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Stringent response in Escherichia coli induces expression of heat shock proteins
J. Mol. Hiol. (1985) 186, 357.-365
Stringent Response in Escherichia coli Induces Expression of Heat Shock Proteins Alan D. Grossman’j-, Wayne E. Taylor2$, Zachary F. Burton2$ Richard R. Burgess2 and Carol A. Gross’11 ‘Department of Bacteriology and ‘McArdle Laboratory for Cancer Research T’niversity of Wisconsin Madison, WI .53T06. TY.S.,J. (Received 9 November 1984, and in revised form 2 July 1985) The rpoD gene (encoding the 70,000 M, sigma subunit of Escherichia coli RNA polymerase) is the most distal gene in an operon that contains three genes. The promoter-proximal gene is rpsl! (encoding ribosomal protein S21) and the middle gene is dnaG (encoding DNA primase). During the stringent response, caused by a deficiency in an aminoaeyl-tRNA, expression of rpsU is decreased, while expression of rpoD is not. This disco-ordinate regulation is due to increased transcription from a minor promoter upstream from rpoD, in the dnaG gene. Transcription from this promoter is also increased during the heat shock response. Expression of other heat shock proteins was found to increase during the stringent response. Thus, the stringent response in E. coli induces expression of heat shock prot’eins. The requirements for this stringent induction of the heat shock proteins differ from those for temperature induction during the heat shock response.
1. Introduction The gene for the 70.000 M, sigma subunit (a7’) of polymerase (rpoD) is the most distal gene in an operon that contains also rpsU and dnaC encoding, respectively, ribosomal protein S21 and DNA primase. Regulatory features of the operon include two promoters upstream from rpsU, three minor promoters within dnaG, a terminator between rpsC! and dnaG and an RNA processing site between dnaG and rpoD (see Fig. 1; Burton et a/., 1983; Lupski et aZ., 1983, 1984). Genes arranged together in an operon are often regulated coordinately under different environmental (Aonditions. However, this is not the case in the complex operon that contains rpoD. The syntheses of S21 and a7’ are re$ited disco-ordinately in response to changes in growth rate (Gausing, 1980; Tshihama & Fukuda, 1980), and temperature shift (Taylor et al., 3984). Differential usage of regulatory &es in the operon could explain this disco-ordinate expression.
Kscherichia coli RNA
7 I’resent address: Harvard University, Biological Laboratories, I6 Divinity Avenue, Cambridge. MA 02138. I1.S.A. $ Prrsent address: Department of Biochemist,ry, I’niversity of Washington. Seattle. WA 98185. U.S.A. 3 I’rrsrnt address: Banting & Best IIepart,ment of Jlrdical Research, University of Toronto. Ontario 8%: 1L6, Canada. I/ Author to whom reprint requestsshould hr addressed.
The disco-ordinate response exhibited by the sigma operon genes following a temperature shiftup results from differential promoter usage during the heat shock response. Upon shift to high temperature, transcription from the major operon promotrrs does not increase. Thus, ribosomal prot.ein S21 does not show a heat shock response. However, transcription, from one of the minor promoters within dnaG shows strong transient activation after temperat’ure upshift. Transcription from this promoter (called rpoDPhs) is responsible for the heat shock response of sigma. Tnduction of rpoljl’hs during the heat shock response is dependent on the rpoH (htpR) gene product (Taylor Strains containing the rpoH165 et al., 1984). mutation have a reduced amount of the rpoH gene product. a,re defective in the heat shock response (Neidhardt & Van Bogelen, 1981; Yamamori & Aura. 1982) and do not induce expression from rpoD1’h.s after temperature upshift (Taylor et al.. 1984). The rpoH gene product is a 32,000 i!IZr sigma factor (03’) that promotes transcription initiation from heat shock promoters in &ro (Grossman it al.,
1984). Tn this paper, we show that expression of the genes in the rpoD operon becomes disco-ordinate during the stringent response and characterize the mechanism of this disco-ordinate regulation. The stringent response (reviewed by Gallant, 1979) is induced when the availability of an aminoacylt,RNA species becomes limiting for protein
357
0
1985 Academic
Press Inc. (London)
Lt)d.
S2l
DNA
prlmase
Slgmo-70
Figure 1. Structure of the rpsl., dnaU. rpol) operon. Determination of the structure and characterization of some features of the operon have been described (Burt,on et al., 1983; Lupski ef al., 1983. 1984: Taylor et cxl., 1984). I’1 and PP are the major operon promoters. tl and t2 are terminators. and Pa. Pb and P,, are minor promoters located in dnaG. PHS is called rpoDPhs in this paper. The arrow (1) indicates the RXA processing site.
synthesis. Under these conditions, ppGpp and pppGpp accumulate and a variety of changes in gene expression occur, including a reduction in the rate of synthesis of stable RNA and ribosomal proteins and an increase in the rate of synthesis of some other proteins. These changes in gene expression do not occur in rel (relaxed) mutants. Studies in vitro have shown that conditions can be found in which ppGpp affects transcription from promoters in the manner expected from results obtained in vivo (Gallant, 1979 and references therein; Glaser et al., 1983; Kajitani & Ishihama, 1984).
Tt has been established that during the stringent response, the synthesis of S21 decreases like that of other ribosomal proteins (Dennis & Nomura, 1974, 1975). We show that under these conditions the differential rate of synthesis of a” slightly increases, although transcripts initiating at the major operon promoters decrease. During the stringent response, the activity of rpoDPhs is induced, allowing the synthesis of 07’ to continue. In addition, synthesis of all of the heat shock proteins that we have measured increases during the stringent response. However, this induction seems to be different from temperature induction during the heat shock response.
2. Materials (a) Bacterial
strains,
and Methods plasmids
and
of synthetic
rates
for
(r) S, nudeasp
individual
proteins
Cells were grown and pulse-labeled as described in the Figure legends. The method for measuring ribosomal protein synthesis was essentially as described (JinksRobertson & Nomura, 1982). Briefly. 14C-labeled cells were added to 3H-labeled samples for standardization. ribosomal proteins were extracted with acetic acid and precipitated with acetone. From 5 to 10 il,,, units of crude ribosomes were added to samples before elect#rophoresis. Two-dimensional polyacrylamide gel electrophoresis was done as described by Howard & Traut (1973). Gels were stained with Coomassie blue and the spot corresponding to S21 cut out, solubilized in 0.5 ml of
mapping
S, mapping was done essentially as described (Burton ef al., 1983). Hybridizations were done in NT,, (v/v) formamide at 50°C for 3 h. S, treatment (5000 units/ reaction) was done at 37°C for 30 min. Protected Dh’A fragments were separated on 5% DNA sequencing gels (Burton et al., 1983). (d) Galactokinase
assays
The different,ial rate of galactokinase synthesis was determined by the slope of a plot of galactokinase activity versus cell optical density at, 450 nm. Galartokinase assays were done as described (McKenney et al., 1981; Taylor et al., 1984), except that deoxycholate (0.3oi,, w/v) was included in the lysis mixture.
media
Strains and plasmids are listed in Table 1. M9 medium (Miller, 1972) was used with glucose (0.426, w/v) as the carbon source. Amino acids (40 pg/ml) were added as indicated. Strains containing plasmids were grown in media with 50 pg ampicillin/ml. (b) Measurement
H,Ot and 0.2 ml of perchlorir acid. and counted in a liquid scintillation counter using a double label rounting program. The relative rate of synthesis was calculated by dividing the 3H/‘4C rat’ IO for a given protein by the 3H/14C ratio in the total extract. The method for measuring synthesis rates of nonribosomal proteins was similar to that for ribosomal proteins, except sample preparation and S-dimensional gel electrophoresis (O’Farrell, 1975) were as described (Grossman et al., 1983). Heat shock proteins are referred to by the gene name (GroE, DnaK, Lon), or by the alphanumeric name (F84.1. C62.5, B25.3, C15.4. C14.7), as described by Neidhardt et al. (1984). In general. synthesis rates are accurate 2 10 to 20?,.
3. Results (a) Plan
of the experimds
The stringent response can be induced either by starving cells for an amino acid or by inactivating an aminoacyl-tRNA synthetase. In all of the experiments reported in this paper, the stringent response was induced by partial inactivation of a temperature sensitive (ts) valyl-tRNA synthetase (vaZSs). Isogenic strains containing either a valS’” or valS + allele were shifted from permissive temperature (285°C) to a partially restrictive temperature (33.5”C), where protein synthesis was inhibited approximately 50% and the stringent response was induced. Under these conditions, the effect of the stringent response on the rate of synthesis of a variety of polypeptides can be 1979, and determined reproducibly (Gallant, references therein). To verify that the changes in
Stringent
Response
Induces
359
Heat Shock in E. coli
Table 1 Strains
used in this work
Strain
Grnotye
(',4(:1707
(‘A(:21 72 (‘9G2173 (‘A(~2 174
fhi, nrgE, his, proA. 1~. thr, rpsL. g&h’. vniS”-Tn 10 (TnlO approximately 50”,, linked to v&S) ( ‘AG 1707 11alS + ( ‘AC: 1707 relA (:A(~1707 arg+ (by Pl transduction) (‘AGl708 arg+ (by Pl transduction) (‘AGl709 arg+ (by PI transduction) A(pro-lac)Xlll, thi, am, rpsL, gulK, trp(am), szlpC”. &S’f-TnlO; F’pro+ luc+ (‘AG2044 valL7 C:AG2044 rpoH16.5 (‘AG2044 rpoHi65. ualS“ thi, argE, his, proA, leu, thr, rpsL, galK, trp(am), supC”, rpoH’, rhg : : TnIO (TnlO approximately 80% linked t o rpoH), calS+-Tn5 (Tnj approximately 504b linked to valS) (‘A(~2171 mlS’s (IAG2171 rpoHl6.i (‘AG2171 rpoH16.5. va1P
l’lasmidcontaining strains
Genotype
(‘A(:1963 ('AG1977 ('AGlOil (‘AGPlO7 (‘AG;! I1 1 (‘.2(:21 13 (‘A(:21 17
(:A(:1707 ( :AG 1709 (‘A(:2045 (‘A02044 (‘.402044 (‘A(:2045 (‘A(:2045
( ‘A( : 170X ( ‘AG 1709 (‘AG 1940 (‘A01941 (‘AGl942 CA02044 (JAG2045 (‘A(~2046 I ( :AGZ047 (:A(:2171
pKWTl2” pKWT 12 a pKWT9 b pKWT5D35’ pKWT51)-56c pKWT5D-35’ pKWT5D56
’
All strains are E. eoli K12. a pKRTl2 contains the 89 base-pair &a1 to Hinf’I fragment with rpoDPhs cloned upstream ofga/K in pKO1 (Taylor pf al., 1984). b pKWTR contains the 179 base-pair Hid to D&I fragment with rpoDPhs cloned into pKO1 (Taylor rt al.. 1984). ’ pKWT5D-35 and pKWT5D-56 contain deletion mutations of rpoDPhs. They are described b! Taylor of al. (1984). and the 5’ endpoints are indicated in Fig. 4.
