A gene encoding a DnaK/hsp70 homolog in Escherichia coli.

Abstract

Eukaryotic organisms have been shown to have multiple forms of hsp70-class stress-related proteins, but only a single family member, DnaK, has been found in prokaryotes. We report here the identification of a heat shock cognate gene, designated hsc, in Escherichia coli. The amino acid sequence deduced from hsc predicts a 65,647-Da polypeptide having 41% sequence identity with DnaK from E. coli, and overexpression produces a protein (Hsc66) with properties similar to DnaK. In contrast to dnaK, however, the hsc gene lacks a consensus heat shock promoter sequence, and expression is not induced by elevated temperature. The hsc gene is located near 54 min on the physical map, immediately upstream of the fdx gene, which encodes a [2Fe-2S] ferredoxin; evidence is presented that the hsc and fdx genes make up a bicistronic operon in which expression of the ferredoxin is coupled to that of Hsc66. The function of Hsc66 is not known, but the coregulation of its expression with that of ferredoxin suggests the possibility of a specific role in association with the ferredoxin protein.

Full text

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Proc. Natl. Acad. Sci. USA
Vol. 91, pp. 2066-2070, March 1994
Biochemisty
A gene encoding a DnaK/hsp79 homolog in Escherichia coli
(chaperone/heat shock cognate protein/ferredoxin operon)
BRENT L. SEATON AND LARRY E. VICKERY*
Department of Physiology and Biophysics, University of California, Irvine, CA 92717
Communicated by Helmut Beinert, November 29, 1993 (receivedfor review May 6, 1993)
ABSTRACT
have multiple forms ofhsp7O-da
only a sngle family member, DnaK, has been found in pro-
karyotes. We report here the Identification of a heat shock
cognate gene,
acid sequence deduced from hsc predicts a 65,647-Da polypep-
tide having 41% sequence identity withDnaKfromE. coil, and
overexpression produces a protein (Hsc66) with properties
smilar to DnaK. In contrast to dnaK, however, the hsc gene
lacks a consensus heat shock promoter sequence, and expres-
sion is not induced by elevated temperature. The hsc gene is
located near 54 min on the physical map, immiaely up-
stream of thefdx gene, which encodes a [2Fe-2S] ferredoxin;
evidence is presented that the hsc and fdx genes make up a
blcistronic operon in which expression of the ferredoxin is
coupled to that ofHsc66. The function ofHsc66 is not known,
but the coregulatio
of its expression with that of ferredoxin
suggests the possibility ofa specific role in association with the
ferredoxin protein.
Eukaryotic organisms have been shown to
stress-related proteins, but
hsc, in Escherichia cofi. The amino
The 70-kDa heat shock proteins (hsp70) and their cognates
(hsc70) make up a ubiquitous, multigene family of highly
conserved proteins, which are involved in diverse protein-
protein interactions (reviewed in ref. 1). They are important
under normal conditions as well as during stress and have
been implicated in a variety ofprocesses including stabiliza-
tion of protein-folding intermediates (2), protein assembly
and disassembly (3), protein secretion (4, 5), and protein
degradation (6). Eukaryotic organisms have been found to
contain multiple hsp70 family members; for example, nine
distinct proteins are produced in Saccharomyces cerevisiae
(3, 7), six have been identified in Drosophila (8), and at least
eight have been described in mammals (9). In contrast, only
a single hsp70-class protein, DnaK, has been reported in
prokaryotes. The most extensively characterized of these
hsp70 proteins is the DnaK protein from Escherichia coli.
DnaK plays a role in the heat shock response (10, 11), but it
is expressed at levels of -1% ofthe cell protein and performs
important cellular functions under nonstress conditions (12-
14). Hybridization analyses in E. coli have revealed only one
gene, dnaK, located near 0.3 min on the genetic linkage map
(15, 16). The presence of a single gene encoding a "stress
70-type" protein in prokaryotes would seem to suggest that
all members ofthe multigene eukaryote hsp70 family evolved
from a single DnaK-like ancestral protein (cf. refs. 17 and 18).
We report here the identification ofa second hsp70-related
gene in E. coli. The genet designated hsc, encodes a protein
of --66 kDa (Hsc66), which shows -40% sequence identity
with DnaK and other hsp70-class proteins. The hsc gene is
found near 54 min on the E. coli chromosome and is located
immediately upstream of the fdx gene, which encodes a
[2Fe-2S] ferredoxin (19, 20). These genes appear to make up
a bicistronic operon in which expression of Hsc66 and
ferredoxin is coregulated.
EXPERIMENTAL PROCEDURES
General Methods. Expression ofHsc66was carriedoutinE.
coli strain MZ-1 (21). Sequencing, bacterial transformation,
and oligonucleotide purification were carried out as described
by Sambrook et aL (22), and (3-galactosidase activities were
determined as described by Miller (23). SDS/PAGE was
carried out according to Laemmli (24). Western immunoblot-
ting was carried out by the method ofTowbin et aL (25) using
enhanced chemiluminescence detection (Amersham).
