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Structure
and Regulation
Saccharomyces
Gene of
cerevisiae*
(Received for publication,
William
R. BoorsteinS
and Elizabeth
and Cellular
53706
Biology
Program
A. CraigSV
and the ?lDepartment
of Physiological
Chemistry,
University
of Wisconsin,
18912
observations.
The nucleotide
sequence(s)
to the GenBankTM/EMBL
505637.
June 4, 1990)
Structure
and Regulation
of a Yeast Heat-shock
Gene
18913
the SSAl
activating sequences
of the SalI-recognition
MATERIALS
S. cereuisiae
Strains-DSlO
112, lysl, lys2, Atrpl,
ura3-52)
AND
METHODS
(MAT
a, hisd-11,
hk3-15,
h&3,leu2was utilized
in all experiments
pre-
provide
a unique restriction
nmpR
ori
CENll
FIG. 1. Plasmids
utilized
to identify
sequences
controlling
SSA4
expression.
A, pWB213,
the plasmid
containing
the SSAI/
1acZ hybrid
gene (used to construct
the 5 deletion
series plasmids).
B, the centromeric
UAS cloning
vector
pWB220,
a derivative
of
pWB213.
pWB220
and pWB213
differ
only in the region between
1acZ and ARSl
sequences.
Genes and other functional
plasmid
sequences are labeled in bold type; the location, size, and orientation
of
genes are indicated
by curved arrows. The two large unshaded
arcs in
each plasmid
represent
sequences
of pBR322
origin. Lightly
stippled
arcs represent
sequences
from the E. coli uric operon. SSA4 sequences
are represented
by solid black arcs. Restriction
enzyme
recognition
sites used in plasmid
constructions
are labeled. The entire nucleotide
sequence
of these vectors
is known
with the exception
of approximately
400 nucleotides
at each end of the CENll
sequence. RI,
EcoRI; Sa, SacI; Sp, SphI; X, XhoI.
were
of deletion
derivatives.
The EcoRI
site upstream
of the SSA4 sequences
was
eliminated
with mung bean exonuclease,
creating
pWB213
(Fig. 1A).
Deletion
derivatives
of the SSA4/lacZ
fusion plasmid,
pWB213,
were created
by cleavage
with SphI followed
by Bal31 exonuclease
treatment
for various
time intervals,
creation
of blunt ends, ligation
of XhoI
linkers
(5-pCCTCGAGG-3),
XhoI cleavage,
and unimolecular
reclosure,
joining
the XhoI linker
at the SSA4 deletion
end
point to a common
XhoI site present
upstream
of the deleted region.
pWB213A-594
was created by inserting
an XhoI linker into the SphI
site and recircularizing
as described
above, without
exonuclease
treatment.
The precise
position
of pWB213A-594
A-234,
and A-174
deletion
end points were determined
by DNA sequence
analysis.
The
remaining
deletion
end points were mapped to within
f3 nucleotides
by polyacrylamide
gel electrophoresis
of small restriction
fragments.
A centromeric
UAS cloning
vector,
pWB220
(Fig. lB), was constructed
by replacing
the SSA4 sequences
of pWB213
with CYCl
promoter
sequences.
Specifically,
the small XhoI to Sac1 region of
pWB213
(containing
the entire SSA4 region and the amino-terminal
portion
of the &Z-coding
region)
was replaced
with the corresponding restriction
fragment
of pLG669-Z
(Guarente
and Ptashne,
1981).
The CYCI sequences
in pWB220
extend from 178 bp upstream
of the
primary initiation site, through the amino-terminal codon. pWB220
also contains
different
linker
and 1acZ fusion
sites from those in
pWB213.
pWB227
and pWB227-R
were constructed
by inserting
a
synthetic
double-stranded
oligonucleotide
matching
the 44 bp indicated in Fig. 8A, flanked
by sequences
necessary
to generate
an XhoI
site and an XhoI
complementary
overhang,
into the XhoI site of
pWB220.
The pWB227
and pWB227-R
inserts
were verified
to be
identical with the native SSA4 sequence. All plasmid constructions
utilized
standard
methods
(Ausubel
et al., 1987).
