(Received for publication, August 16, 1995; and in revised form, November 2, 1995)
From the
The Saccharomyces cerevisiae SIS1 gene encodes an
essential heat shock protein with similarity to the Escherichia
coli DnaJ protein. In sis1-85 and sis1-86 mutants, the sis1 RNA is induced to high levels at room
temperature and without heat shock. The presence of wild type SIS1 in the sis1-85 mutant represses the overexpression
of SIS1-85 protein. Furthermore, overexpression of wild type SIS1
reduces the -galactosidase activity expressed from a SIS1:lacZ fusion. These results suggest that SIS1 negatively regulates its
own expression. The autoregulation of SIS1 transcription is
mediated through a 39-base pair cis-element containing the SIS1 heat shock element plus additional flanking sequences on one side.
Although SIS1 transcription is constitutive, it is transiently
induced upon heat shock. In addition, SIS1 transcription is
regulated by SSA (a class of HSP70 proteins) function. The elevated
transcription of SIS1 in ssa1 ssa2 mutants is
mediated solely through the SIS1 heat shock element.
Therefore, the SIS1 autoregulatory element is different from
the SSA-responsive element, suggesting that the mechanism
involved in autoregulation of SIS1 is distinct from regulation
of SIS1 by SSA proteins.
All organisms respond to environmental stresses, such as heat
shock, by the rapid and transient increase in the rate of synthesis of
a group of proteins collectively called the heat shock proteins (Craig et al., 1994; Lindquist and Craig, 1988). The SIS1 gene encodes an essential DnaJ homologue in the yeast Saccharomyces cerevisiae (Luke et al., 1991). The
DnaJ protein of Escherichia coli was shown to be a member of
the heat shock proteins (Lindquist and Craig, 1988), and the rate of
synthesis of E. coli DnaJ protein increases 10-fold after a
heat shock from 30 to 43 °C (Bardwell et al., 1986). The
increase in DnaJ expression is rapid and appears to be
regulated primarily at the transcriptional level (Georgopoulos et
al., 1994). In E. coli, a mutation in the gene encoding
any one of three heat shock proteins, DnaJ, DnaK, or GrpE, results in
the elevated expression of itself and many other heat shock genes at
low temperatures (Straus et al., 1990). This autoregulation is
achieved at least partially via a negative feedback mechanism where the
synthesis and stability of a heat shock transcriptional activator
is regulated (Georgopoulos et al., 1994,
Straus et al., 1990).
In eukaryotic cells, the
transcription of heat shock genes is normally regulated through a
promoter sequence termed the heat shock element (HSE). ()The
HSE, which is composed of a 5-bp unit nGAAn alternating with another
5-bp unit nTCCn (Amin et al., 1988; Xiao and Lis, 1988), has
been highly conserved during evolution (Bienz and Pelham, 1987). The
heat shock factor binds to the HSE and is required to activate the
transcription of certain heat shock genes not only in response to
stress but also under conditions of normal growth (Sorger, 1990). In
yeast, some heat shock proteins perform essential functions and are
constitutively expressed. Other heat shock proteins are expressed,
sometimes transiently, only upon a shift to a higher temperature (Craig et al., 1994). Transcriptional regulation of budding yeast HSP70 homologues has been the focus of many studies. SSA1, which encodes one of the SSA class of HSP70 proteins,
autoregulates its own transcription via one of its HSEs (Stone and
Craig, 1990). In addition, the expression of many heat shock genes,
such as SSA3, SSA4, HSC82, and HSP26, is highly induced in ssa1 ssa2 mutants
(Boorstein and Craig, 1990b, 1990c; Lindquist and Craig, 1988).