the valS’” strain were due to induction of the stringent response, identical experiments were performed on an isogenic valS’” relA strain and/or the isogenic wild-type strain (valS+, rel+). Strains containing a relA mutation do not induce the stringent response upon limitation of an aminoacyltRNA. (b) Synthesis
of S27 and Rigma during stringent response
the
We determined the rate of synthesis of both 0” and S21 under our standard conditions of partial limitation for charged valyl-tRNA. In the r&4 + strain, the rate of synthesis of S21 and other ribosomal proteins decreased about SOq/b,while that. of a70 increased slightly (Table 2). As expected. the rate of synthesis of the ribosomal proteins was increased in the isogenic r&l strain (Table 2). Our results for ribosomal proteins were consistent with those reported in the literature (Dennis & Komura, 1974, 1975). This experiment established t,hat expression of rpsli and rpoD was disco-ordinate during the skngent response.
(c) Transcription
from operon promoters stringent response
during
the
We used an S, mapping experiment t,o compare the amount of RNA originating from t,he operon promoters Pl and P2 at the permissive (28.5”C) and restrictive (33.5”C) temperatures. RNA was prepared from isogenic reZA+ and reld strains from cultures growing at 28.5”C or 20 min after the cultures were shifted to 33.5”C. The RNA was mapped with a DNA probe with a 5’ labeled end about 280 bases from promoter PI (see Fig. 2(c)). Upon shift to the restrictive temperature, transcripts from both Pl and P2 decreased in the reZ+ strain (Fig. 2(a), lanes 1, 2, 6 to 8), but not in the rel- strain (Fig. 2(a), lanes 3 to 5, 9 to 1I). This experiment indicated that during the stringent, response, kanscription from both major operon promoters was inhibited. We repeated the S, mapping experiment with a DNA probe with a labeled 5’ end that extended into the dnaG coding region (see Fig. 2(c)). As found above, during the stringent response, transcripts from both Pl and P2 were decreased in the rel+ (Fig. 2(b). lanes 1 to 3, 7 to 9) but not the rel-
-4 Il. Grossman
et al.
VOlS’~
“O/S’*
28.5=‘C
IO
-
40
IO
213.5OC
33.5oc
20
40
IO
20
40
IO
20
40
IO
ugRNA
20
40
33.5oc
IO
20
40
4
5
6
IO
20
40
-
20
40
PI P2-
2
3
4
5
6
7
8
9
IO
II
I23
78
9
IO
II
(bl
(‘1)
700bp
Figure 2. S, nucleasr mapping of I’1 and I’2 during the stringent response. The strategy for S, mapping and the I)robes used are indicated in (c). Probe A was end-labeled at the Hind111 site in rpsl,’ and probe B was end-labeled at the XhoT site in dnaO. DNA probes and RKA samples were prepared as described (Burton et aZ., 1983). Strains CAGl940 (~/dS’“, rrl+) and CAGl942 (valS’“. rel-) were grown in M9 glucose supplemented wit,h vitamins and amino acids. When the vulture reached an A,,, value of 0.2 to 0.4. RPiA was isolated from cells grown at 28.5”C or 20 min after shift to 33~5°C. The amount of RNA used in each hybridization is indicated over each lane. Increasing amounts of Rh’A gave proportional increases in the amount of the DNA fragment that was protected, indicating that D?u’A was in excess. is probably a processed RNA species and has Transrripk initiating at Pl and P2 are indicated. The band below “P2” been discussed (Burton it al., 1983). bp. base-pairs.
strain (Fig. 2(b). lanes 4 to 6. 10 and 11). Thus, the efficiency of termination at tl was not dramatically affected during the stringent response. At least. at the RNA level, dnaG appears t’o be under stringent current.ly unable t,o measure control. We are synthesis of the dna.G gene product. (primase) because it is present at such a low level in the cell. (d) Expression from the heat shock promoter upstream from rpoD (rpoDPhs) during the stringent response We followed activity of the heat shock promoter upstream of rpoD by using a fusion of rpoDPhs to the galactokinase structural gene in the pKOl plasmid system (McKenney et al.. 1981; Taylor et from al.. 1984). Expression of galactokinase
rpoL)Phs was induced approximately sevenfold following partial limitation for charged valyl-t,RSA on (Fig. 3). The increased expression was dependent was the. stringent response because no increase detect’ed in the isogenic relA strain (Fig. 3). Expression from the galK or tat promoters fused to t,he galactokinase structural gene in t,he same plasmid system was not stimulated during the stringent response (data not shown). Thus, t,he observed increase was a property of rpoDI-‘hs and not, an artifact of the cloning system. (e) Synthesis
of other heat shock proteins stringent response
Because transcription from during the stringent response,
during
the
rp0DPh.c was induced we asked whether t,he
Stringent
Response
Induces
361
Heat Shock in E. coli
Table 2 Synthesis
of S21 and o”’ during stringent
IIrlative
rel+
the
response
rate of synthesis
(33.5:‘28.5)
a
?dS”
I
Absorbance
ot 450 nm
Figure 3. Activity of’ rpoDPhs during the stringent rrsponse. Strains CA61963 (vaZP, reZ+) and C’AG1977 and were grown as ( oa LS’” , rrl-) each contain pKWT12 described in Fig. 2. except ampicillin (50 pg/ml) was included in the media. Cells were grown at %5”c and shift’ed to 335°C at the point indicated by arrows (1t). Samples were taken and assayed for galactokinase activity as described (Taylor it al., 1984). (0). CAGl963: (0) CAG1977.
rate of synthesis of other heat shock proteins also increased. We measured t)he rates of synthesis of four heat. shock proteins (GroEL, DnaK, F84.1, CSS.S), and of elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) during steady-state growth at 28.5Y or 15 minutes after shift to 33.5”C. A decreased rate of synthesis of EF-Tu and EF-Ts indicates that the stringent response is occurring (Blumenthal et al., 1976; Furano & Wittel, 1976; Reeh et al., 1976). Shift of the valS’” strain to the restrictive temperature induced the stringent response as judged by the behavior of EF-Tu and EF-Ts and resulted in a three- to ninefold induction of synthesis of heat shock proteins (Table 3, column 1). This increase was dependent on induction of the stringent response because it was not observed in the isogenic reZA strain (Table 3, column 2). In fact, synthesis of heat shock proteins was decreased in the relA strain. When wild-type (vu&+) cells were shifted from 28.5”C to 33*5”C, only slight (about 1.3-fold) induction of the heat shock proteins was observed (Table 3, column 3). Upon correction for the slight induction exhibited by wild-type cells due to temperature shift, we find that the heat shock proteins were induced two- to sevenfold during the stringent response (Table 3, column 4). We call this stringent induction. (f) Induction mutant
of heat shock proteins in an rpoH during the stringent response
The transient overproduction of heat shock proteins after temperature upshift requires the rpoH (htpR) gene product’ (Neidhardt & Van
I’roteilr
7-d +
rrl -
S%l b Sigma-70’ I,1 b
O.“% I ,48 0. I8
Im 0.78 I.71
a Strains (‘A(:1940 (zYLS’“. rrl+) and (‘4G194% (raki’“. w-) were grown at 28~5°C in M9 glucose supplemented with vitamins and amino acids except lysine and arginine. IThen cultures reached an A,,, value of 0.2 to 04. samples (3.5 ml) were pulselabeled at either 28.5”(’ or 20 min after shift to 33.5”(’ with “0 p(‘i 13H]arginine/ml (18.3 (li/mmol) and 60 p(‘i j3H Ilvsine/ml (A0 (‘i:mmol) for 1.5 min and chased with excess ar&ine and Iysinr for Imin. b For ribosomal protein determinations, A samplr of cells labeled with “(‘-labeled amino acids was added for standardization to cells from 3.0 ml of each ‘H-labeled sample and the double-labeled mixtures were analyzed as described in Materials and Methods. Relative rates of svnthesis (33.5/28.5) are given as the ratio 1(3H/‘4(‘) protein % - (3H:‘4(‘) total protein 1 at 33.5’Y’ divided by the same ratio at 28.5”(’ ’ For (1” determinations. a sample of ‘%labeled crlls for standardization was added t,o cells from 0.5 ml of each 3H-labeled sample and the double-labeled mixtures were analyzed as described in Materials and Methods. Relative rates of synthesis are given as the ratio [(3H/35S) sigma i (3H/35S) total protein] at 33.5”(’ divided by the same ratio at .)X..‘“(‘.
