Synthesis of functional bacteriophage T4-delayed early mRNA in the absence of protein synthesis.

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Copyright( 1975 AmericanSocietyforMicrobiology Printedin U.S.A.

Synthesis

of

Functional

Bacteriophage T4-Delayed

Early

mRNA

in

the

Absence of Protein Synthesis

JEFFREY W. MORSE' ANDPAUL S. COHEN*

University of Rhode Island, Department ofMicrobiologyand Biophysics,

Kingston,

Rhode Island 02881

Received forpublication4February 1975

When Escherichia coli B207 is grown either

aerobically

or under limited aerobic conditions, pretreatedwith chloramphenicol to block protein synthesis,

and then infected with bacteriophage T4, the phage RNA whichaccumulates, termed "immediate early" (IE), contains thetranscripts ofalimited number of prereplicative genes. Among the transcripts which accumulate is the mRNA which serves as a template for deoxycytidylate

hydroxymethylase

(HMase) synthesis. Among the prereplicative gene transcripts which do not accumulate under these conditions are deoxycytidine triphosphatase

(dCTPase),

a-glucosyl

transferase (a-gt), and deoxynucleotide kinase (kinase); these genes have been termed"delayed early" (DE). In contrast, whenproteinsynthesis isinhibited by

depleting

aerobically

grown E. coli B207 of

K+,

both IE and DE T4 RNA

accumulate, but these transcripts donot contain functional HMase, dCTPase, a-gt, orkinase mRNA's. However, if E. coli is grown underconditions of limited aeration and then depleted of K+ prior to T4 infection, the T4 RNA which accumulates contains both IE and DE transcripts and functional HMase,

dCTPase, and a-gt mRNA's. Functional kinase mRNA does not accumulate under these conditions. The results of these experiments indicate that the

synthesis of functional DE RNA in the absence of simultaneous protein synthesis, depends on the

physiological

condition of the cells and the way in

which protein synthesis is inhibited. In addition, data is presented which

suggests that extensive transcription of DE genes in the absence of protein

synthesis results in the inhibition oftranscription ofcertain IE genes.

Several laboratorieshave

reported

the results

ofexperiments whichshowthatalimited num-ber of T4 early genes, termed "immediate

early" (IE) genes, are transcribed in cells

infected in the presence of chloramphenicol (CM) (4, 14, 19, 28, 30, 35). In contrast, "de-layed early" (DE) genes are expressed shortly

after normal infection, butare not transcribed

inthe presence ofCM (4, 14, 19, 28, 30, 35). Recentlyit has been shown thatthree groups

ofIE genes canbe distinguished on thebasisof

the time at whichthey stop being expressed in vivo. GroupA genes are transcribed during the firsthalf of the early period, group B during the

entireearly period, and group Cthroughout the entire latent period (H. J. Witmer, In Progress inMolecular andSubcellularBiology,inpress).

There also are at least two groups of DE genes. The first group,perhapsrepresentativeof

the majority of DE genes, appears to be tran-scribed as extension products ofIE genes and

'Present address: University of Connecticut Health

Center, Department of Biochemistry, Farmington, Conn. 06032.

330

would,therefore,initiate at IE promotors (2, 23, 24). Transcription of this group of genes does

not take place in the presence of CM in vivo, but does occur in infected cells pretreated with the amino acid analogue 5-methyl tryptophan (14, 19), starved for a required amino acid (A.

Baros, and H. J. Witmer, Arch. Biochem.

Biophys., in press), or allowed brief periods of normal RNA and protein synthesis (8, 25, 26). Treatment with amino acid analogues,

starva-tion for arequiredaminoacid,orbrief periods of normalRNA and protein synthesis, which allow small but significant amounts of protein

synthe-sis(19, 25, 26), suggest that transcription of this group of DE genes either requires ribosome movement along a large portion of the RNA transcripts distal to the IE portions or that smallamountsofspecific protein(s) are synthe-sized immediately after infection which some-how prime these DE genes for transcription. The second group of DE genes (sometimes referredtoas"quasilate" genes) requires signif-icant amounts of T4 RNA and protein for

transcription, presumablyforthe synthesisof a

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mRNA SYNTHESIS

phage-specific proteinnecessary during the ini-tiation step (8, 25, 26).

In the present investigation, we present

evi-dence which shows that DE genes which are formed as extensions of IE transcripts can be synthesized in the complete absence of protein synthesis invivo. However, the transcription of thesegenesdependsonthephysiological condi-tion ofthe cells both during growth and infec-tion and onthe way in which protein synthesis is inhibited.

MATERIALS AND METHODS

Bacteria and bacteriophage. Escherichia coli B207, a mutant of E. coli B which cannot

concen-trate and retain K+ normally from the growth

medium (21), wasused in this study. Bacteriophage

T4amBL292 (gene 55),amaturation-defectivemutant ofT4, synthesizes exclusivelyIE and DE T4 mRNA inE. coli B (3, 34). T4RNA isolated from amBL292-infected E. coli Bwasused inDNA-RNA

hybridiza-tion experiments. E. coli PA 505MSI, kindly pro-vided by J. M. Buchanan, requires diaminopimelic

acid for growth and was used for preparation ofthe lysozyme assay substrate. As in all our previous work, wild-type bacteriophage T4 B was used in all other experiments.

