JOURNAL OF BACTERIOLOGY, June 1978, p. 1133-11400021-9193/78/0134-1133$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 134, No. 3

Printed in U.S.A.

Transient Regulation of Protein Synthesis in Escherichia coliUpon Shift-Up of Growth Temperature

T. YAMAMORI, K. ITO, Y. NAKAMURA,t AND T. YURA*

Institute for Virus Research, Kyoto University, Kyoto 606, Japan

Received for publication 23 February 1978

Synthesis of total cellular proteins ofEscherichia coli was studied upon transferof a log-phase culture from 30 (or 37) to 42°C. Cells were pulse-labeled with[3H]leucine, and the labeled proteins were analyzed by gel electrophoresis in thepresence of sodium dodecyl sulfate. The rates of synthesis of at least five proteinchains were found to increase markedly (5- to 10-fold) within 5 min aftertemperature shift-up and gradually decrease to the new steady-state levels, incontrast to the majority of proteins which gradually increase to the steady-statelevels (about 1.5-fold the rate at 30°C). Temperature shift-down did not causeany appreciable changes in the pattern of protein synthesis as detected by thepresent method. Among the proteins greatly affected by the temperature shift-upwere those with apparent molecular weights of 87,000 (87K), 76K, 73K, 64K, and61K. Two of them (64K and 61K) were found to be precipitated with specificantiserum against proteins that had previously been shown to have an adenosinetriphosphatase activity. The bearings of these findings on bacterial adaptation tovariation in growth temperature are discussed.

Although extensive work has been devoted toelucidate the elementary processes of geneexpression and its regulation in Escherichia coli,relatively little is known about the gross regu-latory mechanisms at the cellular level. To ob-tain an integral picture of gene expression duringcell growth, it would be important to know, forexample, how many proteins are regulated intheir synthesis in response to a change in growthconditions. Changes in synthesis of cellular pro-teins have been studied under conditions ofamino acid limitation ("stringent control") (3,16) or under conditions that give rise to lesionsin DNA (6, 18).

It has been established that the relative con-tent of macromolecules does not change appre-ciably when bacterial cells are grown at differenttemperatures in steady state, despite the widevariation in growth rate (12). However, littleattention has been paid to macromolecular syn-thesis during transitions in growth temperature.The present paper reports some new features ofregulation of E. coli protein synthesis that havebeen revealed by experiments involving transferof a log-phase culture from a low to a hightemperature.

MATERLALS AND METHODSMaterials. Prototrophic E. coli K-12, strain W3350,

was used in most experiments. L-['H]leucine (58

t Present address: Institute of Medical Science, Universityof Tokyo, Tokyo, Japan.

Ci/mmol) and DL-[methylene-'4C]tryptophan (52mCi/mmol) were obtained from the RadiochemicalCentre, Amersham, England. Recrystallized acrylam-ide and sodium dodecyl sulfate (SDS) were purchasedfrom Wako Chemicals Co. (Osaka, Japan) and NakaraiChemicals Co. (Kyoto, Japan), respectively. Brij 58was obtained from Atlas Chemicals, and pancreaticdeoxyribonuclease and ribonuclease were from SigmaChemical Co. (St. Louis, Mo.). Lysozyme and sucrosewere obtained from Seikagaku-Kogyo Co. (Tokyo, Ja-pan) and Schwarz/Mann (Orangeburg, N.Y.), respec-tively.

Pulse-labeling of cells and analysis of proteinby SDS-gel electrophoresis. Cells were grown inmedium E (17) supplemented with 0.5% glucose tomid-log phase (about 2 x 108 cells per ml) and wereshifted to a higher or lower temperature as indicatedfor each experiment. One-milliliter portions of theculture were removed at appropriate intervals, pulse-labeled with 10 ,uCi of L-[3H]leucine for 3 min, andchased with 200 jig of L-leucine and 50 ,ug of L-isoleu-cine per ml for 2 min unless otherwise indicated. Pulse-labeled cells were chilled in ice and mixed with anequal volume of 10% trichloroacetic acid. The precip-itates were collected by centrifugation and washedtwice with cold 5% trichloroacetic acid and once withacetone. The precipitates were dissolved in SDS sam-ple buffer [0.0625 M tris(hydroxymethyl)amino-methane-hydrochloride (pH 6.8)-2% SDS-10% glyc-erol-10 mM dithiothreitol, 0.001% bromophenol blue]and heated at 100°C for 3 min. Electrophoresis wascarried out on 15-cm-long, 1-mm-thick slab gels (5%stacking gel and 11.2% separation gel) using the dis-continuous buffer system of Laemmli (11). Gels werestained with Coomassie brilliant blue. For detection of3H-labeled proteins, fluorographs were taken by ex-

