MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 3262-3267
Vol. 10, No. 6
0270-7306/90/063262-06$02.00/0 Copyright © 1990, American Society for Microbiology
Transcriptional Regulation of SSA3, an HSP70 Gene from Saccharomyces cerevisiae WILLIAM R. BOORSTEINl* AND ELIZABETH A. CRAIG1' 2
Molecular and Cellular Biology Program' and Department of Physiological Chemistry,2 University of Wisconsin-Madison, Madison, Wisconsin 53706 Received 23 October 1989/Accepted 28 February 1990
The SSA3 gene of Saccharomyces cerevisiae, a member of the HSP70 multigene family, is expressed at low levels under optimal growth conditions and is dramatically induced in response to heat shock. Sequences coinciding with two overlapping heat shock elements, located 156 base pairs upstream of the transcribed region, were necessary and sufficient for regulation of heat induction. The SSA3 promoter was also activated in an ssalssa2 double-mutant strain. This increase in the expression of SSA3 was mediated via the same upstream activating sequences that activated transcription in response to heat shock.
Virtually all organisms studied to date respond to environmental stresses, such as heat shock, by the induction of a conserved set of proteins known as the heat shock proteins or HSPs (for a review, see reference 10). A cis-acting, heat-activated transcriptional regulatory sequence, the heat shock element (HSE), first defined in Drosophila melanogaster (24), has been identified in many species including yeast, plants, and mammals (for a review, see reference 6). A trans-acting heat shock transcription activation factor, HSF, which interacts directly with the HSE sequence, has been shown specifically to activate transcription of heat shock genes both in vitro and in vivo (33, 36, 37). HSF from stressed and nonstressed Saccharomyces cerevisiae exhibits comparable binding affinities for HSEs (18, 30). HSF is activated posttranslationally by a mechanism likely to involve phosphorylation (29, 31). The yeast S. cerevisiae contains a family of at least nine genes encoding 70-kilodalton heat shock proteins (HSP70s) (for a review, see reference 11). SSA3 and SSA4 are the only HSP70 genes that are expressed at extremely low levels under optimal conditions and are rapidly induced in response to stress (35). SSAI and SSA2, the other two members of the SSA subfamily, are expressed at substantial levels under nonstressed growth conditions. ssalssa2 double mutants are viable at 30°C and constitutively thermotolerant (able to survive brief exposure to temperatures that are normally lethal) but are temperature sensitive for growth (12). Here we analyze the regulation of SSA3 transcription under stressed and nonstressed conditions in wild-type strains. We also examine the effects of ssal and ssa2 mutations on the regulation of the SSA3 promoter. Analysis of SSA3 and flanking DNA. The DNA sequence of the portion of the genomic clone SSA3H that encodes the amino terminus of the SSA3 protein (Ssa3p) and a contiguous 5'-flanking region was determined (Fig. 1). Amino acids 6 to 16 (predicted from the DNA sequence [Fig. 1B]) comprise a highly conserved amino-terminal domain that is identical to other S. cerevisiae HSP70s (28) as well as to those of distantly related species (16, 17, 25). Both primer extension and Si nuclease analyses indicate that SSA3 transcripts are heterogeneous at the 5' termini; 21 apparent 5' ends were identified by both techniques (Fig. 2). All of the most abundant 5' ends, comprising more than 85% of the *