Kogelrn, 1981; I-amamori & Yura. 1982). rpoH encodes a 32,000 M, sigma factor (Oar) that promotes transcription initiation from heat shock promoters. Tneficient suppression (by SUJ)( “‘) of the
Table 3 Synthesis
Relative
of heat shock prote%ns thP stringent response rates of synthesis calS’s
Protein FH4. I DnaK Gr0EL (w2.6 EF-Tu EF-Ts
during
(33.5/28.5)
a
1’s/As+
rfd+
w-
rd -
Induction ratio b
8.89 3.27 z2.80 6.47 0.46 0.37
0.49 0.41 0.57 0.84 1.02 1.22
1.3:’ 099 1.31 1.41 0% 0%
673 3.%7 Lt.14 4.59 0.48 0.39
a Strains CAG1707 (valS’“, rel+), CAG1708 (valS+, rel+). and ( ‘AQ 1709 (vaN”. rel-) were grown at 28.5”(’ in MS glucose supplemented with vitamins and amino acids except methionine. At an A45O value of 0.2 to 0.4, samples (0.5 ml) were pulselabeled at 28~5°C or I5 min after shift to 33.5”C with 60 /.G [35SJmethionine/ml (adjusted to 60 pCi/14 ng) for I.5 min and chased with excess methionine for 2 min. ‘H-labeled cells were added to 358-labeled samples for standardization and the doublelabeled mixtures were analyzed as described in Materials and Methods. Relative rates of synthesis (33.5/28.5) are given as the ratio [(35S/3H) protein X + (35S/3H) total protein] at 33.5”(’ divided by the same ratio at 28.5”C. b The induction ratio is the ~x&P rel+ ratio divided by the &S+ w/+ ratio.
362
A. 11. Grossman
et,
al.
Table 4 A’trinyenf
response
Relative
rat,es of synthesis rpoIi
Protein F84. I I,Wl (:roEL ( ‘62.5 1325.3 (‘14.i+(‘15.4 EP-Tu EF-Ts
in the rpoH 165 mutant (33.5idX.R)
a
+
rpoH16G
IulS +
7dS”
Ratio
1 .%ti 1.14 1.87 1.86 0.92 1.33 I.10 I a4
15.9 3.05 5.04 9~65 4.86 1.53 0.74 0.58
12.6 44P3 2.70 5.19 52-i 1m 0457 0.56
b
mlS +
ids”
0437 1.17 1.43 1.16 1.15 1.13 1.07 04x4
Ratio
ti.4 2.64 2.93 4.19 2.83 244 0.74 0.56
b
7-r 2.46 2.38 3.58 246 2.16 069 0.57
a Strains CAG2171 (v&S’+, rpoH+), (‘AG2172 (&SC. rpoH165), (‘A(:2173 (ovals”. rpoH +). and (‘A(:21 74 (mLS’“. rpoHlG) were grown as described for Table 2. except that only Iysine was left out of the amino acids. At an -4,,, value of 0.2 to 0.4. samples (1.0 ml) were pulse-labeled at 18.5”(’ or 14 rnin after shift to 33.5”C with 100 pc(‘i [3H]1ysinejm1 (60 Ci/mmol) for 3 min and chased with excess Irsinr for 1 min. “S-labeled cells were added to the 3H-labeled samples for normalization and the double-labeled mixtures were analyzed as described in Materials and Methods. Relative rates of svnthesis (33.,5/Z&5) are given hy the ratio 1(3H/35S) protein X + (3H/3SS) total protein] at 33.5”C &vi&d bg the same ratio at W5”c’. ’ These ratios are the ratio for ~ulS” divided by the ratio for valS+.
nonsensemutation reduces the amount of a3’ in the cell, causes ts growth (Cooper & Ruettinger, 1975), abolishes the heat shock response (Neidhardt & Van Bogelen, 1981; Yamamori & Yura, 1982) and causes a defect in proteolysis (Baker et al., 1984). We measured the synthesis rates of heat shock proteins in the rpoH165 mutant during the stringent response. To our surprise, we found that heat shock protein synthesis was still induced during the stringent
response (Table 4). Induction of some heat shock proteins was reduced. but by no means was the reduction as severe as the absence of induction during the heat shock response. In addition, transcription from the C62.5 gene promoter, rpoDPhs, and the promoters for DnaK, was induced during the stringent response in the rpoH165 mutant) as measured by gaZK fusions (Table 5, Fig. 4). Because it. was unexpected that, stringent induction still occurred in the rpoHZ65 mutant, we
rpoHl65
Table 5 AYringent Relative
rrsponse
in
the
rpoHl65 mutant
rates of transcription
(33~5158~5) a
rpoH + Promoter dtULKl’1 dllUKP:? (‘62.5 gene rpo1)Ph.s (ial IA(~
Plasmid pIY401 c.e pm1404 c.f plx:420c.~ pKWT,51)-56 pKG1900’~h pKM1 I’.’
no.
d
V&S+
/Yds’”
I +5 1 I .3 I.0 I.0 1.o
r, 6.9 9.7 5x 1 .o 14)
rpoH 16.i Ratio 3 6.9 7.5 5.8 1 .o 1 .o
b
PUlLS+ 1 2 2.S I4
Ids” 2.3 9.5 7.5 7.6
Ratio
b
2.3 4.5 3.3 74
a Relative rates of transcription (33.5/28.5) are given by the ratio of the differential rate of galactokinase synthesis after shift to 33.5”(’ divided by the differential rate of galactokinasr synthesis at 28.5”(’ before t.emperature shift (see Mat,erials and Methods). b These ratios are the ratio for ~nlS” divided by the ratio for wlS+. ’ This plasmid was carried in strains (‘A(+2171 (w/S’. rpoH+). (‘AG”li:! (wS”“. rpoH+). (‘,4(:2173 (rw/S+. rpoHl/S). (‘A(:2174 (r~1S”“. rpoH1U). d This plasmid was carried in strainb (‘AG2044 (WE/S+. rpoH+). CAG2045 (znW”, rpoH ‘). (‘AC:2046 (t-nlS+, rpolll6.i). (1AG2047 (v&“~, PpoHlM). ’ pD(‘401 contains the 150 base-pair Hinfl fragment with dnrzKP1 cloned upstream from grrlK in pKO1 ((‘owing PI al.. 1985).. ’ pDC404 contains the I45 baw-pair IfincII-Elscl fragment with dnaKP2 cloned up&earn from g&K in pKO1 ((‘owing rt ~1.. 1985). 8 pD(‘420 contains the 225 base-pair Al?) fragment with the (‘62.5 promoter region cloned upst,ream from go/R in pKO1 ((‘owing rt a/., 1985). h pK(:lQOO contains the gal promoter cloned into pKOl and is desrribed in McKenney rf al. (1981). ’ pKMI 1 contains the Inr promoter cloned into pKO1 and is described by McKennev et al. (1981). j This plasmid was carried in strains (!A(:1707 (v&““, rpoH+) and CA01708 (pals+: rpoH+)
Stringent Response Induces Heat Shock in E. coli
363
PHS
5’
-TC
T
-__-- I pKWT5D-56 Stringmt
inductii
(2)
44
“W? inductm
(t%)
8.2
ACMATAATGCC
I
-_---
1----_
I
pKWT5D-35
pKWT9
4-8
(DdeI)
4.6 N.D.
Figure 4. Stringent induction and temperature induction of rpoDPhs deletion mutants. For stringent induction. the palsy strains CAG2072 (pKWTS), CAG2113 (pKWT5D-35), and CAG2117 (pKWT5D-56) were grown as described for Fig. 3, except that proline was left out of the media. Galactokinase assays were done on samples from cells growing at 30°C or at various times after shift to 34°C. Stringent induction ratios are the rate of galactokinase synthesis at 34°C divided by the rate at 30°C. Experiments for heat induction were the same as for stringent induction except the UZZS+ strains CAG2107 (pKWT5D-35) and CAG2111 (pKWT5D-56) were used and the cultures were shifted from 30°C to 42Y’. Heat induction of pKWT9 was not determined (X.D.) in this experiment or in this strain background. However, in other strain backgrounds, the rpoDPhs in pKWT9 was induced similarly to pKWT5D-56. The arrow (t) indicates the endpoints of each deletion and the broken lines (- - -) under the sequences indicate the direction in which the DlrjA is deleted. The construction and characterization of these plasmids has been described in detail (Taylor et al., 1984). The 5’ ends of the transcripts were determined by S, mapping and are indicated by arrows (1) above the sequence. The - 10 and - 35 regions of the promoter are indicated. The sequence hyphens have been omitted for clarity.
repeated these experiments in a different strain background with similar results. In addition, we verified that the strains carried the rpoHl65 allele, were defective in the heat shock response, and were defective in proteolysis at 30°C and 42°C (A. D. Grossman, unpublished results). Apparently, the reduced level of a32 in the rpoH165 strain is sufficient to block the heat shock response and to cause a defect in proteolysis but is not sufficient to block stringent induction.
