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?????
???. 13: 1505?1518 (1997)
Regulation of Ribosome Synthesis in Yeast
RUDI J. PLANTA
Department of Biochemistry and Molecular Biology, IMBW, BioCentrum Amsterdam, Vrije Universiteit,
de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Received 4 September 1997; accepted 10 September 1997
Yeast 13: 1505?1518, 1997.
??? ????? ? Saccharomyces cerevisiae; bibosome biogenesis; ribosomal RNA; ribosomal proteins; gene regulation
CONTENTS
Introduction
Transcription of the ribosomal RNA genes
Expression of the ribosomal protein genes
Acknowledgement
References
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INTRODUCTION
The formation of functional ribosomes is a highly
complex process requiring the interplay of a large
number of molecular reactions. Ribosome formation in eukaryotic cells involves the participation
of three different RNA polymerases for the production of four different rRNAs and about 80
different ribosomal proteins (r-proteins), (almost)
all of them present in a single copy per ribosome.
Although the various stages in the production of
the different ribosomal constituents, as well as
their assembly into active ribosomes, occur in
different cellular compartments, normally growing
cells do not contain free pools of most of the
ribosomal components. Therefore, the expression
of the various ribosomal genes must be subject to
tight coordinate control to ensure the production
of equimolar amounts of the different rRNA and
r-protein constituents. In addition, cells can adjust
the expression of the whole set of ribosomal genes
in a concerted fashion in response to variations
in environmental conditions, such as changes
*Correspondence to: R. J. Planta, Department of Biochemistry
and Molecular Biology, IMBW, BioCentrum Amsterdam, Vrije
Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The
Netherlands. Tel: (+31) 20 4447548; fax: (+31) 20 447553;
e-mail: brink@chem.vu.nl
Contract grant sponsor: Netherlands organization for Scientific
Research.
CCC 0749?503X/97/161505?14 $17.50
? 1997 John Wiley & Sons, Ltd.
in nutritional conditions or temperature etc.,
which alter the demand for protein biosynthetic
capacity.
In yeast, this regulation of expression is effected
almost entirely at the level of transcription25,38 (see
review by Raue? and Planta54). This makes high
demands upon the transcriptional apparatus, the
more so since the r-protein mRNAs in yeast all
have a relatively short half-life.26 The transcription
of the more than 100 units of rRNA genes in yeast
represents about 60% of the total transcription of a
growing cell. Furthermore, the transcripts of the
137 genes encoding the 78 r-proteins52 belong, in
spite of their short half-life, to the most abundant
mRNAs in the yeast cell.66 Therefore, the regulation of ribosome synthesis is a key process for
cellular viability.
Although a vast amount of information has
been collected on the structure and function of
ribosomal genes in yeast, we still do not fully
understand the regulatory mechanisms controlling
the expression of theses genes. Almost nothing is
known about the transduction of the environmental signals, and the final receptors of these
signals. However, recent findings have opened the
door to a better understanding of the mechanisms by which the rate of ribosome formation is
adjusted to the physiological requirements of the
cell, although there is still a long way to go before
a complete picture will be obtained.
TRANSCRIPTION OF THE RIBOSOMAL
RNA GENES
Regulation of transcription of the rRNA genes
may be central to the intricate process of ribosome
?. ?. ??????
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Figure 1. Organization of the rRNA genes of the yeast Saccharomyces cerevisiae. At the top, a part of the chromosomal tandem
array of 2150 rDNA units is shown. One rDNA unit is enlarged. Regions encoding the rRNA genes are indicated by black bars.
Open bars represent transcribed spacers and non-transcribed spacers (ETS, external transcribed spacer; ITS, internal transcribed
spacer; NTS, non-transcribed spacer). The Pol I and Pol III transcripts are indicated by arrows. At the bottom, the cis-acting
elements involved in regulation of Pol I transcription are enlarged. The enhancer and promoter are indicated by striped boxes. The
Pol I transcript proceeding to the main terminator within the enhancer is represented by a stippled arrow, which part is rapidly
processed (at sites indicated by the vertical arrows) to form the mature end of the 26S rRNA. The black ovals indicate binding sites
for the protein Reb1.
biosynthesis in response to environmental conditions. In Saccharomyces cerevisiae the rRNA
genes are arranged in a tandem array of 140?