1133

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1134 YAMAMORI ET AL.

posing Fuji X-ray films to the scintillator-treated gelslabs (1).To determine the rates of total protein synthesis

during temperature shift, cells were grown in the pres-ence of DL-["4C]tryptophan (0.1 ,uCi/ml plus 20,ug ofunlabeled L-tryptophan per ml) for several generationsand pulse-labeled at each point with [3H]leucine (1,uCi/ml and 1 pg/ml) for 1 min, followed by mixingwith an equal volume of cold 10% trichloroacetic acid.Samples were filtered through Whatman GF/C glassfiber filters and washed three times with 5% trichlo-roacetic acid and twice with ethanol, and acid-insolu-ble radioactivities were determined by a liquid scintil-lation counter. The ratio of 3H to '4C incorporationwas taken to represent the rate of protein synthesis ateach point.Immunoprecipitation of ATPase. Cells were

treated with lysozyme-ethylenediaminetetraacetateand Brij 58, digested with pancreatic deoxyribonucle-ase and ribonuclease, and then disrupted by sonictreatment. Crude extracts were treated with antiseraagainst adenosine triphosphatase (ATPase), essen-tially as described previously (14). ATPase was pre-pared by following a purification procedure for RNApolymerase and was separated from the latter enzymeby high-salt glycerol gradient centrifugation andDNA-cellulose column chromatography (8). Rabbitantisera were prepared by a published procedure (10).

Fractionation ofcell lysate into membrane andsoluble fractions. Cells obtained from a 20-ml cul-ture were converted to spheroplasts, disrupted soni-cally, and centrifuged through 15% sucrose-3 mM eth-ylenediaminetetraacetate with a cushion of 70% su-crose-3 mM ethylenediaminetetraacetate at 60,000rpm for 30 min in a Spinco 65 rotor, as describedpreviously (9). A membrane fraction was collectedfrom the 15 to 70% sucrose interphase region, and thesoluble fraction was collected from the upper region ofthe 15% sucrose layer (9). The pooled soluble andmembrane fractions were treated with 10% trichloro-acetic acid, and protein precipitates were collected,washed with 5% trichloroacetic acid and acetone, dis-solved in the sample buffer, and subjected to electro-phoresis.

RESULTSEffect oftemperature shift-up on the rate

ofprotein synthesis. When a log-phase cultureof E. coli K-12, strain W3350, was transferredfrom 30 to 420C, the growth rate, as determinedby optical density measurement, increased about1.5-fold (Fig. 1). To examine the rate of proteinsynthesis during temperature shift-up, cells thathad been labeled with [14C]tryptophan werepulse-labeled with [3H]leucine at various timesbefore or after the temperature shift, and theratio of 3H to 14C incorporation was calculated.Since the 14C radioactivity should be propor-tional to protein content (cell mass) of the cul-ture, the ratios thus obtained would representthe rates (per cell mass) ofbulk protein synthesisat the time of pulse-labeling. Figure 2 shows thatthe rate of protein synthesis increases within 30

10080-60-

>1

.6 40*

20

i0

30 °C 42°C

-30 0 30 60Time (min)

FIG. 1. Growth of E. coli cells upon temperatureshift-up. A log-phase culture of strain W3350, grownin medium E containing 0.5% glucose at 30°C, wasdivided into two parts (zero time) and shaken furtherat 30°C (0) or 42°C (0). Growth was monitored bymeasuring turbidity with a Klett-Summerson color-imeter (no. 54 filter).