Pasmids. Theplasmid p66-Fdx, usedto overexpress Hsc66,
contained the hsc andfdx genes and flanking regions derived
from clone DT10 originally isolated from anE. colUB genonuc
library (19, 20).t AnEcoRI fragment containing the insertwas
clonedintopBS(+/-) (Stratagene)toyieldtheplasmidpDT10
and into pAblue (27) to yield the plasmid pADT10. Five
hundred and four base pairs of 5' flanking DNA were deleted
from pADT10 by digesting with Nco I and Hindu, and the
overhangs were filled in with Klenow DNA polymerase and
ligated. The resulting plasmid, p66-Fdx, contained the hsc
gene, including 188bpof5' flankingDNA, undercontrolofthe
APL promoter.
Plasmid p66-Lac, used for analyses of Hsc66 expression,
was constructed by amplifying the region ofpDT10 contain-
ing the N-terminal nine amino acids of Hsc66 and 690 bp of
upstream sequence. PCR primers were constructed such that
the 5' end of the PCR product would contain an EcoRI site
and the 3' end would contain an Sma I site to allow fusion of
the hsc coding sequence in-frame with a lacZ coding se-
quence in pMLB1034 (28). The PCR product was digested
with EcoRI and Sma I, and the 730-bp fragment was ligated
to pMLB1034 that had been digested with EcoRI and Sma I.
Plasmid pFdx-Lac, used for promoter analyses, was con-
structed using PCR to amplify a region from pDT10 including
=z900 bp upstream of the fdx coding region and the bases
encoding the first 11 amino acids offerredoxin. PCR primers
were designed such that the 3' end ofthe amplified sequence
would contain a BamHI site. The PCR product was cleaved
with Acc I, at a site located 91 bp upstream ofthefdx coding
sequence, andBamHI, and the resulting 124-bpfragment was
ligated to the 2.4-kbEcoRI-Acc I fragment ofpDT10 contain-
ing the remaining upstream sequences ofDT10. This fragment
was then ligated to the 3.1-kb EcoRI-BamHI fragment of
pMLB1034. The resulting pFdx-Lac contains 2.5 kb of se-
quence upstream of the fdx gene followed by the sequence
encoding the first 11 amino acids offerredoxin fused in-frame
to
3-galactosidase beginning at codon 8. Upstream deletion
derivatives of pFdx-Lac shown in Fig. 5 were prepared by
*To whom reprint requests should be addressed.
tThe sequence reported in this paper has been deposited in the
GenBank data base (accession no. U05338).
tThe hsc andfdxgenes are also present in E. coliK12 and are located
in A clones 7F8 and 5E10 (20) in the miniset library isolated by
Kohara et al. (26).
2066
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payment. This article must therefore be herebymarked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 91 (1994)
2067
digesting pFdx-Lac with the appropriate restriction enzymes,
isolating the large vectorfragmentby agarose electrophoresis,
and ligating the purified deletion construct.
Protein Expression and Purification. MZ-1 cells trans-
formed with plasmid p66-Fdx were grown in Terrific broth
(22) toOD6wo
0.5, induced by heating to 420C for 2 hr, and
subsequently grown overnight at 370C. Cells were harvested
by centrifugation and disrupted by French press. Protein
extracts were fractionated by anion-exchange chromatogra-
phy and molecular sieving chromatography on Sephacryl
S-300. The description of a more complete purification pro-
cedure will be published (L. W. Goodman and L.E.V.).
Primer Extension. Primerextension analysis was carried out
using a synthetic oligonucleotide complementary to nucleo-
tides 22-41 of the hsc coding sequence. The oligonucleotide
was labeled at the 5' end using ('y-32P]ATP and hybridized to
50 ug of total RNA isolated from E. coli strain DH5a previ-
ously transformed with pDT10. RNA was isolated from late-
logarithmic phase cells by breaking with glass beads in the
presence of hot (650C), water-saturated phenol. The mixture
was vortexed for 30 s followed by incubation at 650C for 30 s;
this cycle was repeated twice. The aqueous phase was ex-
tracted with chloroform and precipitated with ethanol. RNA
samples were resuspended in RNase-free water and treated
with RNase-free DNase. Hybridizations and primer exten-
sions were performed using the Promega primer extension kit
following the included protocol. After extension with reverse
transcriptase, samples were digested with RNase A for 30 min
at 370C and extracted with phenol. RNA-DNA hybrids were
precipitated with ethanol, denatured, and subjected to elec-
trophoresis on a 6% polyacrylamide sequencing gel. Unla-
beled oligonucleotide served as a primer for the sequencing
ladder comparison.
RESULTS AND DISCUSSION
Identification ofthe hsc Gene. An open reading frame of1848
bp encoding a possible hsp70-class protein was detected
during sequencing ofDNA in the 5' flanking region ofthefdx
gene ofE. coli (Fig. 1). Analysis ofcodon usage with theE. coli
codon bias (29) shows aclearpreference forthis readingframe,
and a sequence resembling a Shine-Dalgarno sequence for
ribosome binding and translation (30) is found immediately
upstream ofthe initiation AUG. Sequence showing similarity
to a -10 consensus promoter sequence is also observed from
-63 to -68 bp, but no -35 region consensus sequence (31) is
apparent.
The translatedDNA sequence ofthis readingframe predicts
a polypeptide of 616 amino acids with a molecular mass of
65,647 Da. A search of the GenBank data base using the
deduced amino acid sequence indicated that the predicted
protein showed homology to prokaryotic and eukaryotic
hsp70-class stress proteins, and among the 100 proteins ex-
hibiting the highest similarity scores, all were either heat shock
or heat shock cognate proteins. Because of the apparent lack
ofaheat shockpromoter consensus sequence in the 5' flanking
region of the gene (cf. ref. 7; see also below), we considered
the gene product to be a heat shock cognate protein and
designated the gene hsc and the predicted protein Hsc66.