(g) DNA sequencesrequired for stringent induction We compared the DNA sequences required for heat induction with those required for stringent induction. The sequence 5’.C-T-G-C-C’-A-Cr-C-~‘-3’ in the region from -44 to - 35 base-pairs upstream from the mRNA start is required for the heat activation of rpoDPhs. This sequence was identified by the deletion mutation contained in plasmid pKWT5D35 that abolishes normal heat induction (Fig. 4; Taylor ct al., 1984). However, this sequence was not required for stringent induction. Induction of rpoDPhs during the stringent response was similar in clones with (pKWT5D-56) and without (pKWT5D35) this sequence (Fig. 4). A deletion mutation ending at - 16 abolished promoter activity and abolished stringent induction. Sequences downstream from + 11 were not required for stringent induction because the promoter in plasmid pKWT9, with the 3’ end at the DdeI site, was induced during the stringent response (Fig. 4). Taken together, these results localize the sequences required for stringent induction to a region of 46 base-pairs located between -35 and + 11 of
rpoDPhs.
4. Discussion Expression of the genes in the rpoD (a”) operon becomes disco-ordinate during both the stringent response and the heat shock response. In both cases, expression of rpoD, the most distal gene in the operon, increases relative to bhat of the promoter-proximal gene, rpsC:. In each case, the mechanism of this uncoupling is the activation of a promoter (rpoDPhs) located upstream from IPOD, in the 3’ end of dnaG, the middle gene in the operon. The heat shock promoter upstream from rpoD was not the only heat shock promoter induced during the stringent response. The bwo heat shock promoters upstream from dnaK. dnaKP1 and dnaKP2. and the heat shock promoter upstream from the gene encoding the heat shock protein C62.5, have been fused to the galactokinase gene (Cowing et al., 1985). In these fusions: galactokinase synthesis increased during the stringent response. In addition: synthesis of all of the heat shook proteins we have measured (DnaK, GroEL, C62.5, F84.1, D25.3, Lon, C14.7+C15.4) was increased during the stringent response. It is likely that in all cases the increased synthesis of heat shock proteins resulted from increased transcription initiation at heat shock promoters. We call this stringent induction. Other researchers have noted changes in the relative rates of synthesis of many proteins during amino acid starvation and the stringent response (Blumenthal et aZ., 1976; Chao, 1977: Furano & Wittel, 1976; O’Farrell, 1978; Reeh et aE., 1976). In some studies, starvation conditions were induced by removing a required amino acid and in other studies starvation conditions were induced using a valP mutation. Because most of those studies were carried out before the heat shock response in E. coli
364
d.
I).
Grossman
\vas well-charac:terize(l, the increased synt,hesis of the heat shock proteins as a class was not recognized. Comparison of the published gels with dat’a on the heat’ shock response and with our data on stringent induction indicat,ed that many of the proteins previously observed to increase during the stringent response were. in fact, heat shock proteins. Thus. the stringent response joins a growing list of “stress” conditions that are known to induce the synthesis of heat shock proteins. IGsides temperature upshift’ and the stringent response, synthesis of heat shock proteins can be induced by ethanol (Neidhardt ct al., 1984; Travers B Mace, 1982), ultraviolet light irradiation (Krueger & Walker, 1984), naladixic acid (Krueger & LI’alker. 1984), coumermycin (Travers & Mace. f 982: Menzel & Cellert,; 1983), and infection with phage lambda (Drahos & Hendrix, 1982: Kochan & Murialdo, 1982). It is not) known how t,hese agents induce heat shock protein synthesis. While the heat shock response and the stringent response both result in increased t)ranscript,ion initiation at heat shock promoters and increased synthesis of heat shock proteins, the requirements for induction are different. relil mutant’s do not accumulate ppGpp after amino acid starvation and are defective in the stringent response and in stringent induction. However, the heal shock response is normal in relil mutants (A. I). Grossman, unpublished results). Tn t,hr course of these studies, we found that the SC122 st,rain that has been used for some of the studies on heat shock (Neidhardt & Van Bogelen, 1981: Neidhardt’ d al.. 1983) has a relaxed phenotype (A. 1). Grossman. unpublished results). When the rpoIl165 nonsense mutation is inefficiently suppressed, resulting in a reduced amount of a32, the mutant is ts for growth (Cooper & Ruettinger, 1975; Tamamori & Yura. 1982), defective in the heat shock response (Neidhardt & Van Bogelen. 1981: Yamamori & Aura, 1982), and defective in proteolysis, even at permissive growth temperature (Baker rt al., f 984). However, the reduced amount of 032 in the mutant did not prevent stringent’ induction (Table 4). Finally, the DNA sequence 5’-C-T-G-C’-C’-A-(‘-(“-(‘-3 located at -44 and -35 base-pairs from the mRNA start of rpoDPhs is required for temperat,ure induct’ion (Taylor et al., 1984) but not for stringent induction. The mechanism by which the synthesis of heat shock proteins is increased during t,he stringent response is clearly different from that during the heat shock response. Because stringent induction occurs in the rpoHl65 mutant, it is possible that 032 is not required for the increased synthesis of heat shock protein during the stringent response. A different o factor could be activated to promote transcription initiation from heat shock promoters under these conditions. We feel that it is unlikely that cr” promotes this transcription. We have been unable to find conditions in vitro in which 0” promotes transcription initiation from heat shock promoters (Cowing et al., 1985). Tn addition, c”
et
al
seemsto inhibit expression of heat shock gerlr’s 111 I*iz~o(A. I>. Grossman Cyr(‘. A. (iross. unpnblished results). However. it remains possible that there is an additional o factor that can prornotfl transcript,ion initiation from heat. shock promoters. Alternatively, a32 may be required for stringent induction. The ryoHZ6;S mut,ant is not dr*void of cr32. Rather. it has a reduced amount of the gem’ product. Stringent induct’ion is not elimirrated in the rpoHl&i mutant; however. the amount of’ induction of some heat, shock proteins and of heat shock gene transcription decreased in the mutant strain (Tables 4 and ;i). These observations arc consistent with a role for a32 In stringent induction. The rpoH765 mutation prevents induction of the heat shock proteins after temperature upshift but has a smaller effect on the increased synt.hrsis of heat shock proteins during the stringent response. If a3* is required for transcript initiation from heat shock promot,ers during stringent induction, then the st,ringent responsemust stimulate transcript ion from t*hesepromoters in a way that is different from the stimulation during the heat shock rc~sponst~. Transcript,ion initiation at heat shock promoters may be regulat’ed. in part,, by a compchtition between 032 and 0” for binding to core R?iA polymerase. or by competition between holoenzymr containing 0 32 (Ea3’) and holoenzyme containing a7’ (Ea”) for binding to heat shock promoters. It is tempting to speculate that during the stringent response, ppclpp (or other small molecules) alters this competition either by inhibiting cr7’ or Ea7’ or by st,imulating u32 or EONS. We are investigating these possibilities. We thank \.lr. IS’alter and Y. N. Zhou for superb technical assistance. I. Slater for typing several drafts of this paper. and B. Sugden, W. Dove and (:. Robrrt,s for their helpful comments on the manuscript. This work was supported by the College of Agricultural and Life Sciences. r’niversity of Wisconsin-Madisotl. grant GM28575 from the Eational Tnstit)utes of Health to
R. Burgessand C. Gross, and grants R01 AI 19635and K04
AI
00573
to (1. Gross. References
Baker, T. A., Grossman,
A. D. & Gross, C. A. Proc.. NUT, 81, 6779-6783. Blumenthal, R. M., Lemaux, P. G., Neidhardt, F. C. $ Dennis,P. P. (1976).Mol. Gen. Genet. 149, 291-296. Burton. Z. F., Gross, C. A., Watanabe, K. K. & Burgess. R. R. (1983). Cell, 32, 335.-349. C!hao, L. (1977). Biochem. Biophys. Res. Conmun. 77, 42-49. Cooper, S. & Ruettinger, T. (1975). ,UoZ. Gen. Genet. 139. 167-176. Cowing, D. W., Bardwefl, *J. C. A., Craig, E. A.. Woolford, C., Hendrix, R. W. & Gross, C. A. (1985). Acad.
hi.,
Proc.
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Acad.
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V.S.A.
82, 2679-2683.
Dennis, P. P. & Nomura, M. (1974). Proc. Nut. Acud. Sci., C.9.A. 71, 3819-3823. Dennis, P. P. & Xomura, M. (1975). Nature (London), 225, 460-465. Drahos. D. J. & Hendrix, R. W. (1982). J. Bacterial. 149, 1050-1063.
Stringent
Response
Induces
Furano. A. V. & Wittel. F. I’. (1976). Cell, 8. 115-122. Gallant. I. A. (1979). Annu. Rev. Genet. 13, 393-415. Gausing. K. (1980). In RiDosomes: Structuw, Function, and Genetics (Chambliss. G., Craven. G. R.. Davies. ,I.. Davis, K.. Kahan, I,. & Nomura. M.. eds), pp. 69:3-718, University Park Press, Baltimore. (ilasrr. G.. Sarmientos. 1’. & Cashel. M. (1983). AVafur~ (London),
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(:rossnian. A. D.. Eurgess. R. R.. Walter. \V. & Gross. (‘. .I. (1983). (‘ell, 32. 151-159. (:roxsman. A. D.. Erickson. .J. LV’. & Gross. (‘. A. (1984). (‘rll. 38. 38% 390. Howard. G. A. 8: Traut. R. R. (1973). FEBS Letters, 29. Iii
Jshihama. 31.
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A. Hr Fukuda.
R,. (1980). Mol. Cdl. Hiochem.
177-196.
.Jinks-Kobert~son, S. Rr Nomura. M. (1982). ,I. Racteriol. 151. 193~-20”. Kajitani, M. & Tshihama, A. (1984). J. Biol. Chem. 259. I’E-. .