200 rDNA units of 9�kb on the long arm of
chromosome XII.49 In addition, R-loop analysis
has revealed the presence in yeast cells of a small
number of circular DNA molecules consisting of
one or more of these rDNA units.44 Since replicative intermediates of these circular molecules have
been observed occasionally, they may play a role in
regulating the number of active rRNA genes.
The genes encoding 17S, 5� and 26S rRNA are
arranged in a pre-rRNA operon, which is transcribed by RNA polymerase I (Pol I) in the
nucleolus (cf. Figure 1). The three genes are separated by two internal transcribed spacers (ITS1
and -2) while external transcribed spacers are
present upstream from the 17S rRNA gene (5*ETS) and downstream from the 26S rRNA gene
(3*-ETS). The ITS and ETS sequences are removed
post-transcriptionally in an ordered series of
steps (reviewed by Venema and Tollervey68). In
S. cerevisiae, unlike in other eukaryotes, each
rDNA unit also contains a 5S rRNA gene which is
located within the non-transcribed spacer regions
separating two consecutive rRNA operons and is
transcribed by Pol III.50 The direction of transcription of the 5S rRNA gene is opposite to that of
? 1997 John Wiley & Sons, Ltd.
the large rRNA operon. A number of additional
5S rRNA genes differing from those in the 9�kb
repeats are located at the telomere-proximal end
of the array,41 but these genes appear to be
transcriptionally inactive.
The cis-acting elements involved in the regulation of Pol I transcription in yeast have been
extensively studied, both in vivo and in vitro. Two
main regulatory elements were found to be present
within the yeast rDNA unit: (1) the Pol I promoter, which is located between nucleotide positions "146 and +8 relative to the transcription
initiation site (+1); 22,30,48 (2) a 170?190 bp long
transcriptional enhancer, located about 2�kb
upstream of the transcription initiation site, i.e.
100?300 bp downstream of the preceding rRNA
operon (see also Figure 1). This enhancer element
can stimulate rRNA transcription about 15?30
fold,9,10 can act bidirectionally, and lies close to
and even partially overlaps a number of 3*-end
generating sites, including the termination site.22,57
Using a system in which tagged (mutated) rDNA
units were integrated into the rDNA locus,31 compelling evidence was obtained that the intergenic
spacer between the enhancer and the promoter of
the yeast rDNA units contains no other transcriptional regulatory elements for Pol I, in contrast to
the situation in other eukaryotes.
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???. 13: 1505?1518 (1997)
?????????? ?? ???????? ?????????
The yeast Pol I promoter has been analysed
in vivo by transformation of yeast cells with
(mutated) rDNA minigenes as well as in vitro using
the same minigenes as templates.30,48 In general,
an rDNA minigene is a size-reduced rRNA
operon, in which the (presumed) Pol I transcription initiation and 3*-end generating sites flank a
reporter sequence, which replaces the interjacent
sequences. Such minigenes have been shown in
several studies to be transcribed correctly both
in vivo and in vitro by Pol I.
In Figure 2A, the structure of the rDNA minigene construct most frequently used in the studies
of the organization of the yeast Pol I promoter is
shown.48 In order to ensure efficient transcription
of this minigene, designated SIRT, a copy of the
rDNA enhancer was included both upstream and
downstream of the artificial transcription unit,
since the enhancer can act bidirectionally.19,31 We
have tried to optimize the transcript stability by
including a relatively large part of the 26S rRNA
gene in the construct and by using a short (18 bp)
reporter oligonucleotide. The SIRT minigene produces a transcript of 681 nucleotides which could
be easily detected in vivo using the reporter oligonucleotide as a probe. With this minigene, 5*- and
3*-deletion mutants as well as linker scanning
mutants (LSM) of the presumed Pol I promoter
were constructed, as described in detail previously.48 The LSMs were obtained by appropriate
combinations of 5*- and 3*-deletion mutants,
joined by a 12 bp scanning linker.
Using the series of the 5*- and 3*-deletion
mutants, it was found that the rDNA promotor in
yeast extends from "146 to +8.30,48 Analysis of
the transcripts derived from the LSMs, both in vivo
and in vitro, revealed that the yeast promoter
consists of three domains (see Figure 2B, C), which
is somewhat different from the two-domain structure found in higher eukaryotes. Domain I ("28
to +8) in yeast appears to be equivalent to the core
promoter element (CPE) found in other eukaryotes, and domain III ("146 to "91) may be
similar to the upstream control element (UCE).