DNA Sequence
Determination
and Analysis-Appropriate
regions
of the SSA4H
plasmid
were subcloned
into M13mp
vectors
and
Sequence
was obtained
across all restriction
and on both DNA strands.
DNA and protein
with University
of Wisconsin
Genetics
Computing
Group programs
(Devereux
et al., 1984).
mRNA
Analysis-RNA
was isolated
by vigorous
vortexing
of S.
cerevisiae
cells suspended
in 0.1 M Tris (pH 7.5), 0.1 M LiCl, 0.01 M
dithiothreitol,
in the presence
of glass beads, 0.5% sodium
dodecyl
sulfate,
phenol,
and chloroform.
Lysis
was followed
by multiple
phenol/chloroform
extractions
and ethanol
precipitations
as described (Ellwood and Craig, 1984). Yields were determined by AzGO ~
and normalized
A
by comparison
of ethidium
bromide-stained
rRNA
following
gel electrophoresis.
A Sau3A to RsaI (position
+147 to +87) fragment
was used as a
DNA
primer
for extension
analysis
of transcripts
from the native
gene. A Sau3A to AuaI (+147 to -402)
fragment
was used as a probe
for Sl analysis
(Fig. 2B). The DNA fragments
were 5 end-labeled
at
the Sau3A termini.
The standard
17-bp lncZ sequencing
primer
5GTAAAACGACGGCCAGT-3
was utilized
for priming
extension
of
SSAl/lacZ
fusion
transcripts.
DNA
restriction
fragments
were hybridized
to RNA in 80% formamide,
0.4 M NaCl following
brief (45s) denaturation
at 80 C; hybridization
reactions
were slowly cooled
from 48 to 37 C over 7 h. Oligonucleotide
primers
were prepared
and
hybridized
under aqueous conditions
in the presence
of poly(A)
RNA
1977).
Primer
extension
reactions
were
performed
in the
Structure
18914
and Regulation
of a Yeast Heat-shock
Gene
5 days of growth
at 23 C. Cells were collected
in siliconized
1.5-ml
microcentrifuge
tubes; cell pellets
were rapidly
frozen
in a -70 C
bath and were stored
at this temperature.
Optimization
of growth
conditions
by using rich medium
resulted
in lower basal and higher
heat-shocked
levels of expression
than observed
in defined,
plasmidselective,
medium
(data not shown).
Plasmid
stability
was routinely
assessed as follows:
an aliquot of cells was plated on rich medium
and
the fraction
of resultant
colonies
that contained
the TRPI marker
carried
on the plasmid
was determined
following
replica
plating
to
the medium lacking
tryptophan.
More than 80% of the cells routinely
retained
plasmid
at the time of sample collection.
fl-Galactosidase
activity
was determined
by microfluorometric
assay utilizing
the substrate
4-methylumbelliferyl-fl-n-galactoside
(to
be described
in detail elsewhere).
Briefly,
cells were suspended
and
permeabilized
in 1 ml of Z buffer
(Miller,
1972) with 0.05% sodium
dodecyl
sulfate,
50 mM P-mercaptoethanol,
1 mM phenylmethylsulfonyl fluoride,
vortexed
vigorously,
diluted
to yield fluorogenic
product in the linear detection
range of the fluorimeter,
and incubated
with 1 mM 4-methylumbelliferyl-/3-n-galactoside
for 15-120
min at
room temperature.
The reactions
were performed
in a final volume
of 200 ~1 in 96-well
microtiter
plates. Fluorescence
of samples
with
extremely
low fi-galactosidase
levels was enhanced
by the addition
of
50 ~1 of 1 M Na2C03
after 2 h of incubation.
Fluorescence
was
determined
using a Titertek
Fluoroskan
I fluorimeter.
fi-Galactosidase activities
are presented
as (f2-l/(tz-1
x AGNnm x u x s)) x C x N,
where fi-, is the increase
in fluorescence
between
times 1 and 2, tzmL
is the time in minutes,
between
the two fluorescence
readings,
Asw nm
is a measure
of cell density
at the time of cell collection,
u is the
volume of the culture
assayed, in milliliters,
and s is correction
factor
for plasmid
stability
that is equal to the fraction
of cells retaining
the
plasmid at the time of cell collection.
