(Received for publication, June 1, 1995; and in revised form, August 4, 1995)
From the
Cadmium-resistant Saccharomyces cerevisiae strain 301N exhibits high basal as well as cadmium-induced expression of the CUP1 metallothionein gene. Since regulation of CUP1 is usually restricted to copper ions, our goal was to identify the factor responsible for the high metallothionein levels in strain 301N. The gene responsible for the observed phenotype is a spontaneously mutated heat shock transcription factor gene (HSF1). A double, semidominant HSF1 mutant with substitutions at codons 206 and 256 within the DNA-binding domain of the heat shock factor (HSF) confers two phenotypes. The first phenotype is elevated transcriptional activity of the HSF mutant (HSF301), which results in constitutive thermotolerance. A second HSF301 phenotype is enhanced binding affinity for the heat shock element (HSE) within the CUP1 5`-sequences, resulting in high basal transcription of metallothionein. The CUP1 HSE is a minimal heat shock element containing only two perfectly spaced inverted repeats of the basic nGAAn block. Cells containing HSF301 are resistant to cadmium salts. The single R206S mutation is responsible for the high affinity binding to the CUP1 HSE. In addition, the R206S HSF substitution exhibits constitutive transcriptional activation from a consensus HSE (HSE2). The F256Y substitution in HSF attenuates the effects of R206S on the consensus HSE2, but not on the CUP1 HSE.
All cells are capable of coping with changes in their
environment, such as exposure to elevated temperatures, toxins, and
oxidants. In response to certain stress conditions, activation of
stress gene expression occurs, resulting in an elevated synthesis of
stress proteins, commonly called heat shock proteins
(hsp)()(1, 2) . That these hsp genes are
induced by a variety of stress conditions implies that they have
broadly protective functions.
The induction of heat shock protein(s) occurs at the level of transcription(3, 4, 5) . Genes encoding the various hsp molecules contain a conserved promoter element, designated a heat shock element (HSE)(1, 6) . The induction of hsp70 in animal cells by heat or metal ions requires only the HSE in the promoter(7, 8) . HSEs contain multiple 5-bp inverted repeats of the sequence nGAAn(9, 10, 11, 12) . The number of 5-bp boxes may range from three to six(9, 10, 11) . A perfect consensus array of three boxes would be the sequence 5`-nGAAnnTTCnnGAAn-3`. Not all HSEs have perfect inverted repeats, but it appears that they have at least two perfect nGAAn boxes(9, 10, 11, 12) . A compilation of 40 naturally occurring HSEs from different organisms revealed that seven contained only three nGAAn blocks, and in each case, these three nGAAn boxes were in combination with additional HSE units, permitting cooperative interactions(12) .
Transcriptional activation of
genes containing heat shock promoter elements is mediated by the heat
shock factor (HSF). Saccharomyces cerevisiae has one HSF
encoded by the HSF1 locus(13, 14) . Yeast HSF
is a trimeric protein reported to bind HSE sequences constitutively at
low temperature(5, 15, 16, 17) .
Within the N-terminal region of the 833-residue yeast HSF polypeptide
is a conserved sequence of 89 residues that is important in binding to
the 5-bp HSE boxes(18, 19) . It is likely that each
subunit of trimeric HSF contacts a separate 5-bp box within a HSE, but
the actual HSFHSE complex appears to contain multiple HSF
trimers(12, 17, 18, 19) .
Furthermore, most stress genes contain multiple HSEs, so additional
interactions can exist between adjacent HSF
HSE complexes. The
interaction of Drosophila HSF with HSEs is known to be highly
cooperative(9, 20) , although in yeast, cooperativity
is not essential as a minimal HSE of three perfect nGAAn units is
functional (21) .
In yeast, the bound HSFHSE complex
is transcriptionally silent until stress
activation(16, 22, 23, 24, 25) .
HSF undergoes a conformation change upon stress activation, but the
mechanism of stress-induced conformational dynamics is
unclear(16, 22, 23, 24, 25) .
In contrast, the activation process in animal cells initially involves
oligomerization to the trimeric state, which already exists in
yeast(1) . Yeast HSF contains domains that function as
constitutive transcriptional activation domains when fused to
heterologous DNA-binding domains(22, 23) . Since these
transactivation domains are not constitutive in HSF at low temperature,
it appears that the normal mode of action of HSF is to hinder the
effectiveness of these domains. This hindrance is relieved upon change
to stress conditions(16) . The regulatory domains of HSF can
even repress the activity of a heterologous transcriptional activation
domain fused in place of its own C-terminal activation
domain(24) .