2.0-

.2 A~~~

Z~~~b 5 /; -----4

& 1.0

0 0 20 30 40 50 60Time(min)

FIG. 2. Effect of temperature shift-up on the rateofprotein synthesis. Cells ofstrain W3350 weregrownin 20 ml of medium E containing 0.5% glucose, L-tryptophan (20 pg/ml), and DL-r4C]tryptophan (0.1,uCi/ml) at 300C, divided into 0.5-ml portions, andshaken further at 420C. At each time before (zerotime) or after the temperature shift, cells were pulse-labeled with 0.5 ,uCi of pHJleucine (1 tiCi/ml and 1pg/ml) for ) min. The labeling was stopped by addingtrichloroacetic acid, and acid-insoluble radioactivi-ties were deterrnined as described in the text. The3H/'4C ratio for each sample was normalized to thatfor the zero-time sample, which was taken as unity.The data from three independent experiments arepresented: (U) experiment 1; (A) experiment 2 (0)experiment 3.

min after the temperature shift and reaches anew steady-state value, which is about 1.5-foldthe rate at 30°C. However, the initial responseof the cells seems to follow more complex kinet-ics; the rate increases markedly, though tran-siently, within a few minutes, decreases some-what, and then increases again to the newsteady-state level. Similar kinetics were ob-

J. BACTERIOL.

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE SHIFT-UP AND PROTEIN SYNTHESIS 1135

tained in several independent experiments, in-cluding those presented in Fig. 2.Transient stimulation of synthesis of spe-

cific proteins upon temperature shift-up. Inthe course of our studies with temperature-sen-sitive mutants of E. coli (20), we noticed that a

temperature shift-up from 30 to 420C causesmarked and transient alterations in the differ-ential synthesis rates of some proteins even in awild-type strain. Thus, we analyzed the proteinssynthesized under these conditions in some de-tail. A culture of strain W3350 was grown inminimal medium at 300C, shifted to 42°C, andpulse-labeled with [3H]leucine at various timesbefore or after the temperature shift. The la-beled cells were directly treated with trichloro-acetic acid, and the proteins precipitated were

analyzed by SDS-gel electrophoresis. Figure 3shows the patterns of 3H-labeled proteins as

obtained by fluorography. It can be seen thatsynthesis of some protein chains relative to thetotal proteins is markedly stimulated shortlyafter the temperature shift-up, reaching themaximum at about 5 min, and then decreasesgradually to a steady-state level comparable tothe preshift level. Proteins affected most underthese conditions are those with apparent molec-ular weights of about 87,000 (87K), 76K, 73K,64K, and 61K. The proteins synthesized aftertemperature shift-up seem to remain stable,since radioactivities associated with these pro-tein bands did not decrease appreciably during"chase" for at least 1 h (data not shown).A rough estimation of the proportion of these

"stimulated proteins" in the total proteins syn-thesized was made from densitometer tracingsof the fluorograms (Table 1). It is clear that theproportion of each of these proteins reaches themaximum (5- to 10-fold stimulation) at about 5min after temperature shift-up, although theextents of stimulation calculated here give onlyapproximations. A temperature shift from 30 to370C (Fig. 3B) or 23 to 34°C (data not shown)gave similar but less striking effects. Tempera-ture shift-down does not seem to cause anyappreciable changes in the pattern of proteinssynthesized (Fig. 4).

Relative rates of synthesis of these and theremaining classes of protein (per cell mass) were

then calculated by multiplying the rate of totalprotein synthesis (Fig. 2) by the proportion (per-centage) each protein class occupies in the totalprotein (Table 1). Figure 5 shows that not onlythe proportion in total protein but also the rel-ative rate (per cell mass) of synthesis increasesabruptly but transiently for those five proteinchains, whereas the synthesis rates of other pro-teins increase gradually during about 30 min

after temperature shift to reach the new steady-state level.