The arbitrary
fluorescence
units
were converted
to Miller
units
(Miller,
1972) by the empirically
derived
constant,
C = 5.7 x 10. N is a normalization
factor
determined by assay of a known
standard
quantity
of fl-galactosidase
to
control
for differences
in temperature
and pH which affect @-galactosidase
activity
and fluorescence
of methylumbelliferone,
respectively.
Selected
samples
were permeabilized
as described
above and
were assayed in parallel
with the substrate
o-nitrophenyl-fl-n-galactopyranoside,
as described
(Slater and Craig, 1987); results of the two
assays were similar,
with the exception
of the low activity
samples
that were below the accurate
detection
limits of the 0-nitrophenyl-@D-galactopyranoside
assay. Data presented
are averages
from two to
six determinations
of at least two independent
transformants.
Individual determinations
were almost always within
20% of the averages
shown here. Occasionally,
high or low values were obtained.
In these
cases, samples
from all treatments
of a single culture
were similarly
affected
(i.e. basal, heat-shock,
and stationary
phase);
thus, patterns
of induction
were highly reproducible.
RESULTS
Characterization
of the SSA4 Gene and Encoded Transcripts-The
SSA4 gene was isolated on a 9-kb genomic
clone.3 We constructed
a restriction
map of the pSSA4H
genomic insert and localized the SSA4 coding region by DNA
hybridization
analysis utilizing fragments of the related SSAI
gene as probes (Fig. 2A and data not shown). The nucleotide
sequence of the coding region, as well as 1.2 kb of flanking
DNA upstream of the gene, was determined
(Fig. 28). A single
large open reading frame with similarity to SSAl was identified. This SSA4 ORF has the potential of encoding a protein
of 642 amino acids with a predicted molecular mass of 69,650
Da and an isoelectric point of 4.86. These values are consistent
with previous estimates based on polyacrylamide
gel electrophoresis analysis of the SSA4 protein, Ssa4p (Werner-Washburne et al., 1987).
While Ssa4p is closely related to hsp70s and noninducible
cognates of distantly related species (68-75% identity to Drosophila and human homologues),
it is most closely related to
the yeast hsp70 Ssalp and Ssa2p, which are 97% identical.
Ssalp and Ssalp are each 642 amino acids in length, although
optimal alignment involves the introduction
of at least three
J. C. A. Bardwell
and E. A. Craig,
unpublished
data.
FIG. 2. Restriction
map and nucleotide
sequence
of SSA4
and flanking
genomic
DNA.
A, restriction
map of the 9.0-kb SSA4
Hind111 fragment,
SSA4H.
The SSA4 protein-coding
region and the
upstream
open reading
frame are represented
by arrows.
The filled
region of the map indicates
the part of the clone whose DNA sequence
Structure
18915
FIG. 3. Comparison
of predicted
complete
SSA4 and SSAI
hsp70
protein
sequences.
Solid bars, colons, or dots between
corresponding
amino acids of the two proteins
indicate
identity,
or high
or low levels of similarity,
respectively.
This alignment
was obtained
by optimizing
matches
while introducing
the smallest
number
of gaps
(represented
by dots in the sequence
lines) according
to the algorithm
of Needleman
and Wunsch
(1970).
Matches
between
nonidentical
amino
acids were weighted
as described
by Gribscov
and Burgess
(1986). Alternative
alignments
can be obtained
with a larger number
of gaps, particularly
in the C-terminal
region,
but these do not
significantly
increase
the similarity.
Ssalp is from Slater and Craig
(1989).
gaps,
indicating
that insertion/deletion
mutations
occurred
during the divergence of these genes (Fig. 3). Ssalp and Ssa4p
are 82% identical; they are 90% similar, accounting
for conservative amino acid substitutions
as described by Gribscov
and Burgess (1986). There is an abrupt decrease in similarity
near the carboxyl termini of the proteins. The amino-terminal
525 amino acids of Ssa4p are 89% identical with Ssalp, while
the carboxyl-terminal
117 amino acids share only 50% idenis shown in B. B, Rⅈ
Bs. BsrEII;
H. HindIII:
HD, HnaI: K, KnnI:
M, MluI; N, NdeI; p, I%tI; Pv, Pⅈ
RI, EcoRI;~$
salI; Sk, SacIt
SC, ScaI; S,o, SphI; St, StuI; X, XhoI. The following
enzymes
do not
cleave the SSA4H
fragment:
ApaI, BarnHI,
BclI, NruI,
PuuI, SacII,
SmaI. B, nucleotide
sequence
of SSA4 and flanking
DNA.