transcripts, map to the dinucleotide TA, consistent with the correlation observed for the CYCI gene between pyrimidinepurine dinucleotides and initiation sites (Fig. 1B) (20). All of the observed 5' ends map between 44 and 83 base pairs (bp) downstream of the 8-bp sequence 5'-TATATAAA-3', which is comprised of two overlapping sequences previously defined to function as yeast TATA boxes (9, 20, 22). There are eight sequences that match at least six of eight conserved nucleotides of the canonical heat shock regulatory element, CNNGAANNTTCNNG, where N represents any nucleotide (Fig. 1B) (5). Four of the HSE-like sequences are present in overlapping pairs, an arrangement often found upstream of heat-inducible genes in yeast and other organisms (for a review, see reference 6). SSA3 promoter dissection. To facilitate the localization of DNA sequences that function in regulating transcription of the SSA3 gene, a chimeric gene was constructed by fusing SSA3 sequences between -764 and codon 3 (Fig. 1B) to the lacZ gene of Escherichia coli in a centromeric vector. The level of expression of the fusion gene in cells grown in rich medium at a constant 23°C temperature was low, only 4 Miller units. Following heat shock, a dramatic increase in ,-galactosidase activity was observed within 30 min. The 5' termini of the fusion transcripts were indistinguishable from those of the native SSA3 gene (data not shown). A progressive series of deletions from the upstream end of the SSA3IlacZ hybrid gene was constructed to localize cis-acting transcriptional regulatory elements (Fig. 3). Deletion of sequences 5' to -166 resulted in SSA3IlacZ fusion genes retaining heat-inducible activity; removal of the two distal HSE-like sequences had no effect on either the basal or heat-inducible levels of expression. Deletion of an additional 12 bp, removing more than half of the overlapping HSEs centered at -156, essentially abolished the heat inducibility of the promoter. The presence of sequences that negatively modulate SSA3 expression was suggested by the increase in basal activity caused by deletion of sequences between -468 and -200 and between -154 and -124. To further define DNA sequences that regulate transcription of the SSA3 gene, fragments of the SSA3 promoter region were tested for their ability to activate the heterologous CYCI promoter (14) in a heat-inducible manner. A 113-bp restriction fragment from SSA3 that included sequences from -236 through -124 was able to mediate a low basal activity (2.4 Miller units) and a rapid, 20-fold heat
Corresponding author. 3262
NOTES
VOL. 10, 1990
§
A
H
Bn Sa N
II
RI
Sp
SSA3
X
Ss
P2
Bs
ORF
I Sn
3263
Ss
Ss X
Ps
P1
Ps Rv
II~~~ Bs Sp N Sn Ri Hp
H
Bm A 1 Kb
B -1090
GCCTT TACAAAATGA
-1075
TGGAAGAAAG GGATGGCGCA AAATTCGAGA ATATTGTTCA CAACTTCAAG GAACGGCAGA TGATGGTTTC TTATCCGAAA ATTGACGAAG ATGATACCTG
-975
SnaB I GTACAATCTT ACCGAGTTTG TGCAGATGGA TAAAATCCGA AAGATAGTAA GGAAAGATGA AAACCAGTTC TCTTACGTAG ATTCTTCGAT GACCACAGTT
-875
CAAGAAAATG AGCTGCTAAA ATCCAGCTTG CAAAAAGCAG GTTCTAAAAT GGAAGCCAAG AATGAAGATG ATCCTGCACA TTCTTTAAAC TATACAGTAA
-775
TAAACTTCAA ATCTAGAGAA GCCATAAGGC CTGGCCATGA AATGGAGGAT TTTTTAGACA AGTCTTACTA CTTGAACACT GTAATGCTAC AAGGAATTTT
-675
TAAAAATTCA AGTAATTATT TTGGGGAGTT GCAGTTTGCG TTCTTAAATG CCATGTTTTT TGGTAACTAC GGGTCGAGTT TGCAATGGCA TGCTATGATC
-575
GAACTGATAT GTTCAAGCGC TACGGTGCCT AAACATATGC TCGATAAATT AGACGAAATC TTATATTATC AGATAAAGAC ATTGCCTGAA CAATACTCAG
-475
ACATCTTGTT GAATGAACGA GTTTGGAATA TTTGTCTGTA TTCGTCATTT CAAAAAAACT CCCTACACAA CACAGAAAAG ATAATGGAAA ACAAATATCC
-375
AGAATTGCTT GGTAAAGACA ATGAAGACGA CGCTCTTATT TACGGTATCA GTGATGAAGA AAGGGATGAC GAGGATGATG AGCACAACCC TACCATTGTT
-275
GGCGGTCTCT ATTACCAAAG GCCATAACGA TCATCGTGCG GCGCTATCAT CAAACGTATT TGACTTGATG CCTATGGAGG TTATGGGTGC CCTTAATTAG
-175
GGATCGCTGT GGAAAGTTAT AGAATATTAC AGAAGCAGCC ACAAGGGTGA CCAGAAGATG GTTAAGGGAT GTATCATATT GCACAATTGG AAACGAATGG +1
Xba I
Sph I
-236
BstE 11 -75
26
107