Intracellular localization of the stimu-lated proteins. In an attempt to localize theaffected proteins to the soluble or membranefraction, lysates of labeled cells were fraction-ated by the method of Ito et al. (9). Figure 6shows the protein patterns of both soluble andmembrane fractions as analyzed by SDS-gelelectrophoresis. It is apparent that all species ofstimulated proteins are found mainly in the sol-uble fraction, although some 64K and/or 61Kproteins are also found in the membrane frac-tion. The amount of the latter proteins in themembrane fraction was estimated to be less thanabout 10% of that found in the soluble fraction.In this particular experiment, two pairs of closelylocated bands (76K/73K and 64K/61K) werenot well separated from each other, presumablydue to some minor differences in electrophoreticconditions. It may also be noted that synthesisof a protein found in the membrane fraction,having a molecular weight slighlty higher thanthat of outer membrane protein I, is appreciablystimulated after temperature shift-up. However,increased synthesis of this protein seems to per-sist at 420C, and it does not belong to the "tran-siently stimulated" class of proteins (Fig. 3).

Identification of the 64K and 61K poly-peptides as subunits of an ATPase. In ourprevious studies on the synthesis of DNA-de-pendent RNA polymerase (13), it was noted thatthe synthesis of a protein that copurifies withthe polymerase and has an ATPase activity(7, 8) is stimulated markedly by temperatureshift-up, just as observed here. We thus exam-ined whether the ATPase protein can be foundamong the proteins shown to be subject to tran-sient stimulation by the temperature shift.Crude extracts of pulse-labeled cells weretreated with antiserum against ATPase, and theresulting precipitates and supernatants weresubjected to electrophoresis on SDS-gels. A pairof bands with molecular weights of 64K and 61Kwas clearly seen in the electrofluorogram ob-tained with antigen-antibody precipitates (Fig.7). In contrast, the supernatant contained littleproteins corresponding to the two bands (datanot shown), indicating that most of these pro-teins had been specifically removed by the an-tiserum. This pair of protein bands exactly co-incides in electrophoretic mobiliy with two ofthe protein chains whose synthesis is stimulatedby the temperature shift.

Figure 7 also shows that radioactivity in theseATPase bands increases upon temperature shift-up, although this experiment permits only roughquantitative comparisons because no correction

VOL. 134, 1978

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1136 YAMAMORI ET AL.

A B1 2 3 4 5 6

_M_014W- -

2 3 4 5 6

m *1 -ow,..

- 94

-68-.60

U._1

*__IS

a

i-

I

4m

-

FIG. 3. Effect of temperature shift-up on the composition of newly synthesized proteins. A culture of strainW3350 was grown in medium E at 30°C to about 2 x 108 cells per ml, divided into two portions, and shiftedto 37 or 42°C. One-milliliterportions were removed before (zero time) or after temperature shift, pulse-labeledwith 10 itCi of fHJleucine, chased with unlabeled leucine and isoleucine, and treated with trichloroaceticacid, and the whole protein mixtures were analyzed by SDS-gel electrophoresis followed by fluorography, asdescribed in the text. Radioactivities applied to the gels were 1.0 x 105 cpm for each column. Films wereexposed for 4 days. (1) zero time (30°C); (2) 5 min; (3) 10 min; (4) 20 min; (5) 30 min; and (6) 60 min after theshift. (A) Shift from 30 to 42°C; (B) shift from 30 to 37°C. The positions of molecular weight markers (94K,68K, and 60K; see reference 9), as well as of the protein chains affected by the temperature shift (87K, 76K,73K, 64K, and 61K), are indicated.

for recovery during antiserum treatment and gel beled ATPase was used as an internal referenceelectrophoresis has been made. It was possible (see 13); an approximately 10-fold stimulation ofto obtain a more quantitative estimate from our the ATPase synthesis was observed at 10 minprevious experiments in which purified 35S-la- after temperature shift-up (13). Although the gel

J. BACTERIOL.

I

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE SHIFT-UP AND PROTEIN SYNTHESIS 1137