Comparison ofHsc66 with DnaK. The protein exhibiting the
highest degree of sequence similarity to Hsc66 is DnaK ofE.
coli, and a comparison ofthe amino acid sequences predicted
for the two proteins is presented in Fig. 2. The alignment
shown yields a sequence identity of 41% and a similarity of
60% over the region ofresidues 17-616 ofHsc66. Similarities
to other hsp70 proteins are also notable: Hsc66 has 36%
identity with bovine hsc70 (32) and 39% identity with yeast
Ssclp over the same region (33). The similarities observed are
especially notable in the N-terminal two-thirds ofthe proteins;
this is the most highly conserved region in hsp70 proteins and
has been identified as an ATPase domain in other forms of
hsp70 (34). A number of the conserved residues have been
shown to be involved inATP binding in bovine Hsc70 (35, 36),
and Hsc66 residues 208-230 show homology to the ATP
binding sites ofprotein kinases (37). These similarities suggest
that, like other hsp70 proteins, Hsc66 may possess ATPase
activity. In addition, Thr-212 ofHsc66 aligns with Thr-199 of
DnaK, a site of autophosphorylation (38), raising the possi-
bility that Hsc66 may also be subject to regulation by phos-
phorylation at this position.
Significant differences between the sequences predicted for
Hsc66 and DnaK, however, are apparent. It was necessary to
introduce several gaps in the sequence of Hsc66 to optimize
the alignment. Alignmentofthe sequences ofHsc66andDnaK
with the structure of the ATPase fragment of bovine Hsc7O
(35, 36) suggests that the regions in which gaps were intro-
duced may correspond to residues near the surface of the
folded protein; thus, these differences may reflect different
surface structural features of Hsc66 compared to DnaK and
bovine Hsc7O in those regions. In addition, Hsc66 is predicted
to have anN-terminal extension not present inDnaK and lacks
17 C-terminal residues found in DnaK. The 16-residue N-ter-
minal extension of Hsc66, which is not present in E. coli
DnaK, is unusual because a similar extension is absent from
the predicted sequences of DnaK proteins found in other
prokaryotes. Some eukaryotic forms of hsp70 contain N-ter-
minal extensions, which function in targeting to, or retention
in, the endoplasmic reticulum ormitochondria(forreview, see
ref. 38), but these do not show sequence similarity to the N
terminus of Hsc66. Moreover, the N-terminal sequence of
Hsc66 does not show homology to known signal sequences of
membrane-bound or periplasmic proteins of E. coli, and its
role remains to be determined. The divergence between the
amino acid sequences of Hsc66 and DnaK in the C-terminal
region is similar to the variability observed in other hsp70
proteins. The C-terminal domain is believed to be involved in
protein recognition (37-39), and the differences observed
suggest that Hsc66 is likely to interact with different target
protein(s) within the cell.
Expression ofHsc66. To establish the identity ofthe hsc gene
product, the plasmid p66-Fdx, containing 188 bp of5' flanking
DNA and the putative coding region, was constructed to
overexpress Hsc66 in MZ-1 cells. Fractionation of extracts
from induced cells revealed a major band of -66 kDa, which
was partially purified by anion-exchange and gel-filtration
chromatography. Fig. 3 Left shows the preparation following
SDS/PAGE and blotting to apoly(vinylidene difluoride) mem-
brane. The major band of -66 kDa was subjected to N-ter-
minal amino acid sequencing fornine cycles and was identified
as Hsc66; the first residue ofthe mature protein was found to
be alanine, indicating that the formylmethionine was removed
as found for other proteins in E. coli which have alanine as the
penultimate N-terminal residue (40). DnaK, which has prop-
erties similar to Hsc66, copurified in the preparation and is
visible as a minor band that migrates with an apparent mo-
lecularmass of -75 kDa, an anomalypreviously reported (41).
Western immunoblot analyses were carried out on the
partially purified preparation to test the relatedness of Hsc66
and DnaK. Antisera to DnaK from three rabbits were sepa-
rately tested, and in no case was cross-reactivity with Hsc66
observed. As shown in Fig. 3 Right, DnaK is readily detected
in the preparation by each antiserum, whereas Hsc66, al-
though present in larger amounts, is not detected, suggesting
that the major epitopes present in DnaK are not conserved in
Hsc66.
Characterizationofthehsc-fdxOperon.The 5' flankingregion
of the hsc gene does not contain sequences resembling the
consensus sequences found in heat shock promoters (-CnC-
ccTTGAA- in the -35 region and -CCCCATnT- in the -10
region), which are recognized by the heat shock o32 factor (7).