VV v V V VV vv V VV Y v vvv AAGGGX X~TGACTG AAATTGGTAG CATAAACATT CTTGTATGTC AATGTTTGTC ACTAAACGGA TAGAATAGGT ACTAAACGCT ACAAAGAAAA XbaI
ATG TCT AGA GCA GTT GGT ATT GAT TTG GGA ACA ACT TAC TCG TGT GTT GCT CAT TTT TCC AAT GAT AGG GTA GAG ATA ATT Met Ser Arg Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Ala His Phe Ser Asn Asp Arg Val Glu Ile Ile
Sin I GCA AAT GAT CAA GGT AAT AGG ACC Ala Asn Asp Gln Gly Asn Arg Thr
130
FIG. 1. Restriction map and partial DNA sequence of SSA3 and flanking genomic DNA. (A) Restriction map of the cloned 5.1-kilobase HindIII SSA3H restriction fragment. The region encoding the SSA3 protein and the portion of a large open reading frame (ORF) identified by DNA sequence analysis are represented by arrows. Abbreviations: A, AvaI; Bm, BamHI; Bn, BanII; Bs, BstEII; H, HindIII; Hp, HpaI; N, NdeI; P1, PvuI; P2, PvuII; Ps, PstI; RI, EcoRI; Rv, EcoRV; Sa, Sacl; Sn, SnaBI; Sp, SphI; Ss, SspI; X, XbaI. The following enzymes did not cleave SSA3H: NheI, Sall, KpnI, SacII, SmaI, MluI, BglII, ApaI, XhoI, and FspI. The DNA sequence of the solid black region of the restriction map was determined and is shown in panel B. (B) DNA sequence of the amino-terminal region of the SSA3 structural gene and 5'-flanking region as determined by the dideoxy-chain termination method (4). The major putative initiation site closest to the SSA3 protein-coding region has been designated + 1. The upstream open reading frame extends from the beginning of the sequences shown here through position -252 (on the same strand as the SSA3 coding region) and has the potential of encoding a protein of at least 308 amino acids (see above; unpublished observations). Neither the upstream open reading frame DNA nor its predicted amino acid sequence is closely related to sequences currently in the GenBank, EMBL, or NBRF databases. Putative SSA3 mRNA initiation sites are indicated by vertical arrow heads (scale: v, V, V, V, from least to most abundant 5' termini). Matches to the canonical HSE consensus sequence CNNGAANNTTCNNG are underlined. The putative TATA element(s) is enclosed in a box. Sequences resembling BAS21PH02 (position -670 through -654) (3), GRFIIRAPI (dashed underline, -198 through -186) (8, 26), and GCN4 (-61 through -56) (2, 15) binding sites were also identified. Three iterations of imperfect 6- and 7-bp repeats in altemating orientations are indicated by arrows above the sequence. Restriction enzyme recognition sites used for cloning and for construction of primers and probes used in transcript mapping are labeled. Position -236, marked above, is the site of the 5' deletion endpoint used for isolating the 113-bp fragment. The predicted amino acid sequence of the amino terminus of Ssa3p is shown below the nucleotide sequence. shock induction, indicating that this fragment contains sequences sufficient to confer the heat-inducible regulation characteristic of SSA3 (Fig. 4A). The only matches on this fragment to the heat shock consensus sequence are those centered at -156. A 32-bp synthetic oligonucleotide containing the -156 HSEs and only four to five flanking nucleotides (Fig. 5) was also able to activate the CYCJIIacZ hybrid gene in response to heat shock, although the induction was smaller than that
observed from the 113-bp fragment (Fig. 4A). This short fragment is a heat shock upstream activating sequence (UASHS), as it is sufficient to mediate heat-inducible transcription in an orientation-independent manner. Both elevated basal expression and a smaller increase in expression following heat shock contributed to the low induction ratio observed for this construct. HSE consensus sequences are essential to UASHS activity. Transcriptional induction driven by the 113-mer was greater
3264
.a
NOTES
C)
C)
E
Stringency: _
S'
1
MOL. CELL. BIOL.