TABLE 1. Change in proportion of each proteinrelative to total protein synthesized upon

temperature shift-up'Change in proportion of protein (%) at time

Protein class (min) after shift

0 5 10 20 30 60

87K 1.3 4.1 1.9 1.6 1.7 1.076K 0.76 4.0 1.9 1.8 1.5 1.473K 0.76 4.0 1.9 2.1 1.7 1.164K 0.47 4.5 2.5 1.8 1.7 1.461K 0.47 4.5 2.5 1.7 1.4 1.187K + 76K + 3.76 21.1 10.7 9.0 8.0 6.073K + 64K+ 61K

a Proportion of each protein class among total protein syn-thesized at various times after temperature shift (30 to 42°C)was estimated from densitometer tracings (Joyce-Loebl den-sitometer MKIII) of the fluorograms presented in Fig. 3. Eachpeak cut out from a tracing paper was weighed, and the weightwas normalized to that of total protein. Since the bands ofthese proteins labeled at 30°C were rather faint and could noteasily be distinguished from other adjacent bands, the valuesat zero time may have been overestimated.

electrophoresis system used in the previousstudies gave a single protein band ofthis ATPase(7, 8, 13), Laemxnli's system, used here, usuallyresolved two protein species with a purifiedATPase preparation (data not shown) and withthe antibody precipitates (Fig. 7). Thus, we con-clude that among the five protein chains stimu-lated transiently by the temperature shift-up,two (64K and 61K) represent subunits of theATPase.

DISCUSSIONThe present study revealed some new features

of regulation of protein synthesis that may bespecifically associated with temperature shift-upin E. coli. When a culture was transferred from30 to 42°C, the rate of bulk protein synthesisappeared to increase to the steady-state levelcharacteristic of the latter temperature within20 to 30 min, exhibiting apparently complexkinetics. The differential synthesis rates of atleast five species of protein chains were found toincrease 5- to 10-fold within 5 min and thendecrease, whereas the rates of synthesis of otherproteins increased gradually only by about50%. The molecular weights of these proteinchains have been estimated to be 87K, 76K, 73K,64K, and 61K on the basis of their mobilities inSDS-gel electrophoresis. Cooper and Ruettinger(4) previously reported that a protein with amolecular weight of 58K is specifically producedat high temperature (42°C); they have detectedthis protein at 15 min after temperature shift-upbut did not follow the time course of its synthe-sis. This protein may correspond to one of theproteins whose synthesis is transiently stimu-

lated. Although West and Emmerson (18) ob-served similar temperature-induced changes inprotein patterns using some mutants of E. coliaffected in cell division, comparable results fora wild-type strain were not presented.Each of the five species of protein chains

seems to exist as soluble protein, although smallportions of 64K/61K polypeptides (ATPase)may be associated with the membrane as well.The latter observation, however, might simply

1 2 3 4 5 612 45

- ur*do Al.1 ,

i'I6i,"-wl 4

FIG. 4. Effect of temperature shift-down on thecomposition of newly synthesized proteins. Experi-mental procedures are the same as in Fig. 3, exceptthat the temperature shift was from 42 to 300C. (1)zero time (42°C); (2) 5 min; (3) 10 nfin; (4) 20 min; (5)30 min; and (6) 60 min after the shift.

VOL. 134, 1978

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1138 YAMAMORI ET AL.

0

0

0 10 20 30 40 60

Time (min)

FIG. 5. Time course ofsynthesis ofstimulatedpro-teins and ofotherproteins upon temperature shift-up.Relative rates (per cell mass unit) of synthesis ofstimulated proteins (sum of the five classes of pro-teins) and oftotalprotein were estimated on the basisof the values presented in Table 1 and Fig. 2 (aver-ages from the three experiments were used). Valuesarepresented as rates relative to that oftotalproteinsynthesis at 300C (zero time), set as 100. Symbols:(5) total protein; (0) stimulated proteins; (0) otherproteins.

reflect the fact that the ATPase has a relativelyhigh molecular weight (9.3 x 105) in its nativeform (7). The proteins or polypeptides beingaffected are not likely to be the components oftranscriptional or translational machinery; RNApolymerase subunits a, /B, and ,B' are not stimu-lated markedly by the temperature shift-up (see13). The molecular weights of all the ribosomalproteins except Si (65,000 in SDS-gels) are lowerthan 60K (19).The 64K and 61K proteins detected here rep-

resent subunits of ATPase whose physiologicalrole is not yet known (7, 8). The differentialsynthesis rate ofthis enzyme protein approachedabout 10% of the total protein synthesizedshortly after temperature shift-up, in goodagreement with previous data (13). The presentsystem of gel electrophoresis resolved two pro-tein bands of the ATPase, suggesting that thisenzyme is composed of two different subunitspecies whose synthesis is coordinately regulatedupon temperature shift-up.