Biochemistry:Seaton andVickery

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2068
Biochemistry: Seaton and Vickery
Proc. Natl. Acad. Sci. USA 91 (1994)
-180
TCAAACGTGT GAAAAAGATG TTTGATACCC GCCATCAGTT GATGGTTGAA CAGTTAGACA ACGAGACGTG GGACGCGGCG GCGGATACCG
-80 (-10)-60
-
TGCGTAAGCT GCGTTTTCTC GATAAA.TGC GAAGCAGTGC CGAACAACTC GAAGAAAAAC TGCTCGATTT TTAATTTCTg GAACTAAAC
30
ATG GCC TTA TTA CAA ATT AGT GAA CCT GGT TTG AGT GCC GCG CCG CAT CAG CGT CGT CTG GCG GCC GGT ATT GAC
Met Ala Leu Leu Gln Ile Ser Glu Pro Gly Leu Ser Ala Ala Pro His Gln Arg Arg Leu Ala Ala Gly Ile Asp
90
CTG GGC ACA ACC AAC TCG CTG GTG GCG ACA GTG CGC AGC GGT CAG GCC GAA ACG TTA GCC GAT CAT GAA GGC CGT
Leu Gly Thr Thr Asn Ser Leu Val Ala Thr Val Arg Ser Gly Gln Ala Glu Thr Leu Ala Asp His Glu Gly Arg
180
CAC CTG CTG CCA TCT GTT GTT CAC TAT CAA CAG CAA GGG CAT TCG GTG GGT TAT GAC GCG CGT ACT AAT GCA GCG
His Leu Leu Pro Ser Val Val His Tyr Gln Gln Gln Gly His Ser Val Gly Tyr Asp Ala Arg Thr Asn Ala Ala
240
CTC GAT ACC GCC AAC ACA ATT AGT TCT GTT AAA CGC CTG ATG GGA CGC TCG CTG GCT GAT ATC CAG CAA CGC TAT
Leu Asp Thr Ala Asn Thr Ile Ser Ser Val Lys Arg Leu Met Gly Arg Ser Leu Ala Asp Ile Gln Gln Arg Tyr
330
CCG CAT CTG CCT TAT CAA TTC CAG GCC AGC GAA AAC GGC CTG CCG ATG ATT GAA ACG GCG GCG GGG CTG CTG AAC
Pro His Leu Pro Tyr Gln Phe Gln Ala Ser Glu Asn Gly Leu Pro Met Ile Glu Thr Ala Ala Gly Leu Leu Asn
390
CCG GTG CGC GTT TCT GCG GAC ATC CTC AAA GCA CTG GCG GCG CGG GCA ACT GAA GCC CTG GCA GGC GAG CTG GAT
Pro Val Arg Val Ser Ala Asp Ile Leu Lys Ala Leu Ala Ala Arg Ala Thr Glu Ala Leu Ala Gly Glu Leu Asp
480
GGT GTA GTT ATC ACC GTT CCG GCG TAC TTT GAC GAT GCC CAG CGT CAG GGC ACC AAA GAC GCG GCG CGT CTG GCG
Gly Val Val Ile Thr Val Pro Ala Tyr Phe Asp Asp Ala Gln Arg Gln Gly Thr Lys Asp Ala Ala Arg Leu Ala
540
GGC CTT CAC GTC CTG CGC TTA CTT AAC GAA CCG ACC GCT GCG GCT ATC GCC TAC GGG CTG GAT TCC GGT CAG GAA
Gly Leu His Val Leu Arg Leu Leu Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Ser Gly Gln Glu
630
GGC GTG ATC GCC GTT TAT GAC CTC GGT GGC GGG ACG TTT GAT ATT TCC ATT CTG CGC TTA AGT CGC GGC GTG TTT
Gly Val Ile Ala Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Leu Arg Leu Ser Arg Gly Val Phe
690
GAA GTG CTG GCA ACC GGC GGT GAT TCC GCG CTC GGC GGC GAT GAT TTC GAC CAT CTG CTG GCG GAT TAC ATT CGC
Glu Val Leu Ala Thr Gly Gly Asp Ser Ala Leu Gly Gly Asp Asp Phe Asp His Leu Leu Ala Asp Tyr Ile Arg
780
GAG CAG GCG GGC ATT CCT GAT CGT AGC GAT AAC CGC GTT CAG CGT GAA CTG CTG GAT GCC GCC ATT GCA GCC AAA
Glu Gln Ala Gly Ile Pro Asp Arg Ser Asp Asn Arg Val Gln Arg Glu Leu Leu Asp Ala Ala Ile Ala Ala Lys
840
ATC GCG CTG AGC GAT GCG GAC TCC GTG ACC GTT AAC GTT GCG GGC TGG CAG GGC GAA ATC AGC CGT GAA CAA TTC
Ile Ala Leu Ser Asp Ala Asp Ser Val Thr Val Asn Val Ala Gly Trp Gln Gly Glu Ile Ser Arg Glu Gln Phe
930
AAT GAA CTG ATC GCG CCA CTG GTA AAA CGA ACC TTA CTG GCT TGT CGT CGC GCG CTG AAA GAC GCG GGT GTA GAA
Asn Glu Leu Ile Ala Pro Leu Val Lys Arg Thr Leu Leu Ala Cys Arg Arg Ala Leu Lys Asp Ala Gly Val Glu
990
GCT GAT GAA GTG CTG GAA GTG GTG ATG GTG GGC GGT TCT ACT CGC GTG CCG CTG GTG CGT GAA CGG GTA GGC GAA
Ala Asp Glu Val Leu Glu Val Val Met Val Gly Gly Ser Thr Arg Val Pro Leu Val Arg Glu Arg Val Gly Glu
1080
TTT TTC GGT CGT CCA CCG CTG ACT TCC ATC GAC CCG GAT AAA GTC GTC GCT ATT GGC GCG GCG ATT CAG GCG GAT
Phe Phe Gly Arg Pro Pro Leu Thr Ser Ile Asp Pro Asp Lys Val Val Ala Ile Gly Ala Ala Ile Gln Ala Asp