The 5
ends of the heat-shock
SSA4 mRNA
map to the double-overlined
nucleotides;
the center
of this region has been designated
+l. The
arrowhead
at position
32 marks the putative
initiation
site for SSA4
transcription
under control
conditions
(see the legend to Fig. 5). Long
bars above the sequence
indicate
occurrences
of matches
to the
canonical
heat-shock
consensus
sequence,
CNNGAANNTTCNNG,
where
N is any nucleotide,
in at least six of the eight conserved
positions.
The only match to the extended
modular
HSE definition
is in the region
of three overlapping
canonical
HSEs
centered
at
-188. The putative
TATA
element and the initiation
and termination
codons
are enclosed
in boxes. A match to the ATF consensus
binding
site is underscored
with a broken line (positions
-103 through
-97).
Positions
of restriction
enzyme
recognition
sites used in cloning
and
in construction
of primers
and probes for mRNA
mapping
are indicated. The amino acid translation
of the incomplete
ORF upstream
of SSA4 is included.
The translation
of the SSA4 ORF is shown
in
Fig. 3.
TAA
00
05
distance
.ccucGGticMcroo
IIIII
IIIIIIII
CCUCCCTTGAAGAAG
10
SSA4
from transcription
1.5
slaR
20
(kb)
SSAI
~=T~~TAA;rT1\CA~~T~~~~T~~~
IIIIIIII
III
-=WGCCMTTGGTGCGGC~TTGAT:
ssA.1
2000
III
2012
FIG. 4. Comparison
of SSA4 to SSAI DNA
sequences.
A,
graphic
matrix
comparison
of the entire
SSA4 and SSAl proteincoding
regions
plus flanking
DNA
(Maize1
and Lenk,
1981).
Wide
bars on the ares indicate
the positions
of the coding regions; initiation
and termination
codons
are labeled. Each dot represents
a match of
17 of 24 adjacent
nucleotides
between
the two sequences.
Diagonal
lines indicate
contiguous
colinear
similarity
between
the two genes.
B, alignment
of SSA4 and SSAl nucleotide
sequences
flanking
the
initiation
(top) and termination
(bottom)
codons.
Vertical
lines indicate identity
between
nucleotides
at corresponding
positions
in the
two genes. Coding
portions
of the genes are in bold type. Numbers
indicate
positions,
as in Fig. 2B, for SSA4 and, as in Slater and Craig
(1987), for SSAI.
Structure
18916
related to entries
in GenBank,
EMBL,
and Regulation
and NBRF
data bases.
B.
A.
--Extension Sl Nuclease
Ss123456
-------
HS:-
A-234
+
-113456J-8910
A-594
+
Standards
E
I
-*
-c
l-
r
e
0
104-5 *
l w
145-9
ma
e.
FIG. 5. Analysis
of SSA4 RNA.
A, Sl and primer extension
analvses
of the native SSA4 RNA isolated
from heat-shocked
DSlO.
S and S are DNA size standards
(pBR322
cleaved
with MspI
and
HaeIII, respectively).
Lanes 1 and 6, controls
containing
the same
quantities
of primer
and probe,
respectively,
used in the mapping
reactions;
Lanes 2 and 5, reaction controls
containing
tRNA,
but no
yeast mRNA;
Lanes 3 and 4, SSA4 primer extension
and Sl nuclease
mapping
reactions
with 13 fig of total RNA per lane. S1 probes
and
extension
primers
were labeled at a common
5 terminus,
therefore,
bona fide 5 ends mapped by each method
should yield comigrating
products.
Unextended
primer
and undigested
probe bands are indicated by arrowheads to the right of A. Higher
resolution
electrophoresis of aliquots
of the samples
shown here suggest the presence
of a
single predominant
5 terminus;
the less abundant
products
from the
two reactions
do not comigrate
(data not shown).