The DnaJ heat shock protein has been conserved in evolution (Caplan et al., 1993). S. cerevisiae is currently known to have eight DnaJ-like proteins: SIS1, YDJ1, SEC63, SCJ1, Zoutin, MDJ1, CAJ1, and XDJ1, all of which contain a region highly similar to the amino-terminal region of bacterial DnaJ (the J-domain) (Luke et al., 1991; Silver and Way, 1993). SEC63 is an integral endoplasmic reticulum membrane protein involved in protein transport across the endoplasmic reticulum (Rothblatt et al., 1989; Sadler et al., 1989). YDJ1 is present in the cytosol and functions to transport polypeptides targeted to both mitochondria and endoplasmic reticulum (Atencio and Yaffe, 1992; Caplan et al., 1992). The SCJ1 gene was identified by its ability, when present on a high copy number plasmid, to cause missorting of a nuclear targeted protein (Blumberg and Silver, 1991). Zoutin was purified from nuclear extracts as a Z-DNA binding protein (Zhang et al., 1992). MDJ1 associates with the inner membrane of mitochondria and is involved in protein refolding and mitochondria biogenesis (Rowley et al., 1994). The functions of CAJ1 and XDJ1 are currently unknown (Mukai et al., 1994; Schwarz et al., 1994). The SIS1 protein is required for the normal initiation of translation and is found associated with ribosomal protein complexes (Zhong and Arndt, 1993).
Although some of the biological functions of eukaryotic DnaJ-like proteins have been identified, little is known about their transcriptional regulation. Among the promoter regions of these yeast DnaJ-like genes, MDJ1 has several weak matches to the consensus heat shock element (Rowley et al., 1994). YDJ1 and SCJ1 only have one copy of nGAAnnTTCn (Blumberg and Silver, 1991; Caplan and Douglas, 1991). However, SIS1 has a well conserved heat shock element (ATATGAACGTTCCAGAAACTTCTGGAAAAAG) containing 5 total units of nGAAn alternating with nTTCn (Luke et al., 1991). In this report, we show that the transcription of SIS1 is transiently increased by heat shock. SIS1 transcription is also induced by a defect in SSA function, and this induction requires only the SIS1 heat shock element. Moreover, SIS1 regulates its own transcription via a specific cis-element that contains the SIS1 heat shock element plus an additional promoter sequence. These findings point to the specificity of the induction of heat shock genes and the multiple mechanisms involved in their transcriptional regulation.
7.5 µg
of RNA was loaded onto each lane of a 1% agarose gel containing 6%
formaldehyde, 1 MOPS (0.02 M morpholinopropanesulfonic
acid, 5 mM sodium acetate, 1 mM EDTA (pH 7.0)). The
gels were blotted onto Bio-Trans nylon membranes. The probes used for
Northern analyses were the 2-kb BstEII fragment of SIS1 from CB547, the 0.9-kb NcoI-SalI fragment of SSA1 from CB2019, the cloned polymerase chain reaction product
encompassing the entire open reading frame of CTT1 from
CB2550, the 1.7-kb MluI-StuI fragment of SSA4 from CB1785, the 1.2-kb EcoRI fragment of HSC82 from CB2313, the 0.6-kb AluI fragment of ACT1 from CB882, and the 2.6-kb PvuII fragment of lacZ from AB174. The blots were washed twice (15 min each) at 24 °C
using 2
SSC with 0.1% SDS and twice (15 min each) at 65 °C
using 0.1
SSC with 0.1% SDS.
Figure 3:
Delineation of the SIS1 autoregulatory element. A, diagrammatic representation of
the constructs and sequences of the SIS1 promoter. The top
line shows the UAS-HIS4:lacZ fusion vector. The
sequences of five oligonucleotides (47, L39, R39, 31, and 23 bp)
corresponding to the SIS1 promoter are listed below the lacZ vector. nGAAn and nTTCn modules are represented by arrows above the oligonucleotides. The preparation of
constructs containing these SIS1 promoter elements is as
described under ``Materials and Methods.'' B, the
effect of SIS1 promoter elements on
-galactosidase
activity expressed from a heterologous promoter. The sis1-85 (CY732) and SIS1 (CY457) strains containing the
constructs represented in A or the vector
UAS-HIS4:lacZ were grown to exponential phase in
SC-uracil medium at 30 °C, and total cell extracts were prepared.
-Galactosidase activities expressed in wild type SIS1 strains (empty bars) and sis1-85 strains (striped bars) were determined. Three independent assays were
carried out.