1140
ATT CTG GTG GGT AAC AAG CCA GAC AGC GAA ATG CTG CTG CTT GAT GTG ATC CCA CTG TCG CTG GGC CTC GAA ACG
Ile Leu Val Gly Asn Lys Pro Asp Ser Glu Met Leu Leu Leu Asp Val Ile Pro Leu Ser Leu Gly Leu Glu Thr
1230
ATG GGC GGT CTG GTG GAG AAA GTG ATT CCG CGT AAT ACC ACT ATT CCG GTG GCC CGC GCT CAG GAT TTC ACC ACC
Met Gly Gly Leu Val Glu Lys Val Ile Pro Arg Asn Thr Thr Ile Pro Val Ala Arg Ala Gln Asp Phe Thr Thr
1290
TTT AAA GAT GGT CAG ACG GCG ATG TCT ATC CAT GTA ATG CAG GGT GAG CGC GAA CTG GTG CAG GAC TGC CGC TCA
Phe Lys Asp Gly Gln Thr Ala Met Ser Ile His Val Met Gln Gly Glu Arg Glu Leu Val Gln Asp Cys Arg Ser
1380
CTG GCG CGT TTT GCG CTG CGT GGT ATT CCG GCG CTA CCG GCT GGC GGT GCG CAT ATT CGC GTG ACG TTC CAG GTC
Leu Ala Arg Phe Ala Leu Arg Gly Ile Pro Ala Leu Pro Ala Gly Gly Ala His Ile Arg Val Thr Phe Gln Val
1440
GAT GCC GAC GGT CTT TTG AGC GTG ACG GCG ATG GAG AAA TCC ACC GGC GTT GAG GCG TCT ATT CAG GTC AAA CCG
Asp Ala Asp Gly Leu Leu Ser Val Thr Ala Met Glu Lys Ser Thr Gly Val Glu Ala Ser Ile Gln Val Lys Pro
1530
TCT TAC GGT CTG ACT GAC AGC GAA ATC GCT TCG ATG ATC AAA GAC TCA ATG AGC TAT GCC GAG CAG GAC GTA AAA
Ser Tyr Gly Leu Thr Asp Ser Glu Ile Ala Ser Met Ile Lys Asp Ser Met Ser Tyr Ala Glu Gln Asp Val Lys
1590
GCC CGA ATG CTG GCA GAA CAA AAA GTA GAA GCG GCG CGT GTG CTG GAA AGT CTG CAC GGC GCG CTG GCT GCT GAT
Ala Arg Met Leu Ala Glu Gln Lys Val Glu Ala Ala Arg Val Leu Glu Ser Leu His Gly Ala Leu Ala Ala Asp
1680
GCC GCG CTG TTA AGC GCC GCA GAA CGT CAG GTC ATT GAC GAT GCT GCC GCT CAC CTG AGT GAA GTG GCG CAG GGC
Ala Ala Leu Leu Ser Ala Ala Glu Arg Gln Val Ile Asp Asp Ala Ala Ala His Leu Ser Glu Val Ala Gln Gly
1740
GAT GAT GTT GAC GCC ATC GAA AAA GCG ATT AAA AAC GTA GAC AAA CAA ACC CAG GAT TTC GCC GCT CGC CGC ATG
Asp Asp Val Asp Ala Ile Glu Lys Ala Ile Lys Asn Val Asp Lys Gln Thr Gln Asp Phe Ala Ala Arg Arg Met
1830
GAC CAG TCG GTT CGT CGT GCG CTG AAA GGC CAT TCC GTG GAC GAG GTT TAA T ATG CCA AAG ATT GTT ATT TTG
Asp Gln Ser Val Arg Arg Ala Leu Lys Gly His Ser Val Asp Glu Val
-160
-140
-120
-100
-40
-20
S-D
1
60
120
150
210
270
300
360
420
450
510
570
600
660
720
750
810
870
900
960
1020
1050
1110
1170
1200
1260
1320
1350
1410
1470
1500
1560
1620
1650
1710
1770
1800
1848Fdx
-
*
Met Pro Lys Ile Val Ile Leu
FIG. 1.
methionine ofHsc66; the initiator methionine for thefdx gene is indicated by Fdx. Possible regulatory sequences are underlined (-10, promoter
sequence; S-D, Shine-Dalgarno sequence), and the proposed site of transcriptional initiation (see Fig. 3) is indicated by an arrow (-*).
Nucleotide sequence and deduced amino acid sequence ofthe hsc gene. DNA sequence numbering begins with the predicted initiator
To test whether other sequences present might function in heat
shock induction ofthe gene, we used the vectorp66-Lac, which
contains the lacZ gene fused in-frame with bases encoding the
first nine amino acids of Hsc66 together with 690 bp of 5'
flankingDNA; control ofexpression ofthe chimericgene is thus
under control of hsc promoter sequences. This plasmid was
introduced intoE. coliJM109 cells, and,f3galactosidase activity
of cell extracts was determined before and after subjecting
cultures to heat shock. No increase in f3-galactosidase activity
was observed following a shift to 46°C or 51°C for up to 30 mi
(data not shown), suggesting that Hsc66 is not induced by heat
shock and is subject to other control mechanisms.