1957. *.
Kochan. *J. $ Murialdo. H. (1982). J. Ha,rteriol. 149. 1166-1170. Krueger. J. H. & LValker. G. (1. (1984). Proc. ‘Vat. Acad. Sri., 1’.A’.A. 81. 1499-1503. Lupski. ,J. R.. Smiley. B. L. & Godson, G. R;. (1983). Mol. Gen.
Genrt.
189.
48-57.
Lupski.
J. R.. Ruiz, A. A. & Godson. G. N. (1984). Mol. c:rn. Urnfd. 195. 391~-401. Edited
Heat Shock in E. coli
365
McKrnney. K.. Shimatake, H.. Court, D.. Schmeissner. I’. 8r Rosenberg, M. (1981). In Gene .4mpZi&ation and Analysis 2 (ChlrlkJlan, J. & Papas. T.. eds). pp. 383-415. Earth Holland Press, Amsterdam. Mrnzel. R. 8: Gellert. M. (1983). Cell, 34. 105-113. Miller. ,I. (1972). Experiments in Molecular Gen,etics, Cold Spring Harbor Laboratory Press. Cold Spring Harbor. Seidhardt, F. (‘. Xr Van Bogelen, R. A. (1981). Biochem. Hiophys. RPS. Commun. 100, 894-900. Seidhardt. F. (‘.. Van Bogelen. R. X. B I,au. E. T. (1983). .I. IZacterioZ. 153. 597-603. Sridhardt. F. C., Van Bogelen. R. A. 8: Vaughn. \‘. (lR84). Annu. Rev. Genet. 18, 295.-329. O’Farrell. I’. H. (1975). ,I. Biol. (‘hem. 250. 1007-4021. O’Farrrll. P. H. (1978). Cell, 14. 5455557. Rwh. S I’ederson. S. & Friesen. *J. 1). (1976). ,lilol. Oc>n. (hut. 149, 279-289. Taylor. N’. E.. Straus, 1). B., Grossman. A. I>.. Burton, %. F.. (+ross. (‘. A. 8r Burgess. R. R. (1984). (‘ell, 38. 3i I -381 Travers. A. ZI. & IMace. H. A. F. (1982). Tn Heat Shock From Bnctrrio to Man (Schlesinger. M.. Ashburner. .\I. Pr Tissi&s. A.. eds). pp. 127-130. (‘old Spring Harbor Laboratory Press, Cold Spring Harbor. Yamamori. T. & Aura, T. (1982). Proc. XQt. =Icnd. Sci., I-.S.A.
by M. Gottesman
79. 860-864.
Stringent Response in Escherichia coli Induces Expression of Heat Shock Proteins Alan D. Grossman’j-, Wayne E. Taylor2$, Zachary F. Burton2$ Richard R. Burgess2 and Carol A. Gross’11 ‘Department of Bacteriology and ‘McArdle Laboratory for Cancer Research T’niversity of Wisconsin Madison, WI .53T06. TY.S.,J. (Received 9 November 1984, and in revised form 2 July 1985) The rpoD gene (encoding the 70,000 M, sigma subunit of Escherichia coli RNA polymerase) is the most distal gene in an operon that contains three genes. The promoter-proximal gene is rpsl! (encoding ribosomal protein S21) and the middle gene is dnaG (encoding DNA primase). During the stringent response, caused by a deficiency in an aminoaeyl-tRNA, expression of rpsU is decreased, while expression of rpoD is not. This disco-ordinate regulation is due to increased transcription from a minor promoter upstream from rpoD, in the dnaG gene. Transcription from this promoter is also increased during the heat shock response. Expression of other heat shock proteins was found to increase during the stringent response. Thus, the stringent response in E. coli induces expression of heat shock prot’eins. The requirements for this stringent induction of the heat shock proteins differ from those for temperature induction during the heat shock response.
1. Introduction The gene for the 70.000 M, sigma subunit (a7’) of polymerase (rpoD) is the most distal gene in an operon that contains also rpsU and dnaC encoding, respectively, ribosomal protein S21 and DNA primase. Regulatory features of the operon include two promoters upstream from rpsU, three minor promoters within dnaG, a terminator between rpsC! and dnaG and an RNA processing site between dnaG and rpoD (see Fig. 1; Burton et a/., 1983; Lupski et aZ., 1983, 1984). Genes arranged together in an operon are often regulated coordinately under different environmental (Aonditions. However, this is not the case in the complex operon that contains rpoD. The syntheses of S21 and a7’ are re$ited disco-ordinately in response to changes in growth rate (Gausing, 1980; Tshihama & Fukuda, 1980), and temperature shift (Taylor et al., 3984). Differential usage of regulatory &es in the operon could explain this disco-ordinate expression.
Kscherichia coli RNA
7 I’resent address: Harvard University, Biological Laboratories, I6 Divinity Avenue, Cambridge. MA 02138. I1.S.A. $ Prrsent address: Department of Biochemist,ry, I’niversity of Washington. Seattle. WA 98185. U.S.A. 3 I’rrsrnt address: Banting & Best IIepart,ment of Jlrdical Research, University of Toronto. Ontario 8%: 1L6, Canada. I/ Author to whom reprint requestsshould hr addressed.
The disco-ordinate response exhibited by the sigma operon genes following a temperature shiftup results from differential promoter usage during the heat shock response. Upon shift to high temperature, transcription from the major operon promotrrs does not increase. Thus, ribosomal prot.ein S21 does not show a heat shock response. However, transcription, from one of the minor promoters within dnaG shows strong transient activation after temperat’ure upshift. Transcription from this promoter (called rpoDPhs) is responsible for the heat shock response of sigma. Tnduction of rpoljl’hs during the heat shock response is dependent on the rpoH (htpR) gene product (Taylor Strains containing the rpoH165 et al., 1984). mutation have a reduced amount of the rpoH gene product. a,re defective in the heat shock response (Neidhardt & Van Bogelen, 1981; Yamamori & Aura. 1982) and do not induce expression from rpoD1’h.s after temperature upshift (Taylor et al.. 1984). The rpoH gene product is a 32,000 i!IZr sigma factor (03’) that promotes transcription initiation from heat shock promoters in &ro (Grossman it al.,
1984). Tn this paper, we show that expression of the genes in the rpoD operon becomes disco-ordinate during the stringent response and characterize the mechanism of this disco-ordinate regulation. The stringent response (reviewed by Gallant, 1979) is induced when the availability of an aminoacylt,RNA species becomes limiting for protein
357
0
1985 Academic
Press Inc. (London)
Lt)d.
S2l
DNA
prlmase
Slgmo-70
Figure 1. Structure of the rpsl., dnaU. rpol) operon. Determination of the structure and characterization of some features of the operon have been described (Burt,on et al., 1983; Lupski ef al., 1983. 1984: Taylor et cxl., 1984). I’1 and PP are the major operon promoters. tl and t2 are terminators. and Pa. Pb and P,, are minor promoters located in dnaG. PHS is called rpoDPhs in this paper. The arrow (1) indicates the RXA processing site.
synthesis. Under these conditions, ppGpp and pppGpp accumulate and a variety of changes in gene expression occur, including a reduction in the rate of synthesis of stable RNA and ribosomal proteins and an increase in the rate of synthesis of some other proteins. These changes in gene expression do not occur in rel (relaxed) mutants. Studies in vitro have shown that conditions can be found in which ppGpp affects transcription from promoters in the manner expected from results obtained in vivo (Gallant, 1979 and references therein; Glaser et al., 1983; Kajitani & Ishihama, 1984).
Tt has been established that during the stringent response, the synthesis of S21 decreases like that of other ribosomal proteins (Dennis & Nomura, 1974, 1975). We show that under these conditions the differential rate of synthesis of a” slightly increases, although transcripts initiating at the major operon promoters decrease. During the stringent response, the activity of rpoDPhs is induced, allowing the synthesis of 07’ to continue. In addition, synthesis of all of the heat shock proteins that we have measured increases during the stringent response. However, this induction seems to be different from temperature induction during the heat shock response.
2. Materials (a) Bacterial
strains,
and Methods plasmids
and
of synthetic
rates
for
(r) S, nudeasp
individual
proteins
Cells were grown and pulse-labeled as described in the Figure legends. The method for measuring ribosomal protein synthesis was essentially as described (JinksRobertson & Nomura, 1982). Briefly. 14C-labeled cells were added to 3H-labeled samples for standardization. ribosomal proteins were extracted with acetic acid and precipitated with acetone. From 5 to 10 il,,, units of crude ribosomes were added to samples before elect#rophoresis. Two-dimensional polyacrylamide gel electrophoresis was done as described by Howard & Traut (1973). Gels were stained with Coomassie blue and the spot corresponding to S21 cut out, solubilized in 0.5 ml of
mapping
S, mapping was done essentially as described (Burton ef al., 1983). Hybridizations were done in NT,, (v/v) formamide at 50°C for 3 h. S, treatment (5000 units/ reaction) was done at 37°C for 30 min. Protected Dh’A fragments were separated on 5% DNA sequencing gels (Burton et al., 1983). (d) Galactokinase
assays
The different,ial rate of galactokinase synthesis was determined by the slope of a plot of galactokinase activity versus cell optical density at, 450 nm. Galartokinase assays were done as described (McKenney et al., 1981; Taylor et al., 1984), except that deoxycholate (0.3oi,, w/v) was included in the lysis mixture.
media
Strains and plasmids are listed in Table 1. M9 medium (Miller, 1972) was used with glucose (0.426, w/v) as the carbon source. Amino acids (40 pg/ml) were added as indicated. Strains containing plasmids were grown in media with 50 pg ampicillin/ml. (b) Measurement
H,Ot and 0.2 ml of perchlorir acid. and counted in a liquid scintillation counter using a double label rounting program. The relative rate of synthesis was calculated by dividing the 3H/‘4C rat’ IO for a given protein by the 3H/14C ratio in the total extract. The method for measuring synthesis rates of nonribosomal proteins was similar to that for ribosomal proteins, except sample preparation and S-dimensional gel electrophoresis (O’Farrell, 1975) were as described (Grossman et al., 1983). Heat shock proteins are referred to by the gene name (GroE, DnaK, Lon), or by the alphanumeric name (F84.1. C62.5, B25.3, C15.4. C14.7), as described by Neidhardt et al. (1984). In general. synthesis rates are accurate 2 10 to 20?,.