Si
Primer Extension M 3
L 2
H
L 4
H 6
5
.
Nuclease
M 8
L 7
L 9
o
10
-
-
-
-
r
-
.
.0
i-
0
0
a
S
.-
139-146
*122-135
U. -
113-116
Ior FIG. 2. Si and primer extension analyses of native SSA3 RNA isolated from heat-shocked cells. S and S' are DNA size standards pBR322 and '1X174, respectively, cleaved with HaeIII. RNA was isolated from DS10 (MATa his3-11,-5 leu2-3,-112 lysi Iys2 SSAI SSA2 Atrpl ura3-52) grown at 23°C to an optical density at 600 nm of 0.4 and subsequently heat shocked at 39°C for 20 min. Lanes 1 and 10, Controls containing the same quantities of primer and probe, respectively, used in the mapping reaction; lanes 2 and 9, reaction controls containing tRNA but no yeast mRNA; lanes 3 to 5 and 6 to 8, SSA3 primer extension and Si nuclease mapping reactions, respectively, with 13 jig of total RNA per lane under low (L)-, medium (M)-, and high (H)-stringency conditions. Hybridization of RNA to DNA restriction fragments was performed in 80% formamide-0.4 M NaCl at three different temperatures following brief (45 s) denaturation at 80°C; high- and medium-stringency hybridizations were performed at 46 and 41°C, respectively, for 7 h; low-stringency hybridization reactions were slowly cooled from 48 to 37°C over 3.5 h and held at 37°C for 2 h. A SinI-to-XbaI (position + 128 to +31) fragment was used as a DNA primer for primer extension analysis. A SinI-to-SnaBI (+128 to -900) fragment was used as a probe for Si analysis. The fragments were 5' end labeled at the SinI termini; therefore, bona fide 5' ends mapped by each method should yield comigrating products. Unextended primer and undigested probe bands are indicated by arrowheads at the lower left and upper right, respectively. The length in nucleotides of the three (arbitrary) size classes of products are indicated at the right. A more precise mapping of the mRNA termini was obtained by higher-resolution electrophoresis, giving the positions noted in Fig. 1B (data not shown). Annealings, hybridizations, extensions, and nuclease digestions were carried out as previously described (4, 7).
[galactosidase activiy -
vw I
3T I*MMM8TXvy L v vv v
-583
-
-38 >
0.03
-AA
-
.