B, primer extensions
of SSA4/lacZ fusion
RNAs
from pWB213n-234
and J-594
transformants
in DSlO under control
(-) and heat-shock
(+) conditions
(Lanes 3-6). Lanes 1 and 2 contain controls: primer only (no reaction)
and primer plus poly(A)
RNA (no yeast RNA), respectively.
A dideoxy
DNA-sequencing
ladder is included
as a size standard
in Lanes 7-10.
The 5 termini
of native and fusion transcripts
were indistinguishable.
The range of sizes of Sl nuclease
and primer
extension
products
are
indicated
in nucleotides.
Comigrating
single faint bands were detected
on long exposures
of extension
products
from
nonheat-shocked
pWB2131-234
and pWB213J-594
transformants
(data not shown).
These
may represent
a very low level of transcriptional
initiation
from position
-+32
(as marked
in Fig. 2H).
Gene
of a Yeast Heat-shock
Structure
and Regulation
of a Yeast Heat-shock
18917
Gene
f3-galactosidase
-801
-188
tl
ATG
-23X
0.2
--T39"C
14
activity
f3943'
70
0.2
12
60
0.1
10
100
0.1
10
100
0.2
11
55
0.7
17
24
0.6
15
25
0.2
0.4
2.0
0.2
0.6
3.0
0.3
0.3
1.0
IacZ
FIG. 6. ,%Galactosidase
activities
from
the SSAsCllacZ
fusion
gene and the promoter
deletion
derivatives.
The solid black lines represent
SSA4 sequences.
The positions
of the initiation
codon, the transcription
initiation
site, and the deletion
end points
are indicated,
pWB21311-234
contains
1 bp beyond
that indicated
identical
with that in the native
gene because
of the addition
of linker
sequences.
Diamonds
represent
partial
matches
to the HSE consensus,
as overlined
in Fig. 2B. The putative
TATA
element
is denoted by T. The plasmid
on the top line, pWB213,
is the parental
plasmid
from which
the deletions
were derived
as described
under
Materials
and Methods.
Deletion
derivative
plasmids,
from
top to bottom,
are named
pWB213A-594
to
pWB213A-61.
pWB213
contains
an 8-bp linker
inserted
at position
-594 of the SSA4 DNA as described
under
Materials
and Methods.
pWB213
also contains
665 bp of vector
sequence
immediately
upstream
of SSA4 that
are not present
in the deletion
derivatives.
All other plasmids
represented
here differ from each other only in the
extent of 5 noncoding
SSA4 sequence
included
(and consequently
the relative
spacing of DNA sequences
flanking
the deletion
end points and the actual junction
sequences).
P-Galactosidase
activities
from transformants
growing
exponentially
at 23 C and following
a l-h 39 C heat-shock
treatment,
are presented
in Miller
units.
Vector
SSA4
DISCUSSION
We have localized
bp region containing
the heat-inducible
UAS of SSA4 to a 44extensive similarity
to sequences that
18918
Structure
and Regulation
of a Yeast Heat-shock
Gene
A.
B
15--
A-594
B.
P-galactosidase
Iz!z!zs
none
0.3
0.2
0.7
44-mer
1.1
13
12
300
360
7. &Galactosidase
activity
from
transformants
of
SSAl/lacZ
fusion
plasmids
under
heat-shock
and control
conditions.
Each pair of curues represents
data from a single exponentially growing
culture
at 23 C; at time 0, an experimental
aliquot was
transferred
to 39 C (solid black symbols)
while a control
was maintained at 23 C (open symbols).
Samples
were collected
at the indicated times and fl-galactosidase
activities
were determined.
&rues
shown are representative
for each plasmid.
/3-Galactosidase
activity
is presented
in Miller
units. A, pWB213A-594.
B, pWB213A-234.
C, pWB227
(circles),
the HSEssar
44-bp oligonucleotide
in pWB220,
as well as a heat-shock
curve only from the UAS vector,
pWB220,
with no insert (triangles).
The plasmids
are illustrated
in Figs. 6 and
8B.