The observation that only a single base separates the termi-
nation codon for hsc and the initiation codon forfdx suggests
that the two genes might function as a bicistronic operon. To
determine whether the genes are cotranscribed, the plasmid

Page 4

Biochemistry: Seaton and Vickery
Proc. Natl. Acad. Sci. USA 91 (1994)
2069
20
40
hoc66: MALLQISEPGLSAAPHQRRLAAGIDLGTTNSLVATVRSGQAETLADHEG
~~~
-
_
_
- _
DnaK:
MGKIIGIDLGTTNSCVAIMDGTTPRVLENAEG
20
60
80
100
RHLLPSVVHYQQQGHSV-GYDARTNAALDTANTISSVKRLMGRSLAD-IQQRY-PH
**
_
DRTTPSIIAYTQDGETLVGQPAKRQAVTNPQNTLFAIKRLIGRRFQDEEVQRDVSI
40
60
120
LPYQFQASENGLPMIETAAGLLNPVRVSADILKALAARATEALAGELDGVVITVPA
-
_
_M
_ _U
MPFKIIAADNGDAWVEVKGQKMAPPQISAEVLKKMKKTAEDYLGEPVTEAVITVPA
100
60
180
YFDDAQRQGTKDAARLAGLHVLRLLNEPTAAAIAYGLDSGQE-GVIAVYDLGGGTF
K
-
80
140
1
K-
_
K
_ _
-
120
140
200
4
YFNDAQRQATKDAGRIAGLEVKRIINEPTAAALAYGLDKGTGNRTIAVYDLGGGTF
160
220
DISILRLSR----GVFEVLATGGDSALGGDDFDHLLADYI----REQAGIPDRSDN
-
_
-
_
DISIIEIDEVDGEKTFEVLATNGDTHLGGEDFDSRLINYLVEEFKKDQGIDLRNDP
220
280
RVQRELLDAAIAAKIALSDADSVTVNVAG----------WQGEISREQFNELIAPL
_
Mm-
_
*
LAMQRLKEAAEKAKIELSSAQQTDVNLPYITADATGPKHMNIKVTRAKLESLVEDL
260
280
320
VKRTLLACRRALKDAGVEADEVLEVVMVGGSTRVPLVRERVGEFFGRPPLTSIDPD
_~_ -
-- - __
VNRSIEPLKVALQDAGLSVSDIDDVILVGGQTRMPMVQKKVAEFFGKEPRKDVNPD
320
340
380
KVVAIGAAIQADILVGNKPDSEMLLLDVIPLSLGLETMGGLVEKVIPRNTTIPVAR
Mm
_
- _
EAVAIGAAVQGGVLTGD--VKDVLLLDVTPLSLGIETMGGVMTTLIAKNTTIPTKH
380
0
440
AQDFTTFKDGQTAMSIHVMQGERELVQDCRSLARFALRGIPALPAGGAHIRVTFQV
E
* _
*-
_
*
SQVFSTAEDNQSAVTIHVLQGERKRAADNKSLGQFNLDGINPAPRGMPQIEVTFDI
440
480
500
DADGLLSVTAMEKSTGVEASIQVKPSYGLTDSEIASMIKDSMSYAEQDVKARMLAE
~U-U
Uam *Mm-
DADGILHVSAKDKNSGKEQKITIKASSGLNEDEIQKMVRDAEANAEADRKFEELVQ
80
500
540
560
QKVEAARVLESLHGALAADAALLSAAERQVIDDAAAHLSEVAQGDDVDAIEKAIKN
_
_
Mm-
_
-
* _
TRNQGDHLLHSTRKQVEEAGDKLPADDKTAIESALTALETALKGEDKAAIEAKMQE
540
560
600
616
VDKQTQDFAARRMDQSVRRALKGHSVDEV
_
_
_
* _
x
LAQVSQKLMEIAQQQHAQQQTAGADASANNAKDDDVVDAEFEEVKDKK
600
620
180
200
260
240
_
-
M
MEn
240
300
-
-
300
340
360
--
.
-
360
400
42
--
_-
400
420
460
*
__ M
*__
460
4
520
_ *
-
_
520
580
*
_
m_
580
638
FIG. 2.
coli Hsc66 and DnaK. Amino acid identities are denoted by a thick
line, and similarities are denoted by a thinner line.
Comparison of the deduced amino acid sequences of E.
pFdx-Lac was constructed (Fig. 4). This plasmid encodes a
ferredoxin-3-galactosidase fusion protein under the control of
promoter elements immediately upstream of the fdx gene as
well as those in the 5' flanking region of the hsc gene. Deletion
derivatives of pFdx-Lac were made using unique restriction
sites at varying distances from the 5' end ofthe insert. Plasmids
were introduced into E. coli JM109 cells, and the cells were
grown to midlogarithmic phase for determination of (3-galacto-
sidase activity levels. Deletion of the EcoRI-HindIII region of
the upstream sequence reduced,(3galactosidase activity by only
7%, whereas deletion of the EcoRI-Nru I region reduced
activity by 94%; no further reduction was observed upon
deletion of bases to within 90 bp of the fdx initiation codon.
These findings suggest that under the growth conditions used
expression of the fdx gene is primarily regulated by promoter
sequences between the HindI and Nru I sites.