vv
,
rrx L:
IKI, 11
%.-
LZJ V
-166 -154 Iv -124
i
L:
=
-93
=
SSA3
L~
-----.-,--,--,,,,--,--,-,
v
vv
-111
Vector
LTVA
4.2 3.7 3.5 6.1 17 16 11 1.1 14 5.8 6.9
S-' -4688}vSSM,SSS/ -236 -200 -175
=
+1 ATG ,...H
-156
-764
_-"'4,. v
lacZ
62 60 61 92 84 80 54 2.1 13 6.3 7.0 < 0.03
15 16 17 15 4.9 5.0 4.9 1.9 0.9 1.1 1.0
Vol. 10, No. 6
0270-7306/90/063262-06$02.00/0 Copyright © 1990, American Society for Microbiology
Transcriptional Regulation of SSA3, an HSP70 Gene from Saccharomyces cerevisiae WILLIAM R. BOORSTEINl* AND ELIZABETH A. CRAIG1' 2
Molecular and Cellular Biology Program' and Department of Physiological Chemistry,2 University of Wisconsin-Madison, Madison, Wisconsin 53706 Received 23 October 1989/Accepted 28 February 1990
The SSA3 gene of Saccharomyces cerevisiae, a member of the HSP70 multigene family, is expressed at low levels under optimal growth conditions and is dramatically induced in response to heat shock. Sequences coinciding with two overlapping heat shock elements, located 156 base pairs upstream of the transcribed region, were necessary and sufficient for regulation of heat induction. The SSA3 promoter was also activated in an ssalssa2 double-mutant strain. This increase in the expression of SSA3 was mediated via the same upstream activating sequences that activated transcription in response to heat shock.
Virtually all organisms studied to date respond to environmental stresses, such as heat shock, by the induction of a conserved set of proteins known as the heat shock proteins or HSPs (for a review, see reference 10). A cis-acting, heat-activated transcriptional regulatory sequence, the heat shock element (HSE), first defined in Drosophila melanogaster (24), has been identified in many species including yeast, plants, and mammals (for a review, see reference 6). A trans-acting heat shock transcription activation factor, HSF, which interacts directly with the HSE sequence, has been shown specifically to activate transcription of heat shock genes both in vitro and in vivo (33, 36, 37). HSF from stressed and nonstressed Saccharomyces cerevisiae exhibits comparable binding affinities for HSEs (18, 30). HSF is activated posttranslationally by a mechanism likely to involve phosphorylation (29, 31). The yeast S. cerevisiae contains a family of at least nine genes encoding 70-kilodalton heat shock proteins (HSP70s) (for a review, see reference 11). SSA3 and SSA4 are the only HSP70 genes that are expressed at extremely low levels under optimal conditions and are rapidly induced in response to stress (35). SSAI and SSA2, the other two members of the SSA subfamily, are expressed at substantial levels under nonstressed growth conditions. ssalssa2 double mutants are viable at 30°C and constitutively thermotolerant (able to survive brief exposure to temperatures that are normally lethal) but are temperature sensitive for growth (12). Here we analyze the regulation of SSA3 transcription under stressed and nonstressed conditions in wild-type strains. We also examine the effects of ssal and ssa2 mutations on the regulation of the SSA3 promoter. Analysis of SSA3 and flanking DNA. The DNA sequence of the portion of the genomic clone SSA3H that encodes the amino terminus of the SSA3 protein (Ssa3p) and a contiguous 5'-flanking region was determined (Fig. 1). Amino acids 6 to 16 (predicted from the DNA sequence [Fig. 1B]) comprise a highly conserved amino-terminal domain that is identical to other S. cerevisiae HSP70s (28) as well as to those of distantly related species (16, 17, 25). Both primer extension and Si nuclease analyses indicate that SSA3 transcripts are heterogeneous at the 5' termini; 21 apparent 5' ends were identified by both techniques (Fig. 2). All of the most abundant 5' ends, comprising more than 85% of the *