Figure 1:
Induction of SIS1 RNA levels. A, SIS1 RNA is induced to high levels in sis1-85 and sis1-86 mutants. Isogenic
strains CY457 (SIS1), CY732 (sis1-85), and
CY733 (sis1-86) were grown to exponential phase in YPD
medium at 24 °C. 5-ml aliquots of culture for each strain were
collected, and total RNA was prepared. The Northern blot was probed for SIS1 and ACT1 RNA. The signals were quantified using
a Fuji imaging plate. The probes used for Northern analysis are as
listed under ``Materials and Methods.'' B,
-galactosidase activity expressed from a SIS1:lacZ fusion
is much higher in sis1-85 and sis1-86 mutants. The isogenic strains CY5026 (SIS1 (SIS1:lacZ)), CY4645 (sis1-85 (SIS1:lacZ)) and CY5020 (sis1-86 (SIS1:lacZ)) were grown to exponential phase in SC-uracil
medium at 24 °C. Duplicate samples for each strain were collected
to determine the
-galactosidase activity. Three independent assays
were carried out. C, SSA1, SSA4, and CTT1 are not highly expressed in the sis1-85 strain at
24 °C. Isogenic strains CY457 (SIS1) and CY732 (sis1-85) were grown to exponential phase in YPD medium
at 24 °C and then shifted to 37 °C. For each strain, 5-ml
aliquots of culture were collected at 24 °C and after 30 min and 90
min at 37 °C. Total RNA was prepared. The Northern blot was probed
for SSA1, SSA4, CTT1, and ACT1 RNA. The
probes used for Northern analysis are as listed under ``Materials
and Methods.''
Figure 2:
The autoregulation of SIS1. A, expression of sis1-85:HA is repressed to
normal levels by wild type SIS1 protein. Strains CY706 (SIS1/sis1-85:HA) and CY732 (sis1-85:HA)
were grown to exponential phase in SC-leucine medium at 24 °C, and
extracts were prepared and loaded onto a 10% SDS-polyacrylamide gel.
The blot was probed with the 12CA5 monoclonal antibody against the HA
tag of SIS1-85:HA. B, high copy number SIS1 gene represses -galactosidase activity expressed from a SIS1:lacZ fusion. Three isogenic strains carrying a high copy
number plasmid with either wild type SIS1 (CY4967) or a
frameshift sis1 gene (CY4968) or without an insert (CY4969)
were grown in SC-uracil-leucine medium to exponential phase at 30
°C. Duplicate samples were collected to determine
-galactosidase activities for each assay. Two independent assays
were performed.
The increased expression of the sis1-85 and sis1-86 genes might be
mediated through the upstream region of SIS1. To test this
possibility, we prepared a SIS1:lacZ translation fusion, where
the upstream region, including the initiation codon of SIS1,
was fused in-frame to the lacZ coding sequence (see
``Materials and Methods''). The -galactosidase activity
expressed from the SIS1:lacZ fusion was assayed in isogenic sis1-85, sis1-86, and wild type SIS1 strains. The SIS1:lacZ fusion gave 6-fold higher
-galactosidase levels in the sis1-85 strain and
5.5-fold higher
-galactosidase levels in the sis1-86 strain as compared to the isogenic wild type strain (Fig. 1B). Therefore, the induction of
-galactosidase activity expressed from the SIS1:lacZ fusion in the sis1 mutants was quantitatively similar to
the induction of sis1 RNA levels in the sis1 mutants.
These results indicate that sequences upstream of the SIS1 initiation codon are responsible for the induction of sis1 RNA levels in sis1-85 and sis1-86 strains. Since the sis1-85 and sis1-86 alleles had similar effects on SIS1 expression,
subsequent experiments were performed in the sis1-85 strain.