3. Results (a) Plan
of the experimds
The stringent response can be induced either by starving cells for an amino acid or by inactivating an aminoacyl-tRNA synthetase. In all of the experiments reported in this paper, the stringent response was induced by partial inactivation of a temperature sensitive (ts) valyl-tRNA synthetase (vaZSs). Isogenic strains containing either a valS’” or valS + allele were shifted from permissive temperature (285°C) to a partially restrictive temperature (33.5”C), where protein synthesis was inhibited approximately 50% and the stringent response was induced. Under these conditions, the effect of the stringent response on the rate of synthesis of a variety of polypeptides can be 1979, and determined reproducibly (Gallant, references therein). To verify that the changes in
Stringent
Response
Induces
359
Heat Shock in E. coli
Table 1 Strains
used in this work
Strain
Grnotye
(',4(:1707
(‘A(:21 72 (‘9G2173 (‘A(~2 174
fhi, nrgE, his, proA. 1~. thr, rpsL. g&h’. vniS”-Tn 10 (TnlO approximately 50”,, linked to v&S) ( ‘AG 1707 11alS + ( ‘AC: 1707 relA (:A(~1707 arg+ (by Pl transduction) (‘AGl708 arg+ (by Pl transduction) (‘AGl709 arg+ (by PI transduction) A(pro-lac)Xlll, thi, am, rpsL, gulK, trp(am), szlpC”. &S’f-TnlO; F’pro+ luc+ (‘AG2044 valL7 C:AG2044 rpoH16.5 (‘AG2044 rpoHi65. ualS“ thi, argE, his, proA, leu, thr, rpsL, galK, trp(am), supC”, rpoH’, rhg : : TnIO (TnlO approximately 80% linked t o rpoH), calS+-Tn5 (Tnj approximately 504b linked to valS) (‘A(~2171 mlS’s (IAG2171 rpoHl6.i (‘AG2171 rpoH16.5. va1P
l’lasmidcontaining strains
Genotype
(‘A(:1963 ('AG1977 ('AGlOil (‘AGPlO7 (‘AG;! I1 1 (‘.2(:21 13 (‘A(:21 17
(:A(:1707 ( :AG 1709 (‘A(:2045 (‘A02044 (‘.402044 (‘A(:2045 (‘A(:2045
( ‘A( : 170X ( ‘AG 1709 (‘AG 1940 (‘A01941 (‘AGl942 CA02044 (JAG2045 (‘A(~2046 I ( :AGZ047 (:A(:2171
pKWTl2” pKWT 12 a pKWT9 b pKWT5D35’ pKWT51)-56c pKWT5D-35’ pKWT5D56
’
All strains are E. eoli K12. a pKRTl2 contains the 89 base-pair &a1 to Hinf’I fragment with rpoDPhs cloned upstream ofga/K in pKO1 (Taylor pf al., 1984). b pKWTR contains the 179 base-pair Hid to D&I fragment with rpoDPhs cloned into pKO1 (Taylor rt al.. 1984). ’ pKWT5D-35 and pKWT5D-56 contain deletion mutations of rpoDPhs. They are described b! Taylor of al. (1984). and the 5’ endpoints are indicated in Fig. 4.
the valS’” strain were due to induction of the stringent response, identical experiments were performed on an isogenic valS’” relA strain and/or the isogenic wild-type strain (valS+, rel+). Strains containing a relA mutation do not induce the stringent response upon limitation of an aminoacyltRNA. (b) Synthesis
of S27 and Rigma during stringent response
the
We determined the rate of synthesis of both 0” and S21 under our standard conditions of partial limitation for charged valyl-tRNA. In the r&4 + strain, the rate of synthesis of S21 and other ribosomal proteins decreased about SOq/b,while that. of a70 increased slightly (Table 2). As expected. the rate of synthesis of the ribosomal proteins was increased in the isogenic r&l strain (Table 2). Our results for ribosomal proteins were consistent with those reported in the literature (Dennis & Komura, 1974, 1975). This experiment established t,hat expression of rpsli and rpoD was disco-ordinate during the skngent response.
(c) Transcription
from operon promoters stringent response
during
the
We used an S, mapping experiment t,o compare the amount of RNA originating from t,he operon promoters Pl and P2 at the permissive (28.5”C) and restrictive (33.5”C) temperatures. RNA was prepared from isogenic reZA+ and reld strains from cultures growing at 28.5”C or 20 min after the cultures were shifted to 33.5”C. The RNA was mapped with a DNA probe with a 5’ labeled end about 280 bases from promoter PI (see Fig. 2(c)). Upon shift to the restrictive temperature, transcripts from both Pl and P2 decreased in the reZ+ strain (Fig. 2(a), lanes 1, 2, 6 to 8), but not in the rel- strain (Fig. 2(a), lanes 3 to 5, 9 to 1I). This experiment indicated that during the stringent, response, kanscription from both major operon promoters was inhibited. We repeated the S, mapping experiment with a DNA probe with a labeled 5’ end that extended into the dnaG coding region (see Fig. 2(c)). As found above, during the stringent response, transcripts from both Pl and P2 were decreased in the rel+ (Fig. 2(b). lanes 1 to 3, 7 to 9) but not the rel-
-4 Il. Grossman
et al.
VOlS’~
“O/S’*
28.5=‘C
IO
-
40
IO
213.5OC
33.5oc
20
40
IO
20
40
IO
20
40
IO
ugRNA
20
40
33.5oc
IO
20
40
4
5
6
IO
20
40
-
20
40
PI P2-
2
3
4
5
6
7
8
9
IO
II
I23
78
9
IO
II
(bl
(‘1)
700bp
Figure 2. S, nucleasr mapping of I’1 and I’2 during the stringent response. The strategy for S, mapping and the I)robes used are indicated in (c). Probe A was end-labeled at the Hind111 site in rpsl,’ and probe B was end-labeled at the XhoT site in dnaO. DNA probes and RKA samples were prepared as described (Burton et aZ., 1983). Strains CAGl940 (~/dS’“, rrl+) and CAGl942 (valS’“. rel-) were grown in M9 glucose supplemented wit,h vitamins and amino acids. When the vulture reached an A,,, value of 0.2 to 0.4. RPiA was isolated from cells grown at 28.5”C or 20 min after shift to 33~5°C. The amount of RNA used in each hybridization is indicated over each lane. Increasing amounts of Rh’A gave proportional increases in the amount of the DNA fragment that was protected, indicating that D?u’A was in excess. is probably a processed RNA species and has Transrripk initiating at Pl and P2 are indicated. The band below “P2” been discussed (Burton it al., 1983). bp. base-pairs.
strain (Fig. 2(b). lanes 4 to 6. 10 and 11). Thus, the efficiency of termination at tl was not dramatically affected during the stringent response. At least. at the RNA level, dnaG appears t’o be under stringent current.ly unable t,o measure control. We are synthesis of the dna.G gene product. (primase) because it is present at such a low level in the cell. (d) Expression from the heat shock promoter upstream from rpoD (rpoDPhs) during the stringent response We followed activity of the heat shock promoter upstream of rpoD by using a fusion of rpoDPhs to the galactokinase structural gene in the pKOl plasmid system (McKenney et al.. 1981; Taylor et from al.. 1984). Expression of galactokinase
rpoL)Phs was induced approximately sevenfold following partial limitation for charged valyl-t,RSA on (Fig. 3). The increased expression was dependent was the. stringent response because no increase detect’ed in the isogenic relA strain (Fig. 3). Expression from the galK or tat promoters fused to t,he galactokinase structural gene in t,he same plasmid system was not stimulated during the stringent response (data not shown). Thus, t,he observed increase was a property of rpoDI-‘hs and not, an artifact of the cloning system. (e) Synthesis
of other heat shock proteins stringent response
Because transcription from during the stringent response,
during
the
rp0DPh.c was induced we asked whether t,he
Stringent
Response
Induces
361
Heat Shock in E. coli
Table 2 Synthesis
of S21 and o”’ during stringent
IIrlative
rel+
the
response
rate of synthesis
(33.5:‘28.5)
a
?dS”
I
Absorbance
ot 450 nm
Figure 3. Activity of’ rpoDPhs during the stringent rrsponse. Strains CA61963 (vaZP, reZ+) and C’AG1977 and were grown as ( oa LS’” , rrl-) each contain pKWT12 described in Fig. 2. except ampicillin (50 pg/ml) was included in the media. Cells were grown at %5”c and shift’ed to 335°C at the point indicated by arrows (1t). Samples were taken and assayed for galactokinase activity as described (Taylor it al., 1984). (0). CAGl963: (0) CAG1977.