transcripts, map to the dinucleotide TA, consistent with the correlation observed for the CYCI gene between pyrimidinepurine dinucleotides and initiation sites (Fig. 1B) (20). All of the observed 5' ends map between 44 and 83 base pairs (bp) downstream of the 8-bp sequence 5'-TATATAAA-3', which is comprised of two overlapping sequences previously defined to function as yeast TATA boxes (9, 20, 22). There are eight sequences that match at least six of eight conserved nucleotides of the canonical heat shock regulatory element, CNNGAANNTTCNNG, where N represents any nucleotide (Fig. 1B) (5). Four of the HSE-like sequences are present in overlapping pairs, an arrangement often found upstream of heat-inducible genes in yeast and other organisms (for a review, see reference 6). SSA3 promoter dissection. To facilitate the localization of DNA sequences that function in regulating transcription of the SSA3 gene, a chimeric gene was constructed by fusing SSA3 sequences between -764 and codon 3 (Fig. 1B) to the lacZ gene of Escherichia coli in a centromeric vector. The level of expression of the fusion gene in cells grown in rich medium at a constant 23°C temperature was low, only 4 Miller units. Following heat shock, a dramatic increase in ,-galactosidase activity was observed within 30 min. The 5' termini of the fusion transcripts were indistinguishable from those of the native SSA3 gene (data not shown). A progressive series of deletions from the upstream end of the SSA3IlacZ hybrid gene was constructed to localize cis-acting transcriptional regulatory elements (Fig. 3). Deletion of sequences 5' to -166 resulted in SSA3IlacZ fusion genes retaining heat-inducible activity; removal of the two distal HSE-like sequences had no effect on either the basal or heat-inducible levels of expression. Deletion of an additional 12 bp, removing more than half of the overlapping HSEs centered at -156, essentially abolished the heat inducibility of the promoter. The presence of sequences that negatively modulate SSA3 expression was suggested by the increase in basal activity caused by deletion of sequences between -468 and -200 and between -154 and -124. To further define DNA sequences that regulate transcription of the SSA3 gene, fragments of the SSA3 promoter region were tested for their ability to activate the heterologous CYCI promoter (14) in a heat-inducible manner. A 113-bp restriction fragment from SSA3 that included sequences from -236 through -124 was able to mediate a low basal activity (2.4 Miller units) and a rapid, 20-fold heat
Corresponding author. 3262
NOTES
VOL. 10, 1990
§
A
H
Bn Sa N
II
RI
Sp
SSA3
X
Ss
P2
Bs
ORF
I Sn
3263
Ss
Ss X
Ps
P1
Ps Rv
II~~~ Bs Sp N Sn Ri Hp
H
Bm A 1 Kb
B -1090
GCCTT TACAAAATGA
-1075
TGGAAGAAAG GGATGGCGCA AAATTCGAGA ATATTGTTCA CAACTTCAAG GAACGGCAGA TGATGGTTTC TTATCCGAAA ATTGACGAAG ATGATACCTG
-975
SnaB I GTACAATCTT ACCGAGTTTG TGCAGATGGA TAAAATCCGA AAGATAGTAA GGAAAGATGA AAACCAGTTC TCTTACGTAG ATTCTTCGAT GACCACAGTT
-875
CAAGAAAATG AGCTGCTAAA ATCCAGCTTG CAAAAAGCAG GTTCTAAAAT GGAAGCCAAG AATGAAGATG ATCCTGCACA TTCTTTAAAC TATACAGTAA
-775
TAAACTTCAA ATCTAGAGAA GCCATAAGGC CTGGCCATGA AATGGAGGAT TTTTTAGACA AGTCTTACTA CTTGAACACT GTAATGCTAC AAGGAATTTT
-675
TAAAAATTCA AGTAATTATT TTGGGGAGTT GCAGTTTGCG TTCTTAAATG CCATGTTTTT TGGTAACTAC GGGTCGAGTT TGCAATGGCA TGCTATGATC
-575
GAACTGATAT GTTCAAGCGC TACGGTGCCT AAACATATGC TCGATAAATT AGACGAAATC TTATATTATC AGATAAAGAC ATTGCCTGAA CAATACTCAG
-475
ACATCTTGTT GAATGAACGA GTTTGGAATA TTTGTCTGTA TTCGTCATTT CAAAAAAACT CCCTACACAA CACAGAAAAG ATAATGGAAA ACAAATATCC
-375
AGAATTGCTT GGTAAAGACA ATGAAGACGA CGCTCTTATT TACGGTATCA GTGATGAAGA AAGGGATGAC GAGGATGATG AGCACAACCC TACCATTGTT
-275
GGCGGTCTCT ATTACCAAAG GCCATAACGA TCATCGTGCG GCGCTATCAT CAAACGTATT TGACTTGATG CCTATGGAGG TTATGGGTGC CCTTAATTAG
-175
GGATCGCTGT GGAAAGTTAT AGAATATTAC AGAAGCAGCC ACAAGGGTGA CCAGAAGATG GTTAAGGGAT GTATCATATT GCACAATTGG AAACGAATGG +1