To determine if the expression of other heat shock or stress-induced genes is activated in the sis1-85 mutant, we compared the RNA levels of SSA1 and SSA4 (which encode HSP70 proteins) and CTT1 (a stress-induced gene that encodes catalase T) (Marchler et al., 1993) in the sis1-85 mutant to those in the isogenic wild type strain. At room temperature, SSA1, SSA4, and CTT1 were not highly expressed in the sis1-85 mutant as compared to the wild type strain (Fig. 1C). Upon heat shock (30 min after a shift to 37 °C), their RNA levels increased (Fig. 1C). These results suggest that the elevated sis1 expression in the sis1-85 mutant at room temperature is not a general stress or heat shock response. Interestingly, SSA4 RNA was induced by heat shock to a higher level in the sis1-85 mutant than in the isogenic wild type strain (Fig. 1C). Moreover, by 90 min at 37 °C, the SSA4 RNA level was maintained at almost the same high level in the sis1-85 mutant, when it was hardly detectable in the wild type SIS1 strain (Fig. 1C). Possibly, the sis1-85 strain could be more sensitive to heat shock.
If SIS1 protein does negatively
regulate its own synthesis, then overexpressing the SIS1 protein might
repress the expression of a SIS1:lacZ fusion.
-Galactosidase activity expressed from the SIS1:lacZ fusion was examined in three strains: one strain contained the
wild type SIS1 gene on a high copy number plasmid YEp13, a
second strain contained the sis1-fs gene (with a frameshift
mutation in SIS1; see ``Materials and Methods'') on
YEp13, and a third strain contained YEp13 without an insert. The wild
type SIS1 gene on a high copy number plasmid specifically
reduced
-galactosidase activity to about 50% of the levels
obtained with either the sis1-fs/YEp13 or the vector YEp13 (Fig. 2B). Therefore, wild type SIS1 protein negatively
regulates its own expression via its upstream region.
To further delineate the SIS1 autoregulatory promoter element, smaller regions of the SIS1 promoter element were tested. Surprisingly, the
-galactosidase expressed from the SIS1 heat shock element
(31 bp containing 5 units of nGAAn alternating with nTTCn, plus 3 bp on
both sides), which lacks 8 bp from both ends of the 47-bp sequence, was
only 2-fold higher in the sis1-85 strain compared to the
wild type SIS1 strain (Fig. 3, A and B). Therefore, the 31-bp SIS1 heat shock element does
not contain all the sequences necessary for SIS1 regulation.
To determine the SIS1 autoregulatory element sequences
present within the 47-bp SIS1 promoter fragment but not
contained within the 31-bp SIS1 heat shock element, we
constructed L39-UAS-HIS4:lacZ and R39-
UAS-HIS4:lacZ reporter plasmids, where the L39-bp and
R39-bp oligonucleotides contain the SIS1 heat shock element
plus eight nucleotides from either the upstream or downstream flanking
region of the 31-bp element (Fig. 3A). Similar to the
47-bp element, the R39-bp SIS1 promoter element gave 6-fold
more
-galactosidase activity in the sis1-85 strain
compared to the isogenic wild type strain (Fig. 3B).
However, like the 31-bp SIS1 heat shock element, the L39-bp
cis-element gave only 2-fold more
-galactosidase activity in the sis1-85 strain as compared to the isogenic SIS1 strain (Fig. 3B). Therefore, the R39-bp SIS1 promoter sequence contains the SIS1 autoregulatory
element, which can mediate the induction of SIS1 in the sis1-85 mutant, comparable to the entire SIS1 promoter (Fig. 1B and Fig. 3B).
To determine if the SIS1 autoregulatory element requires
the entire SIS1 heat shock element, we prepared the 23-UAS-HIS4:lacZ construct, which contains only 2 basic
heat shock units: one each of nGAAn and nTTCn (a part of the SIS1 heat shock element) (Fig. 3A). The
-galactosidase levels expressed from the 23-bp SIS1 promoter sequence were very low in both the sis1-85 and SIS1 strains, almost the same as those expressed from
the original
UAS-HIS4:lacZ construct (Fig. 3B). These results suggest that a 23-bp SIS1 promoter sequence containing part of the SIS1 heat shock
element plus eight nucleotides on the downstream flanking region is not
sufficient for regulation by SIS1.