FIG.
regulate
stress-induced
transcription
of heat-shock
genes
from distantly
related eukaryotic
species. These HSEaa,+,
sequences alone mediate a very similar pattern of transcription to those regulated by the isolated HSEs of SSA3 and
SSAl (Boorstein
and Craig, 1990b; Slater and Craig, 1987).
Therefore,
the role of HSEs in contributing
to the high
expression of the SSAl and SSA3 promoters under nonstress
conditions
(in which the HSE-containing
SSA4 promoter
is
not active) appears to result from differences in regions of the
promoters
distinct from the HSEs (Slater and Craig, 1987;
Boorstein and Craig, 1990a). For example, expression of SSA3
in stationary
phase is dependent upon a distinct promoter
element, UASPDS, but is further enhanced by HSEssa3 (Boorstein and Craig, 1990a).
The difference in regulation
between the SSA subfamily
genes, resulting
from different
promoter
composition,
may
provide an explanation
for the maintenance
of similar genes
over a long period of evolutionary
time; the gene products
may be functionally
equivalent, multiple genes being required
for an appropriate
pattern of expression and/or rapidity
of
TABLE
Activity
of the SSA4
versus
promoter
and UASHs
an SSAlSSA2
strain
&Galactosidase
Hybrid
ssolssa2
gene
SSAl/lacZ
A-594
UAS/CYCl/lacZ
No insert
HSEd
in an ssalssa:!
activity
SSAlSSAP
23 C
t39 C
23 C
122
122
0.2
0.3
185
0.2
162
0.2
1.1
o /3-Galactosidase
activity
was determined
in DS16 and DSlO under
indicated
conditions
as described
in Fig. 6.
* pWB213A-594.
The promoter
region of this plasmid
is depicted
in Fig. 6.
pWB220.
The promoter
region of this plasmid
is depicted
in Fig.
8B.
d pWB227.
The pWB220
derivative
containing
the 44-bp HSE,sA,
oligonucleotide
in the native orientation,
as depicted
in Fig. 8B.
li0
180 240
Time (minutes)
*3ocr
UAS,
60
3%
Insert
44-mer/Reverse
activity
Structure
and Regulation
of a Yeast Heat-shock
Gene
18919
TABLE II
1.
elements
from
Saccharomyces
cerevisiae
genes
4, Slater and Craig, 1987, and
and Finkelstein,
1984, and McDaniel
et al., 1989; line 6, Piper et al., 1988;
et al., 1987; line 9, Rose et al., 1989; line 10, Nicolet
and Craig, 1989; line
2. SSAJ
3. SSAZ-HSEP
(22-mer)
4. SSAl-HSE3
+, B
B
B, M
M
M
5. HSP82
6. PGK
7. iJBI4
TAATTAGGGATCGCTGTGGAAAGTTATAGAATATTACAGAAGCAG
-----TTTT&ZAGA&ZGECATCGGC
TGTAAACTZCAGAACATTCTAGAAAGA
AGTTTCGCAGAACTTTTT=TT;CTTTTTTTCTAGAACGCCGTGGAAGAA
---=
ATCGAAGGTTCTGGAATGGCGGGAAAGGGTTTAGTACCA
TGTGTAEZGTTCTAGAATAATCCTGGATAA
-~~~~
8. Consensus
Frequent:
Core:
Examples
Rare:
9. KAR2
ND
10. STIl
11. SSAI-HSE2
12. SSAI-HSE2+
(15-mei)
(9-mer)
ND
+
TA
cg
TA
..E..TTC..GAA,
==
RY
wy
RY
TA
cg
..TTC..GAA..TTC
==ac
RY
wy
ATAGAACCTTCTGGAAATTTCACC
ATC~GTGAAGATTCGT~GT~TAGAACA~CAGAAAAA
-c-cc
ttaggctcgaCCAGj&CGCGTTCCATCtcgagcaga
aggctcgAG&CGEtcgagcag
+ or -, sequence
is or is not sufficient
to function
as a heat-inducible
UAS, respectively;
M, sequence
has been
shown to function
in heat-inducible
regulation
by mutational
analysis;
B, sequence
is known
to act as a proteinbinding
site, and in particular
as a site of HSF binding
in the case of lines 3 and 4; ND, function
of HSE-like
region of these heat-inducible
promoters
has not been determined.