Xba I
Sph I
-236
BstE 11 -75
26
107
VV v V V VV vv V VV Y v vvv AAGGGX X~TGACTG AAATTGGTAG CATAAACATT CTTGTATGTC AATGTTTGTC ACTAAACGGA TAGAATAGGT ACTAAACGCT ACAAAGAAAA XbaI
ATG TCT AGA GCA GTT GGT ATT GAT TTG GGA ACA ACT TAC TCG TGT GTT GCT CAT TTT TCC AAT GAT AGG GTA GAG ATA ATT Met Ser Arg Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Ala His Phe Ser Asn Asp Arg Val Glu Ile Ile
Sin I GCA AAT GAT CAA GGT AAT AGG ACC Ala Asn Asp Gln Gly Asn Arg Thr
130
FIG. 1. Restriction map and partial DNA sequence of SSA3 and flanking genomic DNA. (A) Restriction map of the cloned 5.1-kilobase HindIII SSA3H restriction fragment. The region encoding the SSA3 protein and the portion of a large open reading frame (ORF) identified by DNA sequence analysis are represented by arrows. Abbreviations: A, AvaI; Bm, BamHI; Bn, BanII; Bs, BstEII; H, HindIII; Hp, HpaI; N, NdeI; P1, PvuI; P2, PvuII; Ps, PstI; RI, EcoRI; Rv, EcoRV; Sa, Sacl; Sn, SnaBI; Sp, SphI; Ss, SspI; X, XbaI. The following enzymes did not cleave SSA3H: NheI, Sall, KpnI, SacII, SmaI, MluI, BglII, ApaI, XhoI, and FspI. The DNA sequence of the solid black region of the restriction map was determined and is shown in panel B. (B) DNA sequence of the amino-terminal region of the SSA3 structural gene and 5'-flanking region as determined by the dideoxy-chain termination method (4). The major putative initiation site closest to the SSA3 protein-coding region has been designated + 1. The upstream open reading frame extends from the beginning of the sequences shown here through position -252 (on the same strand as the SSA3 coding region) and has the potential of encoding a protein of at least 308 amino acids (see above; unpublished observations). Neither the upstream open reading frame DNA nor its predicted amino acid sequence is closely related to sequences currently in the GenBank, EMBL, or NBRF databases. Putative SSA3 mRNA initiation sites are indicated by vertical arrow heads (scale: v, V, V, V, from least to most abundant 5' termini). Matches to the canonical HSE consensus sequence CNNGAANNTTCNNG are underlined. The putative TATA element(s) is enclosed in a box. Sequences resembling BAS21PH02 (position -670 through -654) (3), GRFIIRAPI (dashed underline, -198 through -186) (8, 26), and GCN4 (-61 through -56) (2, 15) binding sites were also identified. Three iterations of imperfect 6- and 7-bp repeats in altemating orientations are indicated by arrows above the sequence. Restriction enzyme recognition sites used for cloning and for construction of primers and probes used in transcript mapping are labeled. Position -236, marked above, is the site of the 5' deletion endpoint used for isolating the 113-bp fragment. The predicted amino acid sequence of the amino terminus of Ssa3p is shown below the nucleotide sequence. shock induction, indicating that this fragment contains sequences sufficient to confer the heat-inducible regulation characteristic of SSA3 (Fig. 4A). The only matches on this fragment to the heat shock consensus sequence are those centered at -156. A 32-bp synthetic oligonucleotide containing the -156 HSEs and only four to five flanking nucleotides (Fig. 5) was also able to activate the CYCJIIacZ hybrid gene in response to heat shock, although the induction was smaller than that
observed from the 113-bp fragment (Fig. 4A). This short fragment is a heat shock upstream activating sequence (UASHS), as it is sufficient to mediate heat-inducible transcription in an orientation-independent manner. Both elevated basal expression and a smaller increase in expression following heat shock contributed to the low induction ratio observed for this construct. HSE consensus sequences are essential to UASHS activity. Transcriptional induction driven by the 113-mer was greater
3264
.a
NOTES
C)
C)
E
Stringency: _
S'
1
MOL. CELL. BIOL.