Recently, evidence has been obtained indicating
that domain II ("76 to "51) must be regarded as
part of a (bipartite) UCE.24 Spacing between the
promoter domains was found to be extremely
important for efficient initiation of transcription.
An insertion of 4 bp between domains I and II,
which distorts the orientation of these domains
by about half a turn of the DNA helix, drastically decreases transcription in vivo.48 Similar
? 1997 John Wiley & Sons, Ltd.
1507
conclusions were drawn from an in vitro analysis.5
Using a set of mutants changing the spacing
between positions "49 to "22, it was shown that
correct spacing between domains II and I is of
critical importance. Furthermore, in the same
study,5 it was shown that the spacing between
domains III and II, although both are part of the
UCE, is critical and that proteins binding to these
domains must be positioned on the same face of
the DNA helix for maximal activity.
At least 25 proteins are involved in the initiation
of transcription of the rRNA operon, i.e. apart
from the 14 subunits of Pol I there are 11 additional factors (see Figure 3). A multiprotein
transcription factor, designated UAF (Upstream
Activating Factor), which is distinct both structurally and functionally from the transcription initiation factor UBF described for other eukaryotes,
binds to the UCE, encompassing domains II and
III of the Pol I promoter. Five proteins were
originally recognized in the UAF, which include
the products of the genes called RRN5, RRN9 and
RRN10, and in addition two uncharacterized proteins, p30 and p18.24 Recent studies showed that
p18 is histone H3, and that in addition histone H4
is also present in UAF (M. Nomura, personal
communication). The nature of p30 is presently
unknown. The presence of H3 and H4 in UAF
suggests that the UAF?rDNA interaction may
involve DNA wrapping around UAF in a similar
way as in nucleosomes. Indeed, the binding of
UAF to the UCE results in the formation of a very
stable complex, committing that rDNA template
to transcription. This complex, together with a
TATA-box binding protein (TBP) (and perhaps
another unidentified factor),62 helps to recruit
other essential factors to the promoter, in particular the so-called core factor (CF) which binds to
the core promoter element, domain I32 (see Figure
3). This CF, consisting of three subunits (encoded
by the genes RRN6, RRN7 and RRN11, respectively), interacts strongly with TBP and, therefore,
appears to be similar to the SL1 factor of metazoan systems. The CF-complex is absolutely
required for correct rDNA transcription and
stimulates, together with an additional factor,
Rrn3p, the recruitment of Pol I to the promoter.76
Rrn3p forms a complex with Pol I, even in the
absence of DNA,76 and is required for the formation of a functional initiation complex. In fact, for
basal transcription, only Rrn3p and CF are required in addition to Pol I. In terms of modulation
of rRNA transcription in response to nutritional
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Figure 2. Dissection of the promoter for RNA polymerase I in yeast. (A) The rDNA minigene construct, SIRT, aligned with a
rDNA unit. Black boxes represent the coding regions of the rRNA genes. Boxes, designated P, E and R are the Pol I promoter,
the enhancer element and the reporter oligonucleotide (ACACCGTCTCGAGAATGC) in the minigene, respectively. Arrows
represent the Pol I transcripts of the chromosomal rRNA operon and the minigene (681 nucleotides long), respectively. The
minigene was inserted in the SacI-site of the vector pEMBL Ye30-� (see ref. 48 for further details). Abbreviations of relevant
restriction enzyme sites: K, KpnI; St, StuI; B, BamHI; Bg, BglII; Bc, BclI; Hp, HpaI; Sc, SacI; Tq, TaqI; X, XhoI. (B) Relative
transcriptional activity and competition ability of the linker scan promoter mutants as compared with the activity of the
non-mutated promoter (WT=100%). The histogram represents the averages of the results (on autoradiograms) from two
independent sets of experiments. The filled bars display the relative activity of the mutant promoters, the open bars the ability of
the mutant promoters to compete with the second template (SIRT�5).30 (C) Organization of the Pol I promoter as interpreted
from the in vivo48 and in vitro30 results. Approximate locations of the three domains I, II and III, are indicated. UCE, Upstream
control element; CPE, core promoter element. Modified Figure, adapted from ref. 30.
changes Rrn3p is a likely target for the extracellular signals. But phosphorylation of UAF
components may also modulate rRNA synthetic
activity in response to environmental signals.
? 1997 John Wiley & Sons, Ltd.