rate of synthesis of other heat shock proteins also increased. We measured t)he rates of synthesis of four heat. shock proteins (GroEL, DnaK, F84.1, CSS.S), and of elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) during steady-state growth at 28.5Y or 15 minutes after shift to 33.5”C. A decreased rate of synthesis of EF-Tu and EF-Ts indicates that the stringent response is occurring (Blumenthal et al., 1976; Furano & Wittel, 1976; Reeh et al., 1976). Shift of the valS’” strain to the restrictive temperature induced the stringent response as judged by the behavior of EF-Tu and EF-Ts and resulted in a three- to ninefold induction of synthesis of heat shock proteins (Table 3, column 1). This increase was dependent on induction of the stringent response because it was not observed in the isogenic reZA strain (Table 3, column 2). In fact, synthesis of heat shock proteins was decreased in the relA strain. When wild-type (vu&+) cells were shifted from 28.5”C to 33*5”C, only slight (about 1.3-fold) induction of the heat shock proteins was observed (Table 3, column 3). Upon correction for the slight induction exhibited by wild-type cells due to temperature shift, we find that the heat shock proteins were induced two- to sevenfold during the stringent response (Table 3, column 4). We call this stringent induction. (f) Induction mutant
of heat shock proteins in an rpoH during the stringent response
The transient overproduction of heat shock proteins after temperature upshift requires the rpoH (htpR) gene product’ (Neidhardt & Van
I’roteilr
7-d +
rrl -
S%l b Sigma-70’ I,1 b
O.“% I ,48 0. I8
Im 0.78 I.71
a Strains (‘A(:1940 (zYLS’“. rrl+) and (‘4G194% (raki’“. w-) were grown at 28~5°C in M9 glucose supplemented with vitamins and amino acids except lysine and arginine. IThen cultures reached an A,,, value of 0.2 to 04. samples (3.5 ml) were pulselabeled at either 28.5”(’ or 20 min after shift to 33.5”(’ with “0 p(‘i 13H]arginine/ml (18.3 (li/mmol) and 60 p(‘i j3H Ilvsine/ml (A0 (‘i:mmol) for 1.5 min and chased with excess ar&ine and Iysinr for Imin. b For ribosomal protein determinations, A samplr of cells labeled with “(‘-labeled amino acids was added for standardization to cells from 3.0 ml of each ‘H-labeled sample and the double-labeled mixtures were analyzed as described in Materials and Methods. Relative rates of svnthesis (33.5/28.5) are given as the ratio 1(3H/‘4(‘) protein % - (3H:‘4(‘) total protein 1 at 33.5’Y’ divided by the same ratio at 28.5”(’ ’ For (1” determinations. a sample of ‘%labeled crlls for standardization was added t,o cells from 0.5 ml of each 3H-labeled sample and the double-labeled mixtures were analyzed as described in Materials and Methods. Relative rates of synthesis are given as the ratio [(3H/35S) sigma i (3H/35S) total protein] at 33.5”(’ divided by the same ratio at .)X..‘“(‘.
Kogelrn, 1981; I-amamori & Yura. 1982). rpoH encodes a 32,000 M, sigma factor (Oar) that promotes transcription initiation from heat shock promoters. Tneficient suppression (by SUJ)( “‘) of the
Table 3 Synthesis
Relative
of heat shock prote%ns thP stringent response rates of synthesis calS’s
Protein FH4. I DnaK Gr0EL (w2.6 EF-Tu EF-Ts
during
(33.5/28.5)
a
1’s/As+
rfd+
w-
rd -
Induction ratio b
8.89 3.27 z2.80 6.47 0.46 0.37
0.49 0.41 0.57 0.84 1.02 1.22
1.3:’ 099 1.31 1.41 0% 0%
673 3.%7 Lt.14 4.59 0.48 0.39
a Strains CAG1707 (valS’“, rel+), CAG1708 (valS+, rel+). and ( ‘AQ 1709 (vaN”. rel-) were grown at 28.5”(’ in MS glucose supplemented with vitamins and amino acids except methionine. At an A45O value of 0.2 to 0.4, samples (0.5 ml) were pulselabeled at 28~5°C or I5 min after shift to 33.5”C with 60 /.G [35SJmethionine/ml (adjusted to 60 pCi/14 ng) for I.5 min and chased with excess methionine for 2 min. ‘H-labeled cells were added to 358-labeled samples for standardization and the doublelabeled mixtures were analyzed as described in Materials and Methods. Relative rates of synthesis (33.5/28.5) are given as the ratio [(35S/3H) protein X + (35S/3H) total protein] at 33.5”(’ divided by the same ratio at 28.5”C. b The induction ratio is the ~x&P rel+ ratio divided by the &S+ w/+ ratio.
362
A. 11. Grossman
et,
al.
Table 4 A’trinyenf
response
Relative
rat,es of synthesis rpoIi
Protein F84. I I,Wl (:roEL ( ‘62.5 1325.3 (‘14.i+(‘15.4 EP-Tu EF-Ts
in the rpoH 165 mutant (33.5idX.R)
a
+
rpoH16G
IulS +
7dS”
Ratio
1 .%ti 1.14 1.87 1.86 0.92 1.33 I.10 I a4
15.9 3.05 5.04 9~65 4.86 1.53 0.74 0.58
12.6 44P3 2.70 5.19 52-i 1m 0457 0.56
b
mlS +
ids”
0437 1.17 1.43 1.16 1.15 1.13 1.07 04x4
Ratio
ti.4 2.64 2.93 4.19 2.83 244 0.74 0.56
b
7-r 2.46 2.38 3.58 246 2.16 069 0.57
a Strains CAG2171 (v&S’+, rpoH+), (‘AG2172 (&SC. rpoH165), (‘A(:2173 (ovals”. rpoH +). and (‘A(:21 74 (mLS’“. rpoHlG) were grown as described for Table 2. except that only Iysine was left out of the amino acids. At an -4,,, value of 0.2 to 0.4. samples (1.0 ml) were pulse-labeled at 18.5”(’ or 14 rnin after shift to 33.5”C with 100 pc(‘i [3H]1ysinejm1 (60 Ci/mmol) for 3 min and chased with excess Irsinr for 1 min. “S-labeled cells were added to the 3H-labeled samples for normalization and the double-labeled mixtures were analyzed as described in Materials and Methods. Relative rates of svnthesis (33.,5/Z&5) are given hy the ratio 1(3H/35S) protein X + (3H/3SS) total protein] at 33.5”C &vi&d bg the same ratio at W5”c’. ’ These ratios are the ratio for ~ulS” divided by the ratio for valS+.
nonsensemutation reduces the amount of a3’ in the cell, causes ts growth (Cooper & Ruettinger, 1975), abolishes the heat shock response (Neidhardt & Van Bogelen, 1981; Yamamori & Yura, 1982) and causes a defect in proteolysis (Baker et al., 1984). We measured the synthesis rates of heat shock proteins in the rpoH165 mutant during the stringent response. To our surprise, we found that heat shock protein synthesis was still induced during the stringent
response (Table 4). Induction of some heat shock proteins was reduced. but by no means was the reduction as severe as the absence of induction during the heat shock response. In addition, transcription from the C62.5 gene promoter, rpoDPhs, and the promoters for DnaK, was induced during the stringent response in the rpoH165 mutant) as measured by gaZK fusions (Table 5, Fig. 4). Because it. was unexpected that, stringent induction still occurred in the rpoHZ65 mutant, we
rpoHl65
Table 5 AYringent Relative
rrsponse
in
the
rpoHl65 mutant
rates of transcription
(33~5158~5) a
rpoH + Promoter dtULKl’1 dllUKP:? (‘62.5 gene rpo1)Ph.s (ial IA(~
Plasmid pIY401 c.e pm1404 c.f plx:420c.~ pKWT,51)-56 pKG1900’~h pKM1 I’.’
no.
d
V&S+
/Yds’”
I +5 1 I .3 I.0 I.0 1.o
r, 6.9 9.7 5x 1 .o 14)
rpoH 16.i Ratio 3 6.9 7.5 5.8 1 .o 1 .o
b
PUlLS+ 1 2 2.S I4
Ids” 2.3 9.5 7.5 7.6
Ratio
b
2.3 4.5 3.3 74
a Relative rates of transcription (33.5/28.5) are given by the ratio of the differential rate of galactokinase synthesis after shift to 33.5”(’ divided by the differential rate of galactokinasr synthesis at 28.5”(’ before t.emperature shift (see Mat,erials and Methods). b These ratios are the ratio for ~nlS” divided by the ratio for wlS+. ’ This plasmid was carried in strains (‘A(+2171 (w/S’. rpoH+). (‘AG”li:! (wS”“. rpoH+). (‘,4(:2173 (rw/S+. rpoHl/S). (‘A(:2174 (r~1S”“. rpoH1U). d This plasmid was carried in strainb (‘AG2044 (WE/S+. rpoH+). CAG2045 (znW”, rpoH ‘). (‘AC:2046 (t-nlS+, rpolll6.i). (1AG2047 (v&“~, PpoHlM). ’ pD(‘401 contains the 150 base-pair Hinfl fragment with dnrzKP1 cloned upstream from grrlK in pKO1 ((‘owing PI al.. 1985).. ’ pDC404 contains the I45 baw-pair IfincII-Elscl fragment with dnaKP2 cloned up&earn from g&K in pKO1 ((‘owing rt ~1.. 1985). 8 pD(‘420 contains the 225 base-pair Al?) fragment with the (‘62.5 promoter region cloned upst,ream from go/R in pKO1 ((‘owing rt a/., 1985). h pK(:lQOO contains the gal promoter cloned into pKOl and is desrribed in McKenney rf al. (1981). ’ pKMI 1 contains the Inr promoter cloned into pKO1 and is described by McKennev et al. (1981). j This plasmid was carried in strains (!A(:1707 (v&““, rpoH+) and CA01708 (pals+: rpoH+)
Stringent Response Induces Heat Shock in E. coli
363
PHS
5’
-TC
T
-__-- I pKWT5D-56 Stringmt
inductii
(2)
44
“W? inductm
(t%)
8.2
ACMATAATGCC
I
-_---
1----_
I
pKWT5D-35
pKWT9
4-8
(DdeI)
