude that either
the Rap1p sites or the 180-bp region will respond to the repression
effected by a failure in the secretory pathway. In a different context,
Neuman-Silberberg et al. (39) also concluded that
Rap1p-binding sites and downstream sequences could play separate roles
in the regulation of RP transcription.
Is TAF145 responsible for the repression of RP gene
transcription?
A recent report suggests that the transcription of
many of the RP genes is particularly vulnerable to a mutation in the
transcription factor TAFII145 (48). Perhaps
TAFII145 is responsible for the effects of the
180-nucleotide region implicated by construct H (Fig. 9B), since this
region contains the TATA box with which TAFII145 is
presumably associated. However, while both IPP1
(48) and ACT1 (our unpublished data) are severely
repressed by ts mutants of TAFII145, they are not repressed
by a ts mutant in the secretory pathway (IPP1; data not
shown). Finally, a genome-wide analysis of the transcriptional effects
of a ts allele of TAFII145 suggests little specificity for
RP genes (17). Thus, it appears that TAFII145 is
not the agent responsible for the repression of transcription of the RP
genes under these conditions.
| |
DISCUSSION |
Turnover of RP mRNA.
The determination of the intrinsic
t1/2 of an mRNA presents several experimental
uncertainties. The most direct but most demanding method is the
approach to equilibrium, which requires not only sensitive
hybridization methods but also the determination of the approach to
equilibrium of the nucleotide pools (12, 23). Alternate
methods require turning off the transcription of all mRNA, using either
inhibitors or the ts allele of rpb1 (reviewed in reference
2). The former have proved inconsistent in yeast. The latter involves three perturbations of the cell: the raising of the
temperature, which brings the heat shock response into play, the
inhibition of any mRNA that might play a direct role in the
t1/2 of a specific mRNA, and the gradual loss of
all the cell's mRNAs, which can change the dynamic of translation and, consequently, of turnover. A more specific approach is the use of a
repressible promoter, such as GAL1, with which one can turn off the expression of a limited number of genes.
It is clear from Fig. 1, 2, and 3, as well as from Table 2, that the
decline of RP mRNA after a temperature shift, in either wt or
sec cells, is similar to that observed when the RP mRNA is
under the control of the GAL1 promoter that is suddenly
repressed. This result has two major implications. It suggests that a
heat shock temporarily represses and a sec mutant
permanently represses RP transcription to nearly the same degree as
glucose does the Gal4p-driven genes. It also suggests that the
intrinsic t1/2 of RP mRNAs can be estimated from
the decline of the mRNA after a heat shock; for most RP mRNAs that
value would be <10 min at 37°C (9, 61). For some genes we
have made an independent determination of the
t1/2, based on approach-to-equilibrium
labelling; for RPL3 it is 13 min, and for RPL30
it is 16.6 min (23). (Note that those two genes were known
as Rp1 and Rp73, respectively, at that time.) This was carried out on
cells growing in a synthetic medium at 23°C with a doubling time of
132 min, and thus the values are likely to be an underestimate compared
to those based on cells growing in YPD at 37°C. Indeed, in our
studies the t1/2 of the GAL1-RPL30
mRNA increases from 5 to 11 min as the temperature is lowered from 37 to 23°C (Table 2).
We suggest, therefore, that the intrinsic t1/2
of the mRNA encoding L30 is between 5 and 7 min at 37°C. Thus, the
decline of this mRNA after cells are shifted from 23 to 37°C can be
ascribed solely to a repression of transcription, without the need to
invoke an activation of turnover (15).
The data shown in Fig. 1 to 3 also suggest that the use of the
rpb1-1 mutant may lead to severely misleading estimates of the t1/2 of mRNAs. This could be either a
general effect due to the pleiotropic consequences of halting all Pol
II transcription or a specific effect on the genes subject to severe repression.
One might ask why the t1/2 of RP mRNAs is so
short, since replacing them at frequent intervals seems an unnecessary
use of resources. Indeed, the t1/2s of the mRNAs
encoding the abundant glycolytic enzymes are much longer
(14). An explanation may be that the level of production of
RPs must be closely monitored (27). Because the RPs
participate in the assembly of a complex structure and because they are
generally strong RNA-binding proteins, an excess of an RP may be far
more deleterious to the cell than an excess of a glycolytic enzyme.
Therefore, it seems likely that there is selective pressure to maintain
a short t1/2 for RP mRNAs in order to more
closely control the relative production of the many RPs.
General considerations regarding transcription of RP genes.
Before discussing our data regarding the control of transcription of RP
genes, we argue on two grounds, magnitude and coordination, for the
potential special nature of RP gene transcription and thus for the
likelihood that it has unusual features.
(i) The transcription of ribosomal proteins is a major portion of the
Pol II activity of the cell. As measured by SAGE analysis (57), RP genes account for 20 of the 30 most abundant
mRNAs; each RP gene is represented, on average, by about 30 to 50 mRNAs per cell (our analysis of data provided by the authors of
reference 57). The 137 RP genes, therefore, would
account for >4,000 of the mRNAs in the cell. A more direct measurement
has recently been reported by Holstege et al. (17). Based on
an estimate of 15,000 mRNAs per cell, they determined that 132 of the
137 RP genes contributed 4,437 mRNAs, about 30% of the total. This is
a reasonable number because the ~15,000,000 ribosomal proteins (~200,000 ribosomes/cell × 78 proteins/ribosome) make up about 15% of the protein mass of the cell, and even more of the protein number, since they average only ~150 amino acids in length. As shown
in Table 2, the t1/2 of an RP mRNA is 5 to 7 min. This value is consistent with our observations of the effect of
heat shock on the mRNA levels of many RP genes (11). It is
also consistent with the data recently reported by Eisen et al.