The other regulatory element in the yeast rDNA
unit, the enhancer, is much less well-defined than
the promoter. The enhancer is a 170?190 bp (depending on the type of rDNA unit) EcoRI-HindIII
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Figure 3. Schematic model of transcription initiation complex assembly at the yeast Pol I promoter (see Figure 2C).
The bent arrow indicates the transcription initiation site. Panels A, B and C show the various protein(s) complexes, and
the order of events in the formation of the preinitiation complex. The UAF complex interacts specifically with UCE, and
subsequently, together with TBP and possibly some unidentified factors,24 recruits the core factor (CF), and this
committed complex (panel B) promotes the formation of a functional initiation complex (panel C) with Rrn3p and Pol
I. Although the contacts of the proteins with DNA shown in panel B and C are not determined precisely, it is clear that
UAF, in contrast to the metazoan UBF, interacts primarily, if not exclusively, with the UCE. Therefore, UAF is not
only structurally, but also functionally, clearly different from UBF. See text for further details. This Figure is a modified
version of Figure 7 in ref. 62 (with permission).
? 1997 John Wiley & Sons, Ltd.
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1510
fragment located about 100 bp downstream of the
preceding rRNA operon. In artificial minigene
constructs, the stimulating effect of the enhancer
was found to be independent of its position or
orientation, although the degree of stimulation is
somewhat influenced by the sequence context.10,43
Moreover, a single enhancer stimulates transcription of both copies of a tandem repeat of two
minigenes, irrespective of its location upstream,
downstream or in between the two genes.19 Also,
by using ?tagged? rDNA units, integrated into the
rDNA locus, it was found that the Pol I enhancers
exert their function in two directions, but mainly
on their two most proximal rRNA operons.31 In
this respect the yeast rDNA enhancer may be
unique among the eukaryotic Pol I stimulating
elements. Attempts to define specific sub-elements
in this rather long enhancer, using either deletion
analysis in minigenes43,72 or mutational analysis of
enhancer sequences in templates transcribed by Pol
I in vitro,35,36 have not resulted in unambiguous
conclusions. Only small deletions or mutations at
the 3*-end of the enhancer segment appear to cause
a drastic reduction in its stimulatory activity.43
Therefore, the main element responsible for enhancer function appears to reside closely upstream
from the HindIII site located about 300 bp downstream from the 3*-end of the 26S rRNA gene,
whereas the other regions of the enhancer are not
essential for full function.
So far, no proteins binding to the enhancer have
been found except the protein called Reb1p.29,46
Both by in vitro gel-retardation analysis and by
genomic footprinting,29 this abundant and ubiquitous protein was shown to bind to a 14-bp region
starting 10 bp downstream from the EcoRI-site
marking the 5*-end of the enhancer fragment. In
some rDNA units another protein, known as
Abf1p, was found to bind about 20 bp downstream of the Reb1p-binding site. However, since
this Abf1p-binding site is not present in all rDNA
units, as cloned in different laboratories,29 the
protein Abf1 does not seem to have any functional
role in the rDNA unit. The Reb1p-binding site
near the 5*-end of the enhancer has an absolutely
conserved 8-bp core (CCGGGTAA) which, interestingly, is also present about 60 bp upstream of
the 5*-end of the Pol I promoter, although in the
opposite orientation.29,46 Gel-retardation assays
showed that the promoter-proximal site has a fourto five-fold higher affinity for Reb1p than the site
within the enhancer.29,46 This finding, and the
observation that termination of Pol I transcription
? 1997 John Wiley & Sons, Ltd.
actually takes place within the enhancer, as will be
discussed below, has led us to propose a model
that envisages a functional linkage between consecutive enhancers and promoters in which the
protein Reb1, either directly or indirectly, plays an
important role.23,31 In this model (see Figure 4),
each rDNA unit is thought to form a loop in such
a way as to bring all Pol I transcription units to
one side, and the intergenic spacers carrying the
5S rRNA genes (transcribed by Pol III) to the
other side of the spatial complex. In this ?looping
out? structure, or ?ribomotor?,23 the enhancerterminator elements are brought into the vicinity
of at least the two neighbouring Pol I promoters.
In this scenario, terminating Pol I molecules and
associated factors can pass directly to the promoter of either the same or the proximal downstream rRNA operon without being released into
the free pool.