4.6 N.D.
Figure 4. Stringent induction and temperature induction of rpoDPhs deletion mutants. For stringent induction. the palsy strains CAG2072 (pKWTS), CAG2113 (pKWT5D-35), and CAG2117 (pKWT5D-56) were grown as described for Fig. 3, except that proline was left out of the media. Galactokinase assays were done on samples from cells growing at 30°C or at various times after shift to 34°C. Stringent induction ratios are the rate of galactokinase synthesis at 34°C divided by the rate at 30°C. Experiments for heat induction were the same as for stringent induction except the UZZS+ strains CAG2107 (pKWT5D-35) and CAG2111 (pKWT5D-56) were used and the cultures were shifted from 30°C to 42Y’. Heat induction of pKWT9 was not determined (X.D.) in this experiment or in this strain background. However, in other strain backgrounds, the rpoDPhs in pKWT9 was induced similarly to pKWT5D-56. The arrow (t) indicates the endpoints of each deletion and the broken lines (- - -) under the sequences indicate the direction in which the DlrjA is deleted. The construction and characterization of these plasmids has been described in detail (Taylor et al., 1984). The 5’ ends of the transcripts were determined by S, mapping and are indicated by arrows (1) above the sequence. The - 10 and - 35 regions of the promoter are indicated. The sequence hyphens have been omitted for clarity.
repeated these experiments in a different strain background with similar results. In addition, we verified that the strains carried the rpoHl65 allele, were defective in the heat shock response, and were defective in proteolysis at 30°C and 42°C (A. D. Grossman, unpublished results). Apparently, the reduced level of a32 in the rpoH165 strain is sufficient to block the heat shock response and to cause a defect in proteolysis but is not sufficient to block stringent induction.
(g) DNA sequencesrequired for stringent induction We compared the DNA sequences required for heat induction with those required for stringent induction. The sequence 5’.C-T-G-C-C’-A-Cr-C-~‘-3’ in the region from -44 to - 35 base-pairs upstream from the mRNA start is required for the heat activation of rpoDPhs. This sequence was identified by the deletion mutation contained in plasmid pKWT5D35 that abolishes normal heat induction (Fig. 4; Taylor ct al., 1984). However, this sequence was not required for stringent induction. Induction of rpoDPhs during the stringent response was similar in clones with (pKWT5D-56) and without (pKWT5D35) this sequence (Fig. 4). A deletion mutation ending at - 16 abolished promoter activity and abolished stringent induction. Sequences downstream from + 11 were not required for stringent induction because the promoter in plasmid pKWT9, with the 3’ end at the DdeI site, was induced during the stringent response (Fig. 4). Taken together, these results localize the sequences required for stringent induction to a region of 46 base-pairs located between -35 and + 11 of
rpoDPhs.
4. Discussion Expression of the genes in the rpoD (a”) operon becomes disco-ordinate during both the stringent response and the heat shock response. In both cases, expression of rpoD, the most distal gene in the operon, increases relative to bhat of the promoter-proximal gene, rpsC:. In each case, the mechanism of this uncoupling is the activation of a promoter (rpoDPhs) located upstream from IPOD, in the 3’ end of dnaG, the middle gene in the operon. The heat shock promoter upstream from rpoD was not the only heat shock promoter induced during the stringent response. The bwo heat shock promoters upstream from dnaK. dnaKP1 and dnaKP2. and the heat shock promoter upstream from the gene encoding the heat shock protein C62.5, have been fused to the galactokinase gene (Cowing et al., 1985). In these fusions: galactokinase synthesis increased during the stringent response. In addition: synthesis of all of the heat shook proteins we have measured (DnaK, GroEL, C62.5, F84.1, D25.3, Lon, C14.7+C15.4) was increased during the stringent response. It is likely that in all cases the increased synthesis of heat shock proteins resulted from increased transcription initiation at heat shock promoters. We call this stringent induction. Other researchers have noted changes in the relative rates of synthesis of many proteins during amino acid starvation and the stringent response (Blumenthal et aZ., 1976; Chao, 1977: Furano & Wittel, 1976; O’Farrell, 1978; Reeh et aE., 1976). In some studies, starvation conditions were induced by removing a required amino acid and in other studies starvation conditions were induced using a valP mutation. Because most of those studies were carried out before the heat shock response in E. coli
364
d.
I).
Grossman
\vas well-charac:terize(l, the increased synt,hesis of the heat shock proteins as a class was not recognized. Comparison of the published gels with dat’a on the heat’ shock response and with our data on stringent induction indicat,ed that many of the proteins previously observed to increase during the stringent response were. in fact, heat shock proteins. Thus. the stringent response joins a growing list of “stress” conditions that are known to induce the synthesis of heat shock proteins. IGsides temperature upshift’ and the stringent response, synthesis of heat shock proteins can be induced by ethanol (Neidhardt ct al., 1984; Travers B Mace, 1982), ultraviolet light irradiation (Krueger & Walker, 1984), naladixic acid (Krueger & LI’alker. 1984), coumermycin (Travers & Mace. f 982: Menzel & Cellert,; 1983), and infection with phage lambda (Drahos & Hendrix, 1982: Kochan & Murialdo, 1982). It is not) known how t,hese agents induce heat shock protein synthesis. While the heat shock response and the stringent response both result in increased t)ranscript,ion initiation at heat shock promoters and increased synthesis of heat shock proteins, the requirements for induction are different. relil mutant’s do not accumulate ppGpp after amino acid starvation and are defective in the stringent response and in stringent induction. However, the heal shock response is normal in relil mutants (A. I). Grossman, unpublished results). Tn t,hr course of these studies, we found that the SC122 st,rain that has been used for some of the studies on heat shock (Neidhardt & Van Bogelen, 1981: Neidhardt’ d al.. 1983) has a relaxed phenotype (A. 1). Grossman. unpublished results). When the rpoIl165 nonsense mutation is inefficiently suppressed, resulting in a reduced amount of a32, the mutant is ts for growth (Cooper & Ruettinger, 1975; Tamamori & Yura. 1982), defective in the heat shock response (Neidhardt & Van Bogelen. 1981: Yamamori & Aura, 1982), and defective in proteolysis, even at permissive growth temperature (Baker rt al., f 984). However, the reduced amount of 032 in the mutant did not prevent stringent’ induction (Table 4). Finally, the DNA sequence 5’-C-T-G-C’-C’-A-(‘-(“-(‘-3 located at -44 and -35 base-pairs from the mRNA start of rpoDPhs is required for temperat,ure induct’ion (Taylor et al., 1984) but not for stringent induction. The mechanism by which the synthesis of heat shock proteins is increased during t,he stringent response is clearly different from that during the heat shock response. Because stringent induction occurs in the rpoHl65 mutant, it is possible that 032 is not required for the increased synthesis of heat shock protein during the stringent response. A different o factor could be activated to promote transcription initiation from heat shock promoters under these conditions. We feel that it is unlikely that cr” promotes this transcription. We have been unable to find conditions in vitro in which 0” promotes transcription initiation from heat shock promoters (Cowing et al., 1985). Tn addition, c”
et
al
seemsto inhibit expression of heat shock gerlr’s 111 I*iz~o(A. I>. Grossman Cyr(‘. A. (iross. unpnblished results). However. it remains possible that there is an additional o factor that can prornotfl transcript,ion initiation from heat. shock promoters. Alternatively, a32 may be required for stringent induction. The ryoHZ6;S mut,ant is not dr*void of cr32. Rather. it has a reduced amount of the gem’ product. Stringent induct’ion is not elimirrated in the rpoHl&i mutant; however. the amount of’ induction of some heat, shock proteins and of heat shock gene transcription decreased in the mutant strain (Tables 4 and ;i). These observations arc consistent with a role for a32 In stringent induction. The rpoH765 mutation prevents induction of the heat shock proteins after temperature upshift but has a smaller effect on the increased synt.hrsis of heat shock proteins during the stringent response. If a3* is required for transcript initiation from heat shock promot,ers during stringent induction, then the st,ringent responsemust stimulate transcript ion from t*hesepromoters in a way that is different from the stimulation during the heat shock rc~sponst~. Transcript,ion initiation at heat shock promoters may be regulat’ed. in part,, by a compchtition between 032 and 0” for binding to core R?iA polymerase. or by competition between holoenzymr containing 0 32 (Ea3’) and holoenzyme containing a7’ (Ea”) for binding to heat shock promoters. It is tempting to speculate that during the stringent response, ppclpp (or other small molecules) alters this competition either by inhibiting cr7’ or Ea7’ or by st,imulating u32 or EONS. We are investigating these possibilities. We thank \.lr. IS’alter and Y. N. Zhou for superb technical assistance. I. Slater for typing several drafts of this paper. and B. Sugden, W. Dove and (:. Robrrt,s for their helpful comments on the manuscript. This work was supported by the College of Agricultural and Life Sciences. r’niversity of Wisconsin-Madisotl. grant GM28575 from the Eational Tnstit)utes of Health to
R. Burgessand C. Gross, and grants R01 AI 19635and K04
AI
00573
to (1. Gross. References
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