Repression of the activation domain(s) appears to involve the DNA-binding domain, the trimerization domain, and the C-terminal conserved sequence. Deletion of the N-terminal 146 codons in HSF1 results in loss of low temperature repression(22) . Constitutive activity is also observed in HSF1 mutations within the DNA-binding domain (residues 167-256), deletions within the oligomerization domain (residues 350-402), or deletions within a C-terminal conserved region (residues 535-551)(22, 23, 24, 25) . A mutation at codon 232 in the DNA-binding domain was shown to yield a 200-fold increase in activity at 26 °C(24) .
In addition to the effects of HSF on expression of hsp genes, HSF is known to affect the expression of the gene CUP1, which encodes metallothionein(26, 27, 28) . The heat shock transcription factor (HSF) is responsible for high basal expression of CUP1 in cells starved for glucose(28) . The 5`-sequence of the CUP1 gene contains a minimal heat shock promoter element (HSE) that mediates both a limited heat shock induction of CUP1 expression with wild-type HSF and CUP1 transcription observed in glucose limitation(26, 28) . In addition, a point mutation within HSF1 can suppress the copper-sensitive phenotype of an ACE1 deletion strain by enhancing basal transcription of CUP1 in glucose-grown cultures(26, 27) .
CUP1 expression is normally regulated by Cu(I) ions through the ACE1 transcription factor(29, 30, 31) . Unlike animal cells, in which metallothionein biosynthesis is regulated by a variety of metal ions, the metalloregulation of yeast metallothionein is copper-specific(30) . However, cadmium-mediated CUP1 expression was observed in one cadmium-resistant strain of S. cerevisiae(32) . This strain, designated 301N(32) , arising from spontaneous mutation, exhibited both high basal CUP1 transcription and cadmium-induced CUP1 expression(33, 34, 35) . The observed cadmium resistance was a consequence of Cd(II) buffering by metallothionein(33) .
To elucidate the basis for the cadmium metalloregulation in strain 301N, we set out to identify the factor responsible for CUP1 expression in this yeast. In this study, we demonstrate that the regulatory factor conferring constitutive expression of CUP1 and cadmium metalloregulation in strain 301N is HSF. A double mutation within HSF1 in sequences encoding the DNA-binding domain leads to elevated basal transcription of CUP1 and constitutive thermotolerance. One dramatic result is that a single mutation at codon 206 (R206S) results in constitutive transcriptional activity of HSF on the consensus HSE-containing promoter, but not on the CUP1 HSE. It is noteworthy that the mutation at codon 256 suppresses the transcriptional activity of the R206S HSF mutant on the consensus HSE, but is without effect on transcriptional activation of the CUP1 HSE. This work clearly demonstrates differential activity of HSF on various HSE sequences.
The 5.7-kb insert was subcloned into the integrating plasmid, YIp5. This vector was linearized and transformed into strain CL7. The resulting strain was crossed with a DTY22 variant. Both haploid strains contained CUP1/lacZ fusion genes. Tetrad analysis revealed a 2:2 segregation of enhanced lacZ expression, implying that the 5.7-kb insert contains sequences capable of up-regulation of CUP1 expression.
The restriction map of each plasmid was unrelated to maps of CUP1 or ACE1, but was similar to that of yeast HSF1. DNA primers specific for S. cerevisiae HSF1 were successful in priming sequencing for each of the three plasmids. From sequence analysis of the entire HSF1 open reading frame, two mutations at codons 206 and 256 (R206S and F256Y) were found. These two codons occur within sequences encoding the DNA-binding domain of HSF. Two other nucleotide differences exist at codons 522 and 831 relative to the HSF1 sequence reported by Wiederrecht et al.(13) , but are identical to the HSF1 sequence reported by Sorger and Pelham(14) .