This hypothesis is supported by the observation
that active rDNA transcription units in Bombyx
mori and Drosophila have been visualized as loops
separated by intergenic spacers.18 A very similar
model has been proposed recently for the spatial
organization of active rDNA transcription units in
mouse.58 These authors suggest that the functional
link between the initiating and terminating region
of the mouse rRNA operons might be mediated by
multimerization of a transcription termination factor, called TTF-I, which, like Reb1p in yeast, binds
upstream of the promoter and at the termination
site of the mouse rRNA operon.58 The mouse (and
human) TTF-I shows a considerable homology
with yeast Reb1p, in particular with respect to the
DNA-binding domains,11 and the two proteins are
considered to be functionally equivalent. It is likely
that Reb1p functions in a similar way by also
forming stable oligomers, but this has not yet been
experimentally tested.
In fact, in vitro transcription analysis has suggested that Reb1p, like TTF-I, can act as a transcriptional terminator of yeast Pol I.33,34 However,
the in vitro system can obviously not account
for all specific features of rDNA transcription in
its natural, chromosomal, context. Therefore, an
in vivo analysis might be more informative with
respect to the actual molecular events in Pol I
transcription. Using various kinds of (mutated)
rRNA minigenes23,57 or ?tagged? rRNA operons
integrated into the rDNA locus,31 evidence was
obtained that Pol I transcription in yeast terminates at position +210 relative to the 3*-end of the
26S rRNA gene (about 100 bp downstream of
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Figure 4. Looping model for the enhancer-stimulated regulation of transcription by Pol I in yeast. Large loops
(thick black lines) represent Pol I operons, whereas the smaller loops represent the intergenic spacer, carrying
the 5S rRNA gene. The Pol I promoters and the enhancers are indicated by filled and open boxes, respectively.
The promoters and enhancers are brought in close proximity to each other by oligomerization of the
DNA-binding protein Reb1p, anchored directly or indirectly to the nucleolar matrix. This figure can only show
a two-dimensional representation of what is in fact a three-dimensional, spatial configuration (Figure designed
and drawn by H. A. Raue?, this laboratory).
the Reb1p-binding site), and that deletion of the
Reb1p-binding region in the enhancer has no effect
on efficient transcription termination, although
it has a slight negative effect on the transcription
of the two neighbouring rDNA units. Using a
different kind of minigene system, Johnson
and Warner20 essentially confirmed the results
? 1997 John Wiley & Sons, Ltd.
obtained by van der Sande et al.57 Upstream of the
proposed termination sites there is a set of very
efficiently working processing sites, generating the
3*-ends of the 26S rRNA and transcripts which are
7?50 nucleotides longer, respectively.23,57 These
processing sites are essential for the efficient formation of the mature rRNAs from the primary
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1512
transcript. All the data together indicate that the
site of termination of Pol I transcription is to some
extent irrelevant, as long as efficient processing of
the primary transcripts at their 3*-ends can take
place, and provided that termination occurs somewhere within the enhancer region to allow an easy
hand-over of the terminating Pol I molecules to
either one of the adjacent promoters (see also
Figure 4). Furthermore, it seems plausible that
transcribing Pol I molecules should not run into
the 5S rRNA gene and, indeed, ?fail safe? terminators have been found by in vivo analysis at a
position around+700, still far upstream of the 5S
rRNA gene.23,57
In the in vivo situation up- and down-regulation
of Pol I transcription may very well be regulated
by the number of rDNA transcription units organized in the proposed loop structure (Figure 4). This
is in accordance with the electron microscopical
observation that rDNA units are either fully transcribed or completely inactive (see e.g. ref. 18). It is
not likely that this type of on- or off-regulation is
exerted by Reb1p directly. This notion is based on
the observation that the Reb1p-binding sites in the
rDNA units are always occupied in vivo as shown
by genomic footprinting, even when yeast cells are
in the stationary phase and the rRNA genes are
not (or hardly) transcribed.29 Moreover, the Reb1protein plays a much more general role in the yeast
cell than solely in the structural organization of the
rDNA units in the nucleolus. Reb1p-binding sites
are present in numerous other locations within the
yeast genome, and many of them are located in the
promoter regions of genes transcribed by Pol II
(see Planta et al.51 for a review, and references
therein). In some cases, the protein stimulates
transcription whereas, in others, it acts as a repressor. According to Chasman et al.,4 Reb1p functions by influencing the chromatin structure and
creating a nucleosome-free region of 230 bp surrounding its binding site. In view of this general
function in yeast gene expression, it is not surprising that the REB1-gene is essential for growth.21
Thus, all the data suggest that, within the rDNA
locus, Reb1p is primarily a structural factor required for efficient Pol I transcription by promoting the interplay of enhancers and promoters with
transcription factors.