It was reported recently that temperature

FIG. 6. Localization of the stimulated proteins.Cells were grown in 40 ml of medium E-glucose at30°C, and a log-phase culture was divided into twoparts; onepart waspulse-labeled at 30°C (columns 1,3, and 4) with 50 ,uCi of [3HJleucine for 3 min andchased with unlabeled leucine (200,ug/ml) and isoleu-

-- f

UW

cine (50 jg/ml) for 2 mi, whereas the other wasshifted to 420C and pulse-labeled after 5 min ofincubation at 420C (columns 2, 5, and 6). The solubleand membrane fractions were prepared as describedin the text, and proteins were solubilized with SDSand applied for SDS-gel electrophoresis. Fluoro-grams were taken after 3 days of exposure. Radioac-tivities of i0' cpm were applied to each column. (1and 2) Total protein; (3 and 5) soluble fraction; (4and 6) membrane fr-action. In the present gel system,the major outer membrane protein I can be seen atthe position of molecular weight = 37K.

J. BACTrERIOL.

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE SHIFT-UP AND PROTEIN SYNTHESIS 1139

1 2 3 4 5 6

-_

64K _61 K-

-i

"Pt.

l--

FIG. 7. Precipitation of 64K and 6lKpolypeptidesby specific antiserum against ATPase. A 5-ml cultureof W3350 was pulse-labeled at 30°C (1, 2, and 3) or 5min after shift to 42°C (4, 5, and 6) with 50 iCi of 3H]leucine and chased with unlabeled leucine-isoleucineas described in the text. Crude extracts were pre-pared, and a portion (2 x 10 cpm) was treated withantiserum against ATPase; the resultingprecipitateswere dissolved in 50 ,ul of SDS sample buffer andanalyzed by SDS-gel electrophoresis. Fluorogramswere taken after 4 days of exposure. (2 and 5) Crudeextract (105 cpm each); (1, 3, 4, and 6) antigen-anti-body precipitates (1, 1,055 cpm in 5 yl; 3, 4,220 cpm in20 ,l; 4, 2,687 cpm in 5 LI; 6, 10,750 cpm in 20 Il).

shift-up of E. coli cells leads to transient accu-mulation of guanosine tetraphosphate, a com-pound implicated as a key substance for thestringent control system (2, 5). Thus, guanosinetetraphosphate may have a role in the temper-ature-induced regulation of protein synthesis.However, starvation for amino acids, duringwhich this nucleotide accumulates, appears toinduce synthesis of proteins apparently differentfrom those reported here (3, 16).The sum of the five (or more, if any of the

protein bands is a mixture of more than onepolypeptide) species of protein chains that aresubject to transient stimulation amounts to asmuch as about 20% of the total protein synthe-sized, and the stimulation ofthese protein chainsseems to be well coordinated. Such a remarkableresponse of bacterial cells to the temperatureshift-up may be significant in considering bac-terial adaptation to variation in growth temper-ature. The present finding that a large propor-tion of such protein is associated with an in-triguing enzyme having ATPase activity mayprovide an important clue for further analysis ofsuch adaptation mechanisms. The present ob-servation at least suggests the existence of anovel regulatory mechanism for gene expressionin E. coli, namely, transient stimulation of spe-cific protein synthesis in response to a suddenchange in growth temperature.

Finally, the results presented in this papershould be borne in mind in future studies ofbacterial mutants, particularly in analysis of pro-tein synthesis with temperature-sensitive mu-tants.

ACKNOWLEDGMENTSWe are grateful to A. Ishihama for critical reading of the

manuscript and to J. Asano and A. Komori for excellenttechnical assistance.