The presence of episomal hsf1-301 in yeast containing wild-type CUP1 conferred resistance to cadmium salts (Fig. 1A). Cells harboring YCpHSF301 were markedly more cadmium-resistant than either control cells or cells harboring YCpHSF (Fig. 1A). This effect is observed in cells at 30 °C, a temperature at which the HSF-mediated stress response is limited. We were unable to determine whether hsf1-301 conferred copper tolerance in these cells as they were wild-type in ACE1, which specifically mediates copper-induced expression of CUP1. To test the effect of hsf1-301 on copper tolerance, the vectors were transformed into ace1-1 cells (DTY23) (Fig. 1B). ace1-1 cells are unable to couple CUP1 expression with the copper concentration. Cells harboring YCpHSF301 were more copper-tolerant than control cells or cells transformed with YCpHSF. When the HSF genes were present on high copy YEp plasmids, wild-type HSF1 conferred limited tolerance, and as expected, hsf1-301 cells exhibited marked copper tolerance.
Figure 1:
Metal resistance of cells
harboring episomal HSF genes. In A, PS145 cells harboring
either TRP1-based YCpHSF or YCpHSF301 were plated on medium in
the presence or absence of 100 µM CdSO. In B, DTY23 (ace1-1) cells harboring URA3-based HSF1 or hsf1-301 plasmids were used. Equal aliquots
of cells were plated on medium at 30 °C in the presence or absence
of 50 µM CuSO
.
To quantify
the transcriptional effect of HSF, PS145 cells harboring the episomal
HSF genes were transformed with a YEp vector containing the CUP1/lacZ fusion gene. Transformants harboring
YCp-based hsf1-301, but not YCp-based HSF1, revealed
high -galactosidase levels at 23 °C, consistent with high
constitutive expression of CUP/lacZ (Fig. 2A). Cells were maintained at 23 °C to
minimize heat activation of HSF. Thus, activity is a measure of
constitutive transcriptional activity of HSF. The difference between
YCpHSF and YCpHSF301 was over 100-fold. Wild-type HSF was able to
transactivate CUP1 expression to an appreciable extent only
when expressed on a high copy plasmid. The presence of hsf1-301 in PS145 cells mimics two key phenotypes of strain 301N, namely
enhanced metal tolerance and high basal CUP1 expression.
Figure 2:
HSF-mediated expression of CUP1/lacZ (A) and
HSE2/CYC1/lacZ (B) in PS145 cells
transformed with different HSF plasmids. Cells were cultured at 23
°C prior to harvesting and quantitation of -galactosidase
activity. Results are averages of three replicate cultures; standard
errors are shown.
A second test of the effects of hsf1-301 on general stress responsiveness was to measure the activity of HSF301 on a consensus HSE sequence. PS145 cells containing either episomal HSF1 or hsf1-301 were transformed with a vector containing a synthetic consensus HSE sequence in place of the upstream activating sequence of a CYC1/lacZ fusion gene(4) . The fusion gene contains a HSE consisting of four nGAAn inverted repeats, the sequence of which is a consensus of HSEs found in a myriad of hsp genes(4) . Both HSF1 and hsf1-301 were able to drive the expression of lacZ from the consensus HSE2 promoter, but hsf1-301 yielded 2-3-fold higher levels of expression (Fig. 2B). No lacZ expression was observed in cells containing a HSE12/CYC1/lacZ fusion gene with a mutated HSE sequence (HSE12) that matched the consensus in only six of eight positions (4, 17) (data not shown). Thus, both HSF and HSF301 transactivate through HSE sequences. Since yeast HSF is known to be constitutively bound to the consensus HSE2 promoter(17) , the enhanced basal transcription observed with HSF301 must arise from elevated transcriptional activation rather than an increased DNA binding avidity.
Metal ions are known to stimulate
the general stress response pathway (1) , and this effect may
contribute to cadmium-induced CUP1 expression in strain
301N(32) . Cd(II) stimulation of CUP1 was found to be
strain-specific. No Cd(II) stimulation of CUP1/lacZ
expression was observed in PS145 cells harboring either hsf1-301 or HSF1. In contrast, significant Cd(II) stimulation of CUP1/lacZ expression was observed in DTY23 cells
harboring episomal hsf1-301 (Fig. 3) or in cells
harboring HSF1 on a high copy plasmid (data not shown). Cells
were incubated with CdSO for 2 h prior to cell harvest.