Very recently, evidence has been presented suggesting that the SIR2 gene product, required for
the maintenance of a repressed chromatin structure that silences transcription at the HM loci and
telomeres, also mediates the silencing of rRNA
? 1997 John Wiley & Sons, Ltd.
genes.1,61 How the active amount of the Sir2protein is influenced by the cellular growth rate,
which determines the number of transcriptionally
active rDNA units is not known. Furthermore,
other factors have been implicated as well in this
silencing process.1,61 Anyhow, S. cerevisiae activates or inactivates rDNA gene copies as needed in
response to growth conditions, and SIR2 appears
to affect this regulatory mechanism.
Several lines of evidence suggest that modulation of rRNA synthesis in response to different
growth conditions can also be the result of alterations in the amount or activity of Pol I (by
covalent modification) and/or transcription factors, such as Rrn3p which is tightly associated
with the polymerase, or components of the UAFcomplex. These alterations would then lead to
changes in transcription initiation by Pol I. A
change in the frequency of transcription initiation
should, however, be coupled to a concomitant
change in transcription elongation, because the
density of Pol I molecules on actively transcribed
rRNA genes is usually the same, as judged from
electron microscopical analysis.
It is clear that we still have to learn a lot more
before we fully understand the various mechanisms
involved in the regulation of rRNA synthesis.
EXPRESSION OF THE RIBOSOMAL
PROTEIN GENES
Yeast ribosomes contain 32 small-subunit and 46
large-subunit r-proteins encoded by 137 genes, because 59 of these genes are duplicated.52 In all cases
studied, both gene copies are functional, although
their expression levels differ often considerably (see
references in Raue? and Planta54). The duplicate
copies encode identical, or virtually identical,
proteins that, with the exception of some acidic
proteins (P1 and P2), are functionally indistinguishable.52 However, their leader and trailer sequences
display substantial differences. A striking feature of
the structure of yeast rp-genes is the frequent presence of an intron (in 99 gene copies) located close
to the 5*-end of the coding region or (in a few cases)
even in the leader sequence. The various yeast
rp-genes are scattered over the entire yeast genome
and they are all independently transcribed.
Despite the difference in gene copy number,
the cellular levels of the mRNAs for the different
yeast r-proteins are approximately the same. Since
the various rp-mRNAs have about the same
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Figure 5. Generalized structure of the two types of rp-gene promoters, containing in addition
to the T-stretch, Rap1p-binding sites (approx. 90%) or Abf1p-binding sites (210% of the
cases). The signalling pathways between environmental signals and the regulation of transcription are largely unknown.
stability,26 this suggests that the rate at which
r-proteins are produced is controlled primarily at
the level of transcription. Over the past decade,
ample evidence supporting this suggestion has
been accumulated, although additional regulation
at the post-transcriptional level, mainly by rapid
degradation of any ribosomal protein produced in
excess,8,65 can occur.
Normally, growing cells contain no free pools of
the r-proteins, with the exception of the acidic
r-proteins. Furthermore, cells can adjust the production of all r-proteins in a concerted fashion to
meet the physiological demands under varying
growth conditions.37,38 Therefore, the transcription of the rp-genes has to be regulated very
precisely and in a strictly coordinate way. Extensive analysis has revealed that two different abundant proteins are involved in the transcriptional
regulation of the rp-gene family. The great majority of the promoters of rp-genes contain (usually
two) binding sites for a protein, designated Rap1p
(repressor/activator protein, also known as TUF),
whereas a few (10) have single binding sites for
Abf1p (Ars binding factor), as reviewed in Mager
? 1997 John Wiley & Sons, Ltd.