A derivative of plasmid pFdx-Lac was also constructed in
which an 8-bp linker was inserted into the Nru I site found at
position 750 ofthe hsc coding sequence. This insertion causes
a shift ofthe reading frame ofhsc and introduces a termination
codon 28 codons after the site oflinker insertion. As shown in
Fig. 4, this frameshift reduced the 3-galactosidase activity of
the fdx-lacZ fusion %7-fold. This finding suggests the possi-
bility that hscandfdx are translationally coupled, with trans-
lation ofthe ferredoxin mRNA dependent on translation ofthe
Hsc66 mRNA.
kDa
1
2
3
4
5
97
-*DnaK*
w
.i
-o
67
*Hsc66 >-
45
FIG. 3.
tially purified preparation of Hsc66 was subjected to SDS/PAGE in
a 10%6 gel, and the gel was blotted to a poly(vinylidene difluoride)
membrane and stained with Coomassie blue to visualize proteins.
Lane 1, molecular mass markers; lanes 2-5, -3Agof total protein.
The membrane containing lanes 1 and 2 shows Coomassie blue-
stained protein bands. The membranes containing lanes 3-5 were
individually probed with antisera to E. coliDnaK obtained from three
rabbits (114, 115B, and 116C, respectively) provided by Graham
Walker (Massachusetts Institute ofTechnology); cross-reacting pro-
teins were detected using a peroxidase-conjugated goat anti-rabbit
second antibody and luminol chemiluminescence exposure to auto-
radiography film.
SDS/PAGE and immunoblot analysis of Hsc66. A par-
To determine the 5' end of the hsc-fdx transcript(s), primer
extension reactions were performed using total RNA isolated
from E. coli cells transformed with plasmid pDT10. Reactions
were primed with an oligonucleotide complementary to nucle-
otides 22-41 within the coding sequence ofhsc orto nucleotides
43-60 within the coding sequence offdx. The results using the
primer within the hsc gene showed a single major transcript
starting 57 bp upstream ofthe initiation ATG (Fig. 5). TheDNA
sequence upstream at positions -68 to -63 (TAAACT) shows
similarity and spacing to that of -10 promoter sequences; no
sequence showing similarity to the -35 consensus promoter
sequence (TTGACG; ref. 31), however, is apparent. Using the
primer within thefdx gene no single, major transcriptional start
site was observed within the resolution of the sequencing gel.
Instead, a band ofmoderate intensity (p4/10thas intense as the
hsc transcript) was seen, which corresponded to a transcrip-
tional start site 21 bp upstream of the fdx initiation ATG;
multiple sites ofweak intensity were also observed along the full
EcoRi
i
-H
HindIll
f
NrnA
Acd
Hscw
coding squenc
jfd4az
J 100%
-H
500 bp
8bp insertion3 rTGA
I
]93%
1 6%
I
I
7%
1
HI
i
1
14%
FIG. 4.
insertions within the plasmid pFdx-Lac were made at the indicated
restriction sites. 3-Galactosidase activities were measured as de-
scribed in Experimental Procedures and are reported as percent
activity of the parent plasmid (100% = 4227 Miller units/mg of
protein).
Promoter analysis of the hsc-fdx operon. Deletions and

Page 5

2070Biochemistry: Seaton and Vickery
12
A G C T
-WAN
3qM
I.,
C
T
G
C
G
A
A
G
C
A
G
T
G
\C
C
FIG. 5.
ofthe hsc gene. A[-ty32P]ATP-labeled primer complementary to the
5' end ofthe hsc gene was hybridized to 50Mgof total RNA isolated
from E. coli strain DH5a transformed with pDT10 and was extended
with reverse transcriptase. Products were analyzed by electropho-
resis on a6% polyacrylamide sequencing gel. Samples in lanes 1 and
2 represent reactions in which the hybridization products were
digested withRNaseA (lane 1) orwere leftundigested (lane 2). Lanes
A, G, C, and T are products ofsequencing reactions using the same
oligonucleotide as primer. The sequence of the antisense strand is
shown on the right, and the putative start site, G at position -57, is
indicated by the arrow.
Primer extension analysis ofthe transcriptional start site
length of the resolving gel (data not shown). These results, in
accordance with the deletion studies described above, suggest
thatunderthe conditions used the primary transcript is initiated
upstream of the hsc gene.
The finding that the hsc and fdx genes are coregulated
suggests that they make up a bicistronic operon. While no
complete consensus promoter sequence can be identified
withinthe 5' flanking region ofthe hsc gene, expression levels
observed with the Hsc66- and ferredoxin-3-galactosidase
fusions and the primer extension results are consistent with
a primary site of transcriptional initiation 57 bp upstream of
the Hsc66 coding sequence. In addition, the presence of a
sequence resembling a p-independent termination signal 120
bp downstream of the fdx gene and the absence of open
reading frames in this region (19) suggest that no additional
genes are encoded in the operon. Additional studies are
needed, however, to define the transcript and its regulation.
TIeFunctionof Hsc66 Hsp7Oproteins participate in avariety
of processes involving protein folding, protein assembly and
disassembly, protein secretion, and protein degradation. The
fumction ofHsc66 in E. coli is not known, but the apparent lack
ofinduction in response to heat shock suggests a role or role(s)
in normal cell metabolism as opposed to stress conditions. The
findingthatthe expressionofferredoxin is coregulatedwith that
of Hsc66 suggests the possibility that Hsc66 may function in
some way with the ferredoxin protein, perhaps assisting in
protein folding or assembly of the iron-sulfur cluster.