Since the R39 SIS1 autoregulatory element contains the SIS1 heat shock
element, it is possible that the heat shock element is necessary for
the induction of SIS1 transcription from the entire SIS1 promoter in the sis1-85 strain. To test this
possibility, we fused the upstream region of SIS1, which was
deleted for the heat shock element, into the lacZ reporter
vector. The induction of -galactosidase expression from the
HSE-SIS1:lacZ fusion in the sis1-85 strain
as compared to the isogenic wild type strain was completely abolished (Fig. 4). Furthermore, the constitutive level of
-galactosidase activity expressed from the
HSE-SIS1:lacZ fusion in the wild type SIS1 strain was dramatically
decreased as compared to that from the SIS1:lacZ fusion (Fig. 4). Therefore, the SIS1 heat shock element is a
dual element that is required not only for the induction of sis1 RNA in the sis1 mutant but also for the relatively high
basal transcription of SIS1 in the wild type strain.
Figure 4:
The requirement of the SIS1 heat
shock element in autoregulation and basal expression of SIS1.
Extracts were prepared from isogenic strains CY4643 (SIS1 (SIS1:lacZ)), CY4644 (SIS1 (HSE-SIS1:lacZ)), CY4645 (sis1-85 (SIS1:lacZ)), and CY4646 (sis1-85 (
HSE-SIS1:lacZ)), which were grown to
exponential phase in SC-uracil medium at 30 °C.
-Galactosidase
activities expressed from wild type SIS1 strains (empty
bars) and sis1-85 strains (striped bars)
were determined. Two independent assays were carried
out.
Figure 5: The heat shock effect on SIS1 promoter elements. Strains CY3998 (47-SIS1-HIS4:lacZ), CY4232 (L39-SIS1-HIS4:lacZ), CY4241 (R39-SIS1-HIS4:lacZ), CY3990 (31-SIS1-HIS4:lacZ), and CY4577 (23-SIS1-HIS4:lacZ) were grown to exponential phase in SC-uracil medium at 24 °C and then shifted to 39 °C. For each strain, 5-ml aliquots of culture were collected at 24 °C and after 15 min or 90 min at 39 °C. Total RNA was prepared. The Northern blot was probed for lacZ, SIS1 (expressed from the chromosomal SIS1 gene), and ACT1 RNA. The probes used for Northern analysis are as listed under ``Materials and Methods.''
To determine whether the heat
shock induction of SIS1 is mediated through the putative SIS1 heat shock element, we determined the effect of heat
shock on the lacZ RNA levels expressed from 47-UAS-HIS4:lacZ, L39-
UAS-HIS4:lacZ, R39-
UAS-HIS4:lacZ, 31-
UAS-HIS4:lacZ, and 23-
UAS-HIS4:lacZ fusions in a wild type strain. We
examined the lacZ RNA levels, and not the
-galactosidase
activities, because heat shock only transiently increases SIS1 transcription. Upon heat shock, the 47-, L39-, R39-, and 31-bp SIS1 promoter sequences, which all contained the SIS1 heat shock element, induced lacZ RNA (Fig. 5). By
contrast, the lacZ RNA expressed from the 23-bp sequence
containing only part of the SIS1 heat shock element (one copy
of nGAAnnTTCn) was not activated at all by heat shock (Fig. 5).
These observations suggest that induction of SIS1 expression
by heat shock requires the SIS1 heat shock element. However,
the R39-bp cis-element gave greater SIS1 RNA induction by heat
shock than any of the other elements (Fig. 5). The cause of this
effect is not known.