and Planta37 (see Figure 5), of which two have, in
addition, a binding site for Reb1p.52
This raises the question of how, despite the
occurrence of at least two different types of promoters, the balance in expression of the more than
130 rp-genes can be achieved. Transcriptional
regulation of the rp-gene family does not seem to
be accomplished by coordinated modulation of the
DNA-binding capacities of Rap1p and Abf1p,
since the in vitro binding activities of the two
proteins remain unchanged, even in the stationary
phase when rp-gene transcription is virtually nil.28
Furthermore, both Rap1p and Abf1p appear to be
involved in many other cellular functions. Rap1p
binds to the upstream regions of many genes, in
particular the heavily transcribed genes encoding
glycolytic enzymes, and in addition to elements
that silence mating-type genes, and to telomeres
(see Shore60 for a review). Abf1p, like Rap1p
appears to function in transcription as either an
activator or repressor, depending upon the context of its binding. Furthermore, Abf1p-binding
sites are also found associated with various
autonomously replicating sequences.2
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1514
The overwhelming importance of Abf1p, Rap1p
(and Reb1p), not only in ribosome synthesis, but
in general chromosome function, has led to extensive investigations aimed at the elucidation of their
mode of action in order to understand their multifunctional character and how they act in concert in
various cellular functions. The three proteins are
similar in that each is an abundant multifunctional
regulatory protein, which is absolutely required for
cellular viability. Their importance is stressed by
the observations that their genes appear to be
constitutively expressed, and that their binding
sites in the genome are occupied in vivo under all
conditions studied.28,29 Curiously, all three genes
carry, in their promoter, binding sites for their own
gene products.15,17,21 At least two of them (Rap1p
and Abf1p) are strong DNA-bending proteins40,70
and all three create, by binding to their cognate
sites, a nucleosome-free region varying from
approximately 70 to 230 bp.7,12,73 All these data
suggest that the three proteins play primarily a
structural role in organizing the chromatin structure in order to allow other regulatory proteins
(gene-specific regulators, like Gcr1p in the case of
glycolytic genes) access to their binding sites (see
Planta et al.51 for a review). This idea is consistent
with the finding that Abf1p-binding sites and
Rap1p-binding sites can functionally replace each
other in the promoter of rp-genes.14 Furthermore,
Abf1p-binding sites and Reb1p-binding sites can
functionally replace each other in different promoters, e.g. in the promoter of the ILV1 gene.56
All the r-protein genes contain, in addition to
the binding sites for Rap1p or Abf1p, a
downstream-located T-rich element in their promoters (see Figure 5). We have shown that both
the T-rich element and the Abf1p- or Rap1pbinding sites are necessary and sufficient for the
characteristic transcriptional upshift response observed when glucose in fermentable amounts is
added to a yeast culture growing on a nonfermentable carbon source.13 The same combination of cis-acting elements is required for a high
level induction of rp-gene transcription in nitrogen
starvation/refeeding experiments.13
T-rich elements are ubiquitous sequences found
in promoters of many yeast genes transcribed by
Pol II. Their activating effect on transcription has
been described as a non-specific one and has been
attributed to either the peculiar structure of
polydT/dA tracts, which may hamper the packaging of promoter DNA in nucleosomes, or to the
binding of protein factors.3,63 Recent experiments,
? 1997 John Wiley & Sons, Ltd.
performed in our laboratory, have indicated that
the T-stretches in the promoters of the rp-genes do
not bind any protein, such as Datin (Dat1p74), but
rather cause a DNA bending, additional to the
DNA bending induced by the binding of Abf1p or
Rap1p (R. L. Lascaris, W. H. Mager and R. J.
Planta, unpublished results). The combined bending, caused by the two cis-acting elements in the
rp-gene promoters, might create a high-affinity
recognition site for a (core) promoter-binding protein, by nucleosome exclusion or phasing. In this
respect, it is interesting that it has been found
recently that efficient transcription of rp-genes in
yeast specifically depends on a TBP-associated
factor, the yTAFII145.59,71 The protein yTAFII145
is the only yTAFII known to directly contact
TBP55 and therefore functions to recruit the other
yTAFIIs into the TFIID complex. Although dispensable for transcription of most yeast genes,
yTAFII145 is specifically required for cell cycle
progression and growth control.71 At high cell
density or following nutrient deprivation, yeast
cells do not divide any more and enter a G0-like
state. In this stationary phase, the levels of
yTAFII145, TBP, and some other yTAFIIs are
drastically reduced.71 These results indicate that
yTAFII145 and other TFIID components have a
specialized role in transcriptional regulation of cell
cycle progression and growth control. As expected
yTAFII145 is required for efficient transcription of
G1-cyclins and certain B-type cyclin genes. However, by applying PCR-based differential display
it was found that another small subset of yeast
genes, in particular rp-genes, is also yTAFII145dependent.59 Promoter-mapping studies revealed
that the region of the gene promoter that renders it
yTAFII145-dependent is not the region containing
the upstream activating sequences, but rather the
core promoter. Elements belonging to the core
promoter include the TATA-box, sequences surrounding the TATA-box, the transcription start
site and sequences downstream of this site. By
mutational analysis of these elements, it was
shown that neither the TATA-box per se nor the
sequences around the transcription start site determine the yTAFII145-dependence, but that the responsive element maps to the region surrounding
the TATA-box.59 No obvious sequence in this
region common to all the rp-genes and other
yTAFII145-dependent genes was found. However,
in yeast gene promoters, such a sequence is very
difficult to define because of the variable distance
between the TATA-box and transcription start
?????