LITERATURE CITED

1. Bonner, W. M., and R. A. Laskey. 1974. A film detectionmethod for tritium labelled proteins and nucleic acid inpolyacrylamide gels. Eur. J. Biochem. 46:83-88.

2. Chaloner-Larsson, G., and H. Yamazaki. 1977. Ad-justment of RNA content during temperature upshiftin Escherichia coli. Biochem. Biophys. Res. Commun.77:503-508.

3. Chao, L. 1977. Gene expression in stringent and relaxedstrains of Escherichia coli during amino acid depriva-tion. Biochem. Biophys. Res. Commun. 77:42-49.

4. Cooper, S., and T. Ruettinger. 1975. Temperature de-pendent alteration in bacterial protein composition.Biochem. Biophys. Res. Commun. 62:584-586.

5. Gallant, J., L. Palmer, and C. C. Pao. 1977. Anomaloussynthesis of ppGpp in growing cells. Cell 11:181-185.

6. Gudas, L. J., and A. B. Pardee. 1976. DNA synthesisinhibition and the induction of protein X in Escherichiacoli. J. Mol. Biol. 101:459-478.

7. Ishihama, A., T. Ikeuchi, A. Matsumoto, and S. Ya-mamoto. 1976. A novel adenosine triphosphatase iso-lated from RNA polymerase preparation of Escherichiacoli. II. Enzymatic properties and molecular structure.

VOL. 134, 1978

I

I

I

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1140 YAMAMORI ET AL.

J. Biochem. 79:927-936.8. Ishihama, A., T. Ikeuchi, and T. Yura. 1976. A novel

adenosine triphosphatase isolated from RNA polymer-ase preparation of Escherichia coli. I. Copurificationand separation. J. Biochem. 79:917-925.

9. Ito, K., T. Sato, and T. Yura. 1977. Synthesis andassembly of the membrane proteins in E. coli. Cell11:551-559.

10. Iwakura, Y., K. Ito, and A. Ishihama. 1974. Biosyn-thesis of RNA polymerase in Escherichia coli. I. Con-trol ofRNA polymerase content at various growth rates.Mol. Gen. Genet. 133:1-23.

11. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

12. Maal0e, O., and N. 0. Kjeldgaard. 1966. Control ofmacromolecular synthesis. W. A. Benjamin, Inc., NewYork.

13. Nakamura, Y., T. Ikeuchi, M. Imai, and T. Yura. 1977.Escape synthesis of RNA polymerase subunits and ter-mination factor rho following induction of prophagelambda in Escherichia coli. Mol. Gen. Genet.150:317-324.

14. Nakamura, Y., and T. Yura. 1975. Evidence for a posi-tive regulation of RNA polymerase synthesis in Esch-

erichia coli. J. Mol. Biol. 97:621-642.15. Nakamura, Y., and T. Yura. 1975. Hyperproduction of

the sigma subunit of RNA polymerase in a mutant ofEscherichia coli. Mol. Gen. Genet. 141:97-111.

16. Reeh, S., S. Pedersen, and J. D. Friesen. 1976. Biosyn-thetic regulation of individual proteins in relA+ andrelA strains of Escherichia coli during amino acid star-vation. Mol. Gen. Genet. 149:279-289.

17. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinaseof Escherichia coli; partial purification and some prop-erties. J. Biol. Chem. 218:97-106.

18. West, S. C., and D. T. Emmerson. 1977. Induction ofprotein synthesis in Escherichia coli following UV or-y-irradiation, Mitomycin C treatment or tif expression.Mol. Gen. Genet. 151:57-67.

19. Wittman, H. G. 1974. Purification and identification ofEscherichia coli ribosomal proteins, p. 93-114. In M.Nomura, A. Tissieres, and R. Lengyel (ed.), Ribosomes.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

20. Yamamori, T., K. Ito., T. Yura, T. Suzuki, and T.lino. 1977. A ribonucleic acid polymerase mutant ofEscherichia coli defective in flagella formation. J. Bac-teriol. 132:254-261.

J. BACTERIOL.

on June 19, 2018 by guesthttp://jb.asm

.org/D

ownloaded from