Figure 6:
The expression of SIS1 in hsp70 and hsp90 mutants. A, SIS1 is
expressed at a high level in ssa1 ssa2 mutants. Isogenic
strains CY3766 (wild type), CY3769 (ssa3 ssa4 #1), CY3770 (ssa3 ssa4 #2), CY3767 (ssa1 ssa2 #1), and CY3768 (ssa1 ssa2 #2) were grown to exponential phase in YPD medium
at 24 °C. 5-ml aliquots of culture were collected for each strain,
and total RNA was prepared. The blot was probed for SIS1, HSC82, and ACT1 RNA. The probes used for Northern
analysis in A, C, and D are as listed under
``Materials and Methods.'' B, the
-galactosidase activity expressed from the upstream region of SIS1 is induced in the ssa1 ssa2 mutant. Extracts
were prepared from strains CY4873 (SSA1 SSA2 (SIS1:lacZ)), CY4875 (ssa1 ssa2 (SIS1:lacZ)), CY4901 (SSA1 SSA2 (HIS4:lacZ)), and CY4902 (ssa1 ssa2 (HIS4:lacZ)), which were grown to exponential phase in
SC-uracil medium at 30 °C. The
-galactosidase activities
expressed from wild type SSA1 SSA2 strains (empty
bars) and ssa1 ssa2 strains (striped bars) were
determined. Two independent assays were performed. C, SIS1 RNA levels are not altered in ssb mutants. Total RNA was
prepared from isogenic strains CY3766 (wild type), CY3051 (
ssb1), CY3206 (
ssb2), and CY3052 (
ssb1
ssb2), which were grown to exponential phase
in YPD medium at 24 °C. The blot was probed for SIS1 and ACT1 RNA. D, the expression of SIS1 RNA in hsc82 hsp82 mutants is not increased. Isogenic strains CY3744 (
hsc82 HSP82), CY3745 (
hsc82
hsp82-4), CY3746 (
hsc82 hsp82-41),
CY3747 (
hsc82 hsp82-33), CY3748 (
hsc82
hsp82-38), and CY3749 (
hsc82 hsp82-2)
were grown to exponential phase in YPD medium at 24 °C. 5-ml
aliquots of culture were collected for each strain, and total RNA was
prepared. The blot was probed for SIS1 and ACT1 RNA.
We further examined whether mutations in other HSP70 genes (e.g. SSB1, SSB2, and KAR2) induce the levels of SIS1 RNA. The levels of SIS1 RNA in ssb and kar2 mutants were similar to the levels in the respective isogenic wild type strains (Fig. 6C and data not shown). Also, the SIS1 RNA levels were not significantly different in strains containing mutations in HSP90 genes (e.g. HSP82 and HSC82) (Fig. 6D), suggesting that the induction of SIS1 RNA is somewhat specific to defects in SSA activity.
The increased level of SIS1 RNA in the ssa1
ssa2 mutant is most likely mediated via transcriptional activation
of the SIS1 promoter. The levels of -galactosidase
activity expressed from the SIS1:lacZ translation fusion in
the ssa1 ssa2 mutant were 5.5-fold higher as compared to the
isogenic wild type strain (Fig. 6B). Because there is
no heat shock element in the HIS4 promoter, the
-galactosidase activity expressed from the HIS4:lacZ construct, where the upstream region of HIS4 was fused to
the lacZ coding region, was not induced in the ssa1 ssa2 mutant (Fig. 6B).
To define the SSA-responsive SIS1 promoter element, we examined the
-galactosidase activity expressed from the 47-, L39-, R39-, 31-,
and 23-bp SIS1 promoter sequences in the
UAS-HIS4:lacZ reporter plasmid in the ssa1 ssa2 mutant versus the isogenic wild type strain. The 47-, L39-, R39-, and 31-bp SIS1 promoter cis-element all gave more than 100-fold
induction in the ssa1 ssa2 mutant as compared to the wild type
strain (Table 2). However, in either the ssa1 ssa2 mutant or the wild type strain, the
-galactosidase activity
expressed from the 23-bp SIS1 promoter element was almost as
low as from the original
UAS-HIS4:lacZ vector (Table 2). Furthermore, deletion of the SIS1 heat shock
element abolished the induction of the SIS1:lacZ fusion in the ssa1 ssa2 mutant (Table 2). These results suggest that
the 31-bp SIS1 heat shock element is both necessary and
sufficient to induce SIS1 RNA in the ssa1 ssa2 mutant. Therefore, the SSA-responsive SIS1 promoter element is different from the SIS1 autoregulatory element, which requires the SIS1 heat
shock element plus additional adjacent sequences.