Since DTY23 cells lack a functional ACE1, the ACE1-dependent,
copper-induced expression of CUP1 is precluded. Transformation
of these cells with YCpHSF301 resulted in appreciable copper
stimulation of CUP1/lacZ. Maximal metal-induced
expression of CUP1 occurred at metal ion concentrations that
were inhibitory to cell growth, so the effect may be a general stress
response. It is unclear why the metal effect is temperature-dependent
and shows a different temperature dependence for copper and cadmium
ions (Fig. 3).
Figure 3:
Metal-induced expression of CUP1/lacZ in DTY23 (ace1-1) cells
transformed with either YEpHSF or YCpHSF301. Cells at A = 0.7 were incubated in the presence
or absence of 50 µM CuSO
or CdSO
for 2 h at the temperatures indicated prior to quantitation of
-galactosidase levels.
Figure 4:
Cadmium tolerance of PS145 cells with
YCp-based vectors containing HSF1 mutants. Cells were plated
on medium at 30 °C containing 100 µM
CdSO.
The
individual HSF mutants were tested for their ability to transactivate
the expression of CUP1/lacZ and
HSE2/CYC1/lacZ fusion genes. Studies with PS145 cells
containing CUP1/lacZ revealed high constitutive
expression of lacZ at 23 °C conferred by HSF301 and both
R206S and V203A HSF mutants (Fig. 5A). Whereas the HSF
double mutant imparted slightly higher metal tolerance to cells
compared with R206S HSF, there was no significant difference between
HSF301 and R206S HSF in activating lacZ expression from the CUP1/lacZ fusion gene. Thus, the single Arg
Ser mutation at codon 206 is largely responsible for the phenotype of
HSF301. The individual F256Y HSF mutant was without effect on lacZ expression from a CUP1/lacZ fusion gene
compared with wild-type HSF (Fig. 5A).
Figure 5: Basal expression of CUP1/lacZ (A) and HSE2/CYC1/lacZ (B) in PS145 cells carrying the YCp-based vectors shown and grown at 23 °C. The results are an average of three replicate cultures; standard errors are shown.
In contrast,
cells containing the HSE2/CYC1/lacZ fusion gene
exhibited markedly enhanced expression of lacZ with R206S HSF
compared with HSF301 (Fig. 5B). This is unlike the
results with CUP1/lacZ, in which the transcriptional
activity of R206S HSF was equivalent to that of HSF301 (Fig. 5A). To confirm that the R206S results were not a
result of a secondary mutation, PS145 cells were retransformed with
HSE2/CYC1/lacZ and HSF genes. Quantitation of
-galactosidase levels revealed similarly high activity as in the
original transformants. In contrast to R206S HSF, V203A HSF was without
effect on HSE2/CYC1/lacZ expression.
Yeast transformants with the various individual mutant HSF genes and the HSE2/CYC1/lacZ fusion gene were tested for heat shock-induced lacZ expression to determine whether R206S HSF was fully active at 23 °C (Fig. 6). Whereas heat shock conditions resulted in induction of lacZ expression for wild-type HSF and F256Y HSF in excess of 10-fold, cells containing R206S HSF exhibited a <2-fold heat shock response. It is also curious that V203A HSF responds so poorly to heat shock (Fig. 6).
Figure 6:
Heat
shock induction of HSE2/CYC1/lacZ (4) in
PS145 cells expressing the HSF molecules shown from genes present on
YCp vectors. Three replicate cultures were either maintained at 23
°C (control) or heat-shocked at 39 °C for 20 min and returned
to 23 °C for 1 h prior to quantitation of -galactosidase
activity. Standard errors are shown.
Figure 7: Gel retardation assay of a radiolabeled CUP1 HSE oligonucleotide using extracts of PS145 cells transformed with either YEpHSF or YEpHSF301. Protein (12 µg) from each extract was used. Lane1 contains only the free CUP1 HSE probe. Lanes 2-4 contain extracts from PS145 cells harboring YEpHSF, and lanes 5-7 contain extracts from PS145 cells with YEpHSF301. In lanes3 and 6, rabbit antiserum to yeast HSF was added to the incubation mixtures prior to electrophoresis, and in lanes4 and 7, control preimmune antiserum was added.
Gel retardation assay of
extracts from cells carrying singly mutated HSF genes showed evidence
of a HSFDNA complex with the CUP1 HSE sequence with the
individual R206S HSF mutant, but not with the F256Y HSF mutant (Fig. 8A). Using a HSE2 oligonucleotide, both the
wild-type and mutant HSF molecules formed DNA-protein complexes as
judged by the gel retardation assay (Fig. 8B). Each
individual mutant HSF protein formed a HSF
DNA complex. A
reproducible difference existed in the extent of electrophoretic
mobility of the HSF
DNA complex for the different HSF molecules.