* HSE region sequences
are shown,
including
all GAA modules,
in alternating
orientation
and separated
by two
nucleotides
(allowing
for a gap of one module,
provided
GAA elements
flanking
the gap are positioned
as if the
gap contained
a GAA sequence)
according
to the HSE definition
of Amin et al. (1988). All sequences
are from
within
400 bp upstream
of the initiation
codons.
Plus strands
are shown. Exact matches
and single mismatches
to
the consensus
core module sequence
(GAA and TTC)
are doubleand single-underlined,
respectively.
Lines l-3,
11, 12: large uppercase
characters
represent
the full extent
of native sequences
tested for UASns
activity;
line 2:
smaller
uppercase
letters
represent
native
SSA3 sequence
beyond
the region shown to be sufficient
for UASns
function;
lines 11 and 12: lowercase
characters
represent
vector sequences
flanking
the SSAI-HSE2
sequences
in
the UAS test constructs.
SSAI-HSE2
22-mer,
15mer,
and 9-mer are from pZJHSE2-26,
pZJHSE2-19,
pZJHSE2-12,
respectively.
Two
example
consensus
sequences,
each consisting
of three adjacent
exact GAA modules,
illustrate
the
sequence
bias at positions
between
GAA blocks.
Nucleotides
that occur frequently
are indicated
on the upper line,
whereas
nucleotides
that occur rarely,
if at all, are shown
below the core modules.
Consensus
sequences
were
derived
from the plus strand
of functionally
defined
HSEs
(lines l-7) only. Consensus
nucleotides
upstream
of
exact GAA blocks that are also downstream
of exact TTC blocks (TTC--GAA)
were derived only from comparably
positioned
nucleotides
in the native
sequences.
Consensus
nucleotides
upstream
of exact GAA blocks,
but not
between
two adjacent
blocks
(NNN--GAA)
were derived
from nucleotides
found upstream
of all exact GAA
modules.
Sequence
bias at positions
upstream
of TTC
sequences
was determined
as described
for positions
upstream
of GAA sequences.
No strong bias was observed
in positions
downstream
of exact GAA or TTC modules
that are not also upstream
of exact core modules.
For line 8, R represents
G or A; Y represents
C or T; W
represents
A or T. Uppercase
letters
in the consensus
sequence
indicate
a greater
degree of sequence
bias than
lowercase
letters.
The requirements
for a functional
HSE are discussed
in the text. As indicated
by the sequences
in lines l-7, arrangements
of GAA and GAA-like
core modules
other than those shown in the consensuses
allow
HSE function.
Heat-shock
Sources:line 1, this work; line 2,
Wiederrecht
et al., 1987; line 5, Farrelly
line 7, Tanaka
et al., 1989, and Ozkaynak
18920
Structure
and Regulation
Gene
via sequences distinct from the HSEs (Stone and Craig, 1990).
Therefore,
it is likely that there are other components, possibly additional
HSPs, in the control loop modulating
the
HSF activity state. In E. coli, mutations in any of three HSP
genes, grpE, dnaJ, or the HSP70 homologue, dnaK, result in
cr3-dependent
increased transcription
of heat-shock
genes4
(Tilly et al., 1983). Since DnaK can interact directly with
DnaJ and GrpE (Sell, 1987; Johnson et al., 1989), analogues
of the latter two proteins may interact with hsp70 in yeast to
control the activation
state of HSF. Perhans the mechanism
by which the primary intracellular
stress stimulus
is transduced, resulting in induction
of the stress response, is conserved between prokaryotes
and eukaryotes,
although
the
mechanism
of activating
RNA polymerase
on heat-shock
promoters
has diverged. Elucidation
of the mechanisms
by
which HSP70 expression controls transcription
of heat-shock
genes will require further definition
of the function of the
HSP70 gene products and the means by which the stress
pathway is activated.
Acknowledgments-We
wish to thank
Jeffrey
Shilling
and the
University
of Wisconsin
Biotechnology
Center
for sequencing
portions
of the SSA4 gene and Diane
Gambill,
Lynn
Manseau,
and
Carolyn
Norris
for comments
on the manuscript.
We also thank
Helen Tu for pHT102
and Dave Stone for the yeast strains
used in
this study.
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