Mutations in SIS1 result in increased levels of sis1 RNA. This finding led us to investigate if SIS1 can regulate its own transcription. Since the sis1-85 and sis1-86 mutations were made by deleting
non-overlapping 66-bp regions of SIS1 (which deletes 22 amino
acid regions in the carboxyl terminus of SIS1) (Luke et al.,
1991), the high levels of sis1-85 and sis1-86 RNAs could be due to three possibilities. One possibility might be
that the 66-bp sequences, which were deleted in sis1-85 or sis1-86, act as a cis-element to control
expression or stability of the SIS1 RNA. A second possibility
was that SIS1-85 and SIS1-86 proteins were gain-of-function
proteins that induced the expression of SIS1. The third
possibility was that the wild type SIS1 protein may act in trans to
negatively regulate its own synthesis, and SIS1-85 and
SIS1-86 proteins lose the function for self-regulation. Our
results showed that the last possibility is correct. The increased
level of SIS1-85 protein can be repressed to a normal level by
the wild type SIS1 protein. Also, overexpression of wild type SIS1
protein (SIS1 gene on a 2µ plasmid) decreases the
-galactosidase levels expressed from a SIS1:lacZ translational
fusion. Furthermore, a specific SIS1 promoter element confers SIS1 regulation to a heterologous promoter.
In the sis1-85 mutant at 24 °C, the levels of SIS1 RNA are highly induced, but the levels of other heat shock genes,
including SSA1, SSA4, and HSC82, ()are not highly induced. In contrast, in the ssa1 ssa2 mutant, the levels of SIS1 RNA as well as the RNA of many
other heat shock genes (e.g. SSA3, SSA4, and HSC82) are highly induced (Boorstein and Craig, 1990b, 1990c;
Lindquist and Craig, 1988). These observations led us to investigate
the specificity of the cis-elements in the SIS1 promoter that
respond to different conditions (e.g. SIS1 function, SSA
function, or heat shock).
Experiments to delineate the SIS1 promoter elements showed that the SIS1 heat shock element
is necessary, but is not sufficient, for SIS1 autoregulation.
Downstream adjacent sequences from the SIS1 heat shock element
(within AATGGGAT) are also required for the induction of SIS1 by the sis1 mutations. Although AATGGGAT has similarity
to the SSA3 post-diauxic shift element (TTAGGGAT) (Boorstein
and Craig, 1990a), the SIS1 RNA was not induced after the
post-diauxic shift. These results suggest that the AATGGGAT
sequence in the SIS1 R39 element is not functionally
equivalent to the SSA3 post-diauxic shift element. Moreover,
the post-diauxic shift elements in the CTT1 promoter are not
highly induced in a sis1-85 mutant at 24 °C (Fig. 1C). Therefore, we propose that the SIS1 heat shock element plus downstream adjacent sequences within
AATGGGAT define a specific SIS1 autoregulatory element.
The SIS1 heat shock element-containing DNA fragments that were
cloned into the HIS4 promoter in place of the normal HIS4 upstream activating sequences were at least 100-fold more active
in the ssa1 ssa2 mutant as compared to the wild type strain.
This amount of induction by a defect in SSA activity is similar to that
reported when the SSA3 and SSA4 heat shock elements
were used as cis-elements (Boorstein and Craig, 1990b, 1990c). That SIS1 is transcriptionally activated in the ssa1 ssa2 mutant via the SIS1 heat shock element supports the
hypothesis that SSA1 and SSA2 are negative regulators of the heat shock
response. In the ssa1 ssa2 mutant, transcription from the
intact SIS1 promoter was induced only about 5-fold (as
measured by -galactosidase activity from a SIS1:lacZ fusion and by RNA levels from the SIS1 gene), while the SIS1 heat shock elements that were cloned into the HIS4 promoter were induced more than 100-fold. These findings suggest
that either the promoter context determines the level of induction of
the SIS1 heat shock element in ssa1 ssa2 mutants or
the intact SIS1 promoter has a specific element that prevents
the very high induction of the heat shock element in ssa1 ssa2 mutants. If such a putative element exists in the SIS1 promoter, it would have to lie at least partially outside the
47-bp sequence because the 47-bp sequence (in place of the HIS4 UAS) was induced over 100-fold in the ssa1 ssa2 mutant.
The SIS1 autoregulatory element requires the SIS1 heat shock element plus additional downstream adjacent sequences.