Note Added In Proof. The hsc gene was independently discovered in
E. coli K12 by Kawula and Lelivelt (42).
We are grateful to G. Wesley Hatfield andJohn Keenerfor helpful
discussions, and we thank Graham Walker for providing antisera to
E. coli DnaK. This work was supported by grants from the National
Institutes of Health.
1.
2.
McKay, D. B. (1993) Adv. Prot. Chem. 44, 67-98.
Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K. &
Hartd, F. U. (1992) Nature (London) 356, 683-689.
3.
Lindquist, S. & Craig, E. A. (1988) Annu. Rev. Genet. 22,
631-677.
Chirico, W. J., Waters, M. G. & Blobel, G. (1988) Nature
(London) 332, 805-810.
Deshaies, R. J., Koch, B. D., Werner-Washburene, M., Craig,
E. A. & Schekman, R. (1988) Nature (London) 332, 800-805.
Chiang, H.-L., Terlecky, S. R., Plant, C. P. & Dice, J. F.
(1989) Science 246, 382-385.
Nover, L. (1991)HeatShockResponse (CRC, BocaRaton, FL).
Craig, E. A., Ingolia, T. D. & Manseau, L. J. (1983)Dev. Biol.
99, 418-426.
Arrigo, A.-P. & Welch, W. J. (1987) J. Biol. Chem. 262,
15359-15369.
Straus, D. B., Walter, W. A. & Gross, C. A. (1989) GenesDev.
3, 2003-2010.
Straus, D., Walter, W. & Gross, C. (1990) Genes Dev. 4,
2202-2209.
Bukau, B. & Walker, G. C. (1989) J. Bacteriol. 171, 2337-2346.
Bukau, B. & Walker, G. C. (1989)J. Bacteriol. 171, 6030-6038.
Herendeen, S. L., VanBogelen, R. A. & Neidhardt, F. C.
(1979) J. Bacteriol. 139, 185-194.
Bardwell, J. C. A. & Craig, E. A. (1984) Proc. Natl. Acad. Sci.
USA 81, 848-852.
Neidhardt, F. C., VanBogelen, R. A. & Vaughn, V. (1984)
Annu. Rev. Genet. 18, 295-329.
Gupta, R. S. & Singh, B. (1992) J. Bacteriol. 174, 4594-4605.
Hughes, A. L. (1993) Mol. Biol. Evol. 10, 243-255.
Ta, D. T. & Vickery, L. E. (1992) J. Biol. Chem. 267, 11120-
11125.
Ta, D. T., Seaton, B. L. & Vickery, L. E. (1992) J. Bacteriol.
174, 5760-5761.
Nagai, K. & Thogersen, H. C. (1984) Nature (London) 309,
810-812.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual (Cold Spring Harbor Lab.
Press, Plainview, NY), 2nd Ed.
Miller, J. H. (1972) Experiments in Molecular Genetics (Cold
Spring Harbor Lab. Press, Plainview, NY).
Laemmli, U. K. (1970) Nature (London) 227, 680-685.
Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl.
Acad. Sci. USA 76, 4350-4354.
Kohara, Y., Akiyama, K. & Isono, K. (1987) Cell 50,495-508.
Brandt, M. E. & Vickery, L. E. (1992) Arch. Biochem. Bio-
phys. 294, 735-740.
Silhavy, T. J., Berman, M. L. & Enquist, L. W. (1984) Exper-
iments with Gene Fusions (Cold Spring Harbor Lab. Press,
Plainview, NY).
Gribskov, M., Devereaux, J. & Burgess, R. R. (1984) Nucleic
Acids Res. 12, 539-549.
Shine, J. & Dalgarno, L. (1974) Proc. Natl. Acad. Sci. USA 71,
1342-1346.
Hawley, D. K. & McClure, W. R. (1983) Nucleic Acids Res.
11, 2237-2255.
DeLuca-Flaherty, C. & McKay, D. B. (1990) Nucleic Acids
Res. 18, 5569.
Craig, E. A., Kramer, J., Shilling, J., Werner-Washburne, M.,
Holmes, S., Kosic-Smithers, J. & Nicolet, C. M. (1989) Mol.
Cell. Biol. 9, 3000-3008.
Chappell, T. G., Konforti, B. B., Schmid, S. L. & Rothman,
J. E. (1987) J. Biol. Chem. 262, 746-751.
Flaherty, K. M., DeLuca-Flaherty, C. & McKay, D. B. (1990)
Nature (London) 346, 623-628.
Flaherty, K. M., McKay, D. B., Kabsch, W. & Holmes, K. C.
(1991) Proc. Natl. Acad. Sci. USA 88, 5041-5045.
Hannink, M. & Donoghue, D. J. (1985) Proc. Natl. Acad. Sci.
USA 82,7894-7898.
McCarty, J. S. & Walker, G. C. (1991) Proc. Natl. Acad. Sci.
USA 88, 9513-9517.
Gething, M.-J. & Sambrook, J. (1992) Nature (London) 355,
33-45.
Flinta, C., Persson, B., Jornvall, H. & von Heine, G. (1986)
Eur. J. Biochem. 154, 193-1%.
Zylicz, M. & Georgopoulos, C. (1984) J. Biol. Chem. 259,
8820-8825.
Kawula, T. H. & Lelivelt, M. J. (1994) J. Bacteriol. 176,
610-619.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Proc. NatL Acad. Sci. USA 91(1994)