(9), in which the mRNA level for most RP genes had declined
to less than 20% of normal by 20 min after a heat shock. Yet, mRNAs
encoding most other genes have a t1/2 of >15
min (17) (based on the use of the rpb1-1 allele,
which admittedly may be misleading). Thus, if RP mRNAs account for 30%
of the total mRNAs yet have a t1/2 that is
substantially shorter than those of most other mRNAs, we are led to
conclude that the RP genes account for nearly 50% of all Pol II
initiation events.
(ii) As would be expected for genes encoding components of a molecular
machine, the 137 RP genes appear to be regulated in lockstep (reviewed
in reference 44). This is true of responses to heat
shock (11, 24), to a defect in the secretory pathway (34 and this paper), to growth conditions such as C
source (16, 22), to levels of cAMP (25, 39), and
to the deprivation of amino acids (37, 62). During the
growth cycle, RP genes are repressed as the cells enter late log phase
(6, 21) and are induced dramatically within 10 min after
stationary cells are diluted into a fresh medium (unpublished data). By
using classical methods no exceptions have been found among the 20 or
so proteins whose mRNAs have been studied or among the 50 or so
proteins whose synthesis has been studied in a few situations
(11). Very recent data from a genome-wide analysis of a few
conditions, e.g., heat shock (9) and diauxie (6),
suggest that none of the RP genes escapes from this coordinate
regulation, although a few genes with apparently intermediate results
will require more direct analysis.
It is intriguing to consider why S. cerevisiae has evolved
to utilize transcription as its primary method to regulate the production of RPs, while both eubacteria (reviewed in reference 64) and vertebrates (reviewed in reference
33) have chosen to regulate RP synthesis largely at
translation, albeit in very different ways. Indeed, a recent
transcriptome analysis of mouse fibroblasts during the transition from
stationary to growth phase found almost no change in the levels of RP
mRNAs (19), although there is a substantial increase in the
rate of RP synthesis (55).
Role of Rap1p in the repression of RP gene transcription.
Rap1p is the major factor activating the transcription of nearly all of
the RP genes, as well as many other genes with abundant transcripts,
such as those encoding elongation factor 1
(EF1) and the enzymes
of the glycolytic pathway. Rap1p is also the primary element of the
complex that silences both the silent mating type loci and genes
adjacent to telomeres (49). Therefore, Rap1p is a likely
candidate for effecting the silencing of the RP genes in response to
stress. Indeed, we find that just 40 bp containing the two Rap1p
binding sites of the RPL30 gene are sufficient to make the
ACT1 promoter respond like an RP gene to a defect in the
secretory pathway (Fig. 5, construct D). Furthermore, in cells carrying
the rap1-17 allele, which encodes a truncated form of Rap1p
that retains both its DNA binding domains and its activation domain but
not its silencing domain, the RP genes are no longer silenced in
response to a defect in the secretory pathway (35), although
temporary silencing in response to a temperature shift still occurs.
This effect of the rap1-17 allele is also true for genes
that have no Rap1p binding sites, such as RPL3
(35) and construct G shown in Fig. 9A (data not shown). This
observation not only implicates Rap1p in the pathway between the
secretory system and the RP genes but also suggests that a different
pathway is used for the repression of RP gene transcription in response to a temperature shift.
Yet several facts suggest that conventional silencing by Rap1p is not
responsible for the repression of RP genes. Abf1p, rather than Rap1p,
is the major transcription factor for several RP genes that are also
repressed in response to a defect in the secretory pathway, e.g.,
RPL3 (Fig. 1). In addition, Rap1p-mediated silencing at
telomeres and at silent mating type loci requires Sir3p and Sir4p
(31), neither of which is necessary for the silencing of RP
genes (Fig. 6). Finally, sequences downstream of the Rap1p binding
sites, adjacent to the promoter of RPL30, will silence the
transcription driven by Gal4p, in response to a temperature shift or to
a defect in the secretory pathway (Fig. 9). These sequences contain the
putative TATA box and presumably bind TBP and its associated TAFs, as
well as the sequences adjacent to the transcription initiation site.
Thus, our attempts to identify the cis-acting sequences of
RP genes that mediate the repression of transcription have led to an
apparent contradiction. Either the Rap1p binding sites or the
promoter-proximal sequences can play such a role. Yet the effect of the
rap1-17 allele suggests that Rap1p is necessary for
silencing in response to a defect in the secretory pathway. Our
conclusion, then, is that for Rap1p to activate the transcription of an
RP gene it must bind to upstream sequences, yet for Rap1p to repress
the transcription of an RP gene it need not bind to the gene directly.
It remains to be seen how this fascinating protein can pull off such a trick.
Whatever the mechanism, the sudden silencing of the RP genes, which
account for 50% of Pol II activity, must have a dramatic effect on the
overall transcriptional economy of the cell. What influence does this
sudden release of transcriptional potential have on the transcription
of other genes?
We are grateful to Josep Vilardell for a close reading of the
manuscript, to Ian Willis, Roy Parker, Allan Sachs, Christine Brown,
and Allan Jacobson for useful discussions, to Jung-Hoon Sohn for
communicating unpublished data, to David Shore, Art Lustig, and Myra
Derbyshire for strains and plasmids, and to Mary Studeny and Saqui Huq
for technical assistance.
This research was partially supported by NIH grants GM25532 to J.R.W.
and CA13330 to the Albert Einstein Cancer Center.
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Abstract
Introduction
Present address: Department of Biochemistry and Molecular
Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Present address: Juvenile Diabetes Foundation International, New
York, NY 10005.