The promoters of both SSA1 and SSA4 contain the
conserved heat shock element similar to the SIS1 heat shock
element (31 bp) that gave about 2-fold induction of -galactosidase
activity in the sis1-85 mutant (see Fig. 3B). The RNA levels of SSA1 or SSA4 increased slightly in the sis1-85 mutant at 24
°C (see Fig. 1C). The lack of high expression of SSA1 and SSA4 in the sis1-85 mutant
may be explained by the absence of specific SIS1 responsive
element in their promoters. By contrast, the increased expression of
many heat shock genes, including SIS1 and SSA4, in
the ssa1 ssa2 mutant could be solely due to the presence of
the conserved heat shock element in their promoters. Therefore, unlike
bacterial DnaJ and DnaK, which are involved in the
autoregulation of the heat shock response by the same mechanism
(Georgopoulos et al., 1994), SIS1 and SSA appear to be differently involved in regulating the expression of
certain heat shock genes.
Among known genes encoding DnaJ-like
proteins in S. cerevisiae, MDJ1 has several weak
matches to the nGAAnnTTCn sequence (Rowley et al., 1994). YDJ1 and SCJ1 only have a copy of nGAAnnTTCn
(Blumberg and Silver, 1991; Caplan and Douglas, 1991). However, one
copy of nGAAnnTTCn is not sufficient to respond to heat shock (Xiao and
Lis, 1988). Consistent with this finding, we found that the 31-UAS-HIS4:lacZ construct containing the SIS1 heat shock element was transcriptionally activated upon heat
shock, but the 23-
UAS-HIS4:lacZ construct containing only
one copy of nGAAnnTTCn did not respond to heat shock at all.
We also
examined SIS1 RNA levels in mutants that are defective in
certain cellular processes, including nuclear transport, protein
synthesis, and protein degradation. Interestingly, the only other
condition where we found increased SIS1 RNA levels (about
2-fold) was in cim3, cim5, and cim3 cim5 mutants.CIM3 and CIM5 possibly
encode components of a 26 S protease complex (Ghislain et al.,
1993). These results raise the possibility that, like bacterial DnaJ,
the SIS1 protein may be involved in the process of protein degradation.
How this would relate to the role of SIS1 for the initiation of
translation (Zhong and Arndt, 1993) is not known.
The induction of SIS1 RNA in the sis1-85 mutant is mediated
through the R39-bp SIS1 promoter sequence (SIS1 autoregulatory element), whereas the induction of SIS1 RNA in the ssa1 ssa2 mutant is via the 31-bp SIS1 heat shock element (SSA-responsive element). The
different cis-element requirements for induction of SIS1 transcription in sis1 mutants versus ssa1 ssa2 mutants most likely reflect the different molecular mechanisms for
transcriptional control. In the autoregulation of the heat shock
response in E. coli, DnaJ/DnaK/GrpE specifically interact with
to control its function (possibly regulating
interaction of
with the holoenzyme RNA polymerase
core) and synthesis (regulating translation and proteolysis)
(Georgopoulos et al., 1994). In budding yeast, SSA protein,
possibly in conjunction with SIS1, may be involved in regulating heat
shock factor for activation from heat shock elements. For SIS1 autoregulation, SIS1 might regulate either heat shock factor or a SIS1 specific trans-acting factor. How could SIS1 regulate
such a transcription factor? We have previously shown that SIS1 is
required for translation initiation (or for a very early translation
elongation step) (Zhong and Arndt, 1993). Perhaps SIS1 regulates the
translation of the transcription factor. Alternatively, that SIS1 RNA levels are induced in cim3, cim5, and cim3 cim5 mutants raises the possibility that SIS1 might
regulate the stability of the transcription factor. In addition,
similar to how E. coli DnaJ functions to convert repA dimers
into repA monomers for binding to the P1 phage origin of replication
(Wickner et al., 1991), perhaps SIS1 regulates (via a
chaperon-like function) the binding of the transcription factor to DNA.
Further delineation of these models will require the identification of
the transcription factor that mediates the autoregulation of SIS1.