It appears that the actual HSF
DNA complex consists of multiple
HSF molecules(12, 22) , so differences in mobility may
arise from variations in the oligomeric complexes that form or from
differences in the individual monomers.
Figure 8: Gel retardation assay with various mutant HSF molecules. In A, gel retardation of the CUP1 HSE oligonucleotide was analyzed with 20 µg of protein from clarified extracts of PS145 cells harboring YEp-based vectors containing the mutant HSF genes shown. In B, gel retardation of the HSE2 oligonucleotide was analyzed with the same cell extracts used in A.
Since gel retardation
studies were carried out with crude yeast extracts in which the actual
HSF protein concentration was undefined, it was important to verify the
conclusion that wild-type HSF did not bind with equal affinity to the CUP1 HSE oligonucleotide. The HSF antiserum did not work in
Western analysis, so competition binding gel shift experiments were
carried out to substantiate this result (Fig. 9). Radiolabeled
HSE2 probe was shifted with extracts from cells expressing either
wild-type HSF or HSF301. Using extracts containing HSF301, the addition
of either unlabeled CUP1 HSE DNA or HSE2 DNA to the binding
reaction prior to addition of protein resulted in a
concentration-dependent competition in binding. The competition
diminished the amount of radiolabeled probe in the retarded
HSFDNA complex (Fig. 9A). In multiple experiments
with HSF301, unlabeled HSE2 or the CUP1 HSE oligonucleotides
were nearly equivalent in competition. In contrast, with extracts
containing wild-type HSF, HSE2 was 5-10-fold more effective than CUP1 HSE DNA as a competitor as determined by densitometry (Fig. 9A). This is further evidence that HSF301 forms a
more avid complex with the CUP1 HSE sequence than wild-type
HSF.
Figure 9:
Competition of wild-type HSF and HSF301
binding to the radiolabeled HSE2 DNA oligonucleotide by unlabeled
oligonucleotides. Only the position of the HSFDNA complex is
shown in the competition studies, and densitometry was used for
quantitation. In A, protein (20 µg) from clarified
extracts of PS145 cells containing either YEpHSF or YEpHSF301 was
incubated with the radiolabeled HSE2 oligonucleotide in the presence of
increasing amounts of unlabeled DNA as specified. In B, the
HSF301 extract was used with radiolabeled HSE2 DNA and increasing
quantities as shown of unlabeled CUP1 HSE, two mutated CUP1 HSEs, or an oligonucleotide duplex containing the binding
site for AMT1. The CUP1 HSE(M1) contained the gapped nTTTn
box. The two consensus nGAAn boxes in the CUP1 HSE were
changed to nGAGn boxes in the HSE(M2)
duplex.
Previous footprinting results on CUP1 suggested that the CUP1 HSE may consist of the sequence CTTCTAGAAGCAAAAAGAG, with the underlined nucleotides being the conserved GAA inverted repeats(28) . HSE sequences do not typically exhibit gapped nGAAn blocks, and the candidate gapped box in the CUP1 HSE is a nonconsensus nGAGn box(10, 12) . To address whether the nGAGn block is a critical part of the CUP1 HSE, three oligonucleotides consisting of altered CUP1 HSE sequences were tested as competitors in a gel shift assay with a cell extract containing HSF301 protein and radiolabeled HSE2 DNA as probe. The first CUP1 HSE oligonucleotide variant contained the gapped GAG trinucleotide substituted by TTT. The gapped nTTTn box duplex failed to compete even at a concentration 100-fold greater than the concentration of CUP1 HSE DNA that exhibited 75% competition (HSE(M1) in Fig. 9B). DNA containing a high affinity binding site for an unrelated transcription factor, AMT1, also failed to compete. Thus, the gapped nGAGn block appears to be an essential part of the CUP1 HSE. In the second CUP1 HSE variant, the gapped nGAGn box was changed to a consensus nGAAn box, and this oligonucleotide duplex appeared equivalent to the wild-type CUP1 HSE sequence as a competitor (data not shown). Each of the three nGAAn boxes in the CUP1 HSE was changed to nGAGn in the third duplex variant, and this oligonucleotide failed to complete (HSE(M2) in Fig. 9B).