Si
Primer Extension M 3
L 2
H
L 4
H 6
5
.
Nuclease
M 8
L 7
L 9
o
10
-
-
-
-
r
-
.
.0
i-
0
0
a
S
.-
139-146
*122-135
U. -
113-116
Ior FIG. 2. Si and primer extension analyses of native SSA3 RNA isolated from heat-shocked cells. S and S' are DNA size standards pBR322 and '1X174, respectively, cleaved with HaeIII. RNA was isolated from DS10 (MATa his3-11,-5 leu2-3,-112 lysi Iys2 SSAI SSA2 Atrpl ura3-52) grown at 23°C to an optical density at 600 nm of 0.4 and subsequently heat shocked at 39°C for 20 min. Lanes 1 and 10, Controls containing the same quantities of primer and probe, respectively, used in the mapping reaction; lanes 2 and 9, reaction controls containing tRNA but no yeast mRNA; lanes 3 to 5 and 6 to 8, SSA3 primer extension and Si nuclease mapping reactions, respectively, with 13 jig of total RNA per lane under low (L)-, medium (M)-, and high (H)-stringency conditions. Hybridization of RNA to DNA restriction fragments was performed in 80% formamide-0.4 M NaCl at three different temperatures following brief (45 s) denaturation at 80°C; high- and medium-stringency hybridizations were performed at 46 and 41°C, respectively, for 7 h; low-stringency hybridization reactions were slowly cooled from 48 to 37°C over 3.5 h and held at 37°C for 2 h. A SinI-to-XbaI (position + 128 to +31) fragment was used as a DNA primer for primer extension analysis. A SinI-to-SnaBI (+128 to -900) fragment was used as a probe for Si analysis. The fragments were 5' end labeled at the SinI termini; therefore, bona fide 5' ends mapped by each method should yield comigrating products. Unextended primer and undigested probe bands are indicated by arrowheads at the lower left and upper right, respectively. The length in nucleotides of the three (arbitrary) size classes of products are indicated at the right. A more precise mapping of the mRNA termini was obtained by higher-resolution electrophoresis, giving the positions noted in Fig. 1B (data not shown). Annealings, hybridizations, extensions, and nuclease digestions were carried out as previously described (4, 7).
[galactosidase activiy -
vw I
3T I*MMM8TXvy L v vv v
-583
-
-38 >
0.03
-AA
-
.
vv
,
rrx L:
IKI, 11
%.-
LZJ V
-166 -154 Iv -124
i
L:
=
-93
=
SSA3
L~
-----.-,--,--,,,,--,--,-,
v
vv
-111
Vector
LTVA
4.2 3.7 3.5 6.1 17 16 11 1.1 14 5.8 6.9
S-' -4688}vSSM,SSS/ -236 -200 -175
=
+1 ATG ,...H
-156
-764
_-"'4,. v
lacZ
62 60 61 92 84 80 54 2.1 13 6.3 7.0 < 0.03
15 16 17 15 4.9 5.0 4.9 1.9 0.9 1.1 1.0