???. 13: 1505?1518 (1997)
?????????? ?? ???????? ?????????
site, and the heterogeneity of start-sites.64 Furthermore, the yTAFII145-dependence may be conferred by multiple elements.
In summary, the expression of rp-genes is regulated by yTAFII145 by interaction of this protein
with a region in the core promoter. Furthermore,
the intracellular levels of yTAFII145 are regulated
by the cellular growth rate, possibly by modification (phosphorylation) or at the level of turnover.
This is very consistent with our observations that
the transcription of the yeast rp-genes is strictly
related to the cellular growth rate.28
All the data discussed above fit the following,
attractive, hypothesis. The strong DNA bending
caused by the combined action of the Rap1p- or
Abf1p-binding sites and the T-stretches makes the
core promoter of the rp-genes free of nucleosomes,
allowing TBP and yTAFII145 access to their binding region in the core promoter. Up- and downregulation of the transcription of the rp-genes is
the result of changes in the intracellular levels of
yTAFII145, which depend on the cellular growth
rate. The signals for the regulation of rp-gene
expression during growth in various media seem to
be mediated by protein kinase A,16,27 although
most details of the signalling pathway must still be
elucidated. It has been suggested that Rap1p (and
Abf1p) constitutes the final target for the signalling.27 However, this is unlikely in view of many
other data presented above (see e.g. refs. 13, 16,
22); rather yTAFII145 and/or TBP may act as the
final target(s) for the growth signal.
Under extreme conditions, such as complete
amino-acid starvation or other blocks of protein
synthesis,16 various stress-conditions, or a defect in
the secretory pathway with consequent interference to membrane biosynthesis,45 there is a complete arrest in transcription of rp-genes. This can
be explained by a defect in the signalling pathway
and, more importantly, by a severe drop in the
levels of yTAFII145, as in the stationary phase,71
under these conditions.
There are several mechanisms that can explain
the balanced production of rRNAs and r-proteins.
Apart from the rapid degradation of any excess
r-protein produced, mentioned previously, and the
lack of proper processing of precursor rRNA in
the absence of a sufficient supply of r-proteins,
there is now the attractive possibility that
yTAFII145 also associates with TBP in the interaction with the Pol I promoter. Furthermore,
the three RNA polymerases share some subunits,39,47,75 thus providing a level at which a
? 1997 John Wiley & Sons, Ltd.
1515
coordinate transcriptional control can be exerted.
Finally, another possible link between rRNA and
r-protein synthesis is suggested by the fact that
several genes encoding polymerase subunits have
a similar promoter structure to that of rp-genes,
including binding sites for Rap1p, Abf1p,
Reb1p.6,39,42,67
ACKNOWLEDGEMENTS
I thank my former PhD students, Drs P. M.
Gonc?alves, G. Griffioen, J. Klootwijk, L. S.
Kraakman, T. Kulkens, W. H. Mager, W.
Musters, C. A. F. M. van der Sande and A. E.
Veenstra for their excellent experimental work,
which has provided the backbone for this review. I
am very grateful to my colleagues Drs M. Nomura
(University of California, Irvine) and M. R. Green
(University of Massachusetts, Worcester) for
sharing their results with me prior to publication. I
am indebted to my colleagues Drs H. A. Raue? and
W. H. Mager for stimulating discussions and technical help with this paper, and to my secretary,
Mrs P. G. Brink, for her invaluable assistance.
Finally, I gratefully acknowledge the generous
support from the Netherlands Organization for
Scientific Research (NWO) via the Netherlands
Foundation for Chemical Research during more
than 30 years.
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