The cadmium tolerance observed in S. cerevisiae strain 301N was found to arise from a semidominant mutation within the DNA-binding domain of HSF. The dominant effect of HSF301 on Cd(II) tolerance arises from HSF301-induced transcription of CUP1-encoding metallothionein. HSF301 confers minimal metal tolerance in S. cerevisiae cells lacking a functional CUP1 (data not shown). Elevated metallothionein levels enable efficient Cd(II) sequestration as a cadmium-metallothionein complex.
A second phenotype of hsf1-301 cells is enhanced transcription activation activity. Wild-type HSF mediates both transient and sustained effects in relationship to stress(16) . The elevated basal activity of HSF301 at low temperatures is observed with a consensus HSE cloned upstream of the CYC1/lacZ fusion gene that was originally used to demonstrate constitutive binding of HSF(17) . In addition, high basal activity of HSF301 at low temperature is indicated by the thermotolerance of cells harboring hsf1-301. The mutations in hsf1-301 appear to result in high basal expression of one or more genes that are critical for thermal tolerance. As mentioned, a V203A substitution in HSF was reported previously to enhance CUP1 expression, but curiously not to give heat tolerance(26, 27) .
Enhanced basal activity of HSF301 is not unique to the 301N mutations in HSF1. Other mutations or deletions within the DNA-binding domain of HSF, the trimerization domain, and the HSF conserved element CE2 result in loss of low temperature repression(23, 24, 25) . Sequences within these three regions of HSF are important in maintaining HSF in a repressed state at 23 °C. Another mechanism for enhanced low temperature activity of HSF301 may be increased efficiency of assembly of the transcription complex(43) . Certain HSF1 mutants are constitutively active(23, 24, 25) . Other mutants have intermediate effects on HSF deregulation in that transactivation is further enhanced by heat shock(23, 24, 25) . The elevated activity of HSF301 is of intermediate effect as transcription of CUP1 is further enhanced by heat shock and metal ions.
The two distinct phenotypes observed with HSF301, namely enhanced low temperature transactivation of multiple genes containing HSE elements and enhanced CUP1 binding, arise from the R206S mutation. There are two dramatic results in the experiments with HSE2/CYC1/lacZ. First, R206S HSF activates HSE2/CYC1/lacZ (but not CUP1/lacZ) expression constitutively. The clear implication is that the R206S HSF molecule is capable of discriminating between HSEs. The second novel result is that HSF301 has reduced transcription activation activity compared with R206S HSF on the HSE2/CYC1/lacZ fusion gene, yet equivalent activity on CUP1/lacZ expression. It appears that the second mutation in HSF301 at codon 256 (F256Y) suppresses the effects of the R206S mutation on HSE2, but not on the CUP1 HSE.
Based on the known structures of Kluyveromyces lactis and Drosophila HSFs(18, 44) , Arg-206 is expected to be a
moderately solvent-exposed residue situated near the start of the
highly irregular second -helix within the DNA-binding domain.
Although the function of this helix in HSF/DNA interaction remains
unresolved, the structure of HSF resembles a family of DNA-binding
proteins with a helix-turn-helix structural
motif(18, 44) . Based on structural comparisons, HSF
helix 3 is predicted to function as the recognition
helix(18, 44) . Residues corresponding to Arg-206 in
CAP, Oct1,
-repressor, and 434 Cro form hydrogen bonds to
phosphates in the DNA backbone(44) . A Glu
Lys mutation
in this codon of the
-repressor resulted in a 600-fold enhancement
in DNA binding affinity, which was attributed to the formation of a new
salt bridge (45) .
The constitutive activity of R206S HSF
was attenuated in the context of the double hsf1-301 mutation.
The F256Y substitution suppresses the activity of the R206S mutation on
HSE2. Phenylalanine is found at codons corresponding to codon 256 in
all known HSF molecules(44) . The distance of Arg-206 and
Phe-256 -carbons in non-DNA-bound HSF is 12.3 Å, and side
chains are separated by 8.9 Å. The presence of a buried Tyr-256
may alter the HSF tertiary fold to compromise the effects of the R206S
mutation. Final understanding of the relationship of Arg-206 and
Phe-256 in the HSF structure will have to await elucidation of the
HSF
DNA costructure.
HSF301 exhibits enhanced binding to CUP1 as reported previously for the V203A HSF mutant(26, 27) . Band shift analysis with the CUP1 HSE oligonucleotide revealed formation of a specific complex with HSF301, but not with wild-type HSF. The absence of a complex formation with wild-type HSF under normal conditions implies that wild-type HSF is not constitutively bound to the CUP1 HSE. A recent report demonstrated that wild-type HSF forms a complex with the CUP1 HSE sequence(28) . To observe a complex, the investigators used an unusually high concentration (30 µg) of partially purified HSF. In the gel shift experiments reported here, we used 12-20 µg of total protein from a crude yeast extract, and under these conditions, wild-type HSF does not bind the CUP1 HSE. In addition, the CUP1 HSE is an inferior competing oligonucleotide to the HSE2 oligonucleotide containing a HSE sequence with four nGAAn inverted sequence repeats.
Most hsp genes contain multiple HSE sequences, with each HSE containing at least three conserved nGAAn inverted repeats(9, 10, 11, 12) . The number of nGAAn repeats usually ranges from three to six(9, 10, 11, 12) . The presence of multiple HSEs within a promoter sequence permits cooperative binding of HSF(9, 12, 17, 20) . A perfect consensus array of three boxes would be the sequence 5`-nGAAnnTTCnnGAAn-3`(9, 10, 11) . Two types of variations of the consensus sequence exist in naturally occurring HSEs. First, limited sequence variation is permitted, and second, interrupted periodic arrangement of nGAAn blocks is tolerated(9, 10, 11) . For example, in Drosophila, three nGAAn blocks that include a one-block gap can be a functional HSE provided that the three blocks are properly oriented and spaced(11) .
The CUP1 5`-sequences contain one consensus HSE, but with only two perfectly spaced nGAAn boxes(26, 27) . Footprinting results indicate that HSF contacts a larger segment of the CUP1 sequences, suggesting that the actual HSE may consist of the sequence CTTCTAGAAGCAAAAAGAG(28) . We verify here that an oligonucleotide containing the two perfect nGAAn repeats, but lacking the gapped nGAGn box, failed to compete with the HSE2 DNA in binding assays with HSF301. GAG is not a common trinucleotide HSE repeat in yeast, but changes in the third position are the least detrimental(10) . The gapped nGAGn box is not a critical factor for the enhanced HSF301 binding to the CUP1 HSE as a gapped nGAAn HSE variant appears equivalent to the wild-type CUP1 HSE sequence in HSF301 binding. Furthermore, the enhanced DNA binding affinity of HSF301 for the CUP1 HSE does not relate to altered specificity as a HSE with all nGAGn boxes fails to bind HSF301.
Two
aspects of the minimal CUP1 HSE appear to be optimal. First,
the arrangement of nGAAn units in the CUP1 HSE appears to be
favorable for activity as it was recently demonstrated that
(nTTCnnGAAn) elements are more active than
(nGAAnnTTCn)
elements(12) . Second, the adenine
upstream of the GAA block in CUP1 is clearly the preferred
nucleotide for this position in S. cerevisiae HSEs(10) . Most naturally occurring HSEs from different
organisms contain more than three nGAAn blocks, and those containing
three nGAAn blocks were typically in combination with additional HSEs,
permitting cooperative interactions (12) . The presence of a
single gapped three-nGAAn block HSE in CUP1 is consistent with
this HSE being a minimal element, and HSF binding may be expected to be
weak. The high affinity binding of HSF301 to this minimal non-consensus
HSE is striking.
The R206S mutation in HSF301 may result in enhanced
DNA affinity by one of several mechanisms. First, the mutation may
permit additional HSFDNA contacts, which stabilize the
interaction. Second, the mutation may confer greater flexibility in
HSF301, permitting enhanced binding avidity to the CUP1 HSE
element with the candidate gapped nGAAn-like array. Third, the R206S
mutation may contribute to higher order assembly beyond the trimeric
state, which may stabilize DNA/protein interaction. The arginine at
codon 206 is not conserved in all HSF molecules, and in fact, a seryl
residue exists at that sequence position in one tomato
HSF(44) . Structural studies on the HSF301 interaction with the CUP1 HSE may be insightful in understanding how HSF contacts
HSEs differentially.