Mediumandgrowthconditions for E. coli B207. The growth medium employed in these studies has been describedpreviously(5,7). Two differentgrowth conditionswereusedinthe present investigation.

(i)Aerobic growth. E. coli B207wasgrownat37C with vigorousshaking inaNewBrunswick G76water bath shakeratvolumeswhichwere nomorethan 10% ofthe growth flaskcapacity. Under these conditions the massdoublingtimewas36 to 42min.

(ii) Limited aerobic growth. Fifty-milliliter

cul-turesofE. coli B207weregrownin250-mlErlenmeyer

flasks at 37C without shaking and were aerated by bubblingairthrough10-ml pipettesintothe cultures

at a constant pressure of 4

lbs/inch'.

Under these

conditions the mass doubling time ofE. coli B207 was between 50 and 60 min, but the kinetics of phage production and the final phage yield did not differ significantly from those observed during T4 infection ofaerobicallygrowncells.

Inhibition ofproteinsynthesis.Proteinsynthesis

wasinhibitedbyeithertreating E. coli B207 withCM

(100

gg/ml

finalconcentration) ordepleting B207 of K+ by washing in K+-free medium as described previously (7, 28). In all the experiments reported

here, CM treatment or K+ depletion inhibited the

rate of protein synthesis by greater than 99%, as

measured by incorporation of

[4C]Ileucine

into

pro-tein (7).

Preparation of DNA and RNA for DNA-RNA hybridization experiments. T4 DNAwas prepared

bythe methodofKaiser andHogness(17).Toprepare

(0 to 12 min) labeled T4 amBL292 RNA (30C),

[6-3H]uracil (0.5

MCi/ml,

0.25

gg/ml),

was added at

thetimeofinfection.At 12min, cells werecollected by centrifugation (2,600 x g) after pouring over an

equal volume of ice. Both radioactive T4 amBL292 RNA andunlabeled competitor RNAs (see text) were extractedby the method of Lembach and Buchanan (19) and were completely alkali and RNase sensitive.

DNA-RNA hybridization. These experiments were performed as previously described (28). Filters

containing5,gofphageDNA were incubated at 37 C in the presence of 2 ug of 'H-labeled T4 amBL292 RNA inatotalvolume of 0.5 ml. Competing nonradi-oactive RNA was added at the indicated concentra-tion at the same time as the radioactive RNA. All experimental points were carried out in triplicate to

ensurethevalidity of these experiments.

Infection and preparation of infected cell ex-tracts.Infection wascarried out ata multiplicity of 5 phage/cell as described previously (7). Under these conditionsgreater than 98% of the cells were infected within 5 min.

Infected cells (50 ml) were collected at the times indicated in the figures, and extracts were prepared as describedpreviously (6).

Enzyme assays. Deoxycytidylate hydroxymeth-ylase(HMase) was assayed by the method of Wiberg andBuchanan (36).Deoxynucleotide kinase (kinase) wasassayed asthymidylate kinase by themethod of Wiberg et al. (37). Deoxycytidine triphosphatase (dCTPase) and a-glucosyl transferase (a-gt) were assayed aspreviously described (8) andlysozymewas assayed by the method of Leutgeb as described by

Schweiger and Gold (31).

Chemicals. [6'HHluracil, L-[1- 1"C ]leucine, [C-formaldehyde, [2- 4C]thymidine 5'-monophosphate, [2-14C deoxycytidine 5'-triphosphate, and uridine diphosphate [U- "4CJglucose, were purchased from NewEngland NuclearCorp. [2,6-'H]diaminopimelic acid was purchased from Amersham/Searle Corp. Tetrahydrofolic acid (gradeIII), ATP,dCTP,UDPG, and 2-mercaptoethanol were purchased from Sigma. Rifampin and egg white lysozyme were purchased from Calbiochem and chloramphenicol was a gift fromParke-Davis andCo.

RESULTS

Hybridization properties of phage RNA

made in the absence of protein synthesis. In the following experiments, we determined

whether T4 mRNA made in K+-depleted cells contained exclusively IE RNA or both IE and DE RNA. As in previous work (28), we took

advantage of the finding that T4 amBL292, a

maturation-defective mutant, makes allspecies ofIE andDE RNA but no late RNA whengrown

on the nonpermissive host, E. coli B (3, 34). Nonradioactive RNAsmadeduringinfectionfor 20 min in the presence of CM (20-min CM-RNA) and RNA made during either a5-minor

20-min infection of K+-depleted cells (5-min

KD-RNA and 20-min KD-RNA, respectively) were individually competed against RNA

iso-lated from T4 amBL292-infected E. coli B207 labeled with [3H

]uracil

from 0 to 12 min after infection

[(0-12)-amRNA].

These experiments 331

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were performed underfourdifferent conditions

of growth and infectionofcells.

(i)Aerobicgrowth-aerobicinfection.Only about one-half ofthe (0-12)-amRNA was

com-peted out by 5-min KD-RNA. That this RNA contains all the IE species of RNA is shown by the facts that the competitive power of 5-min KD-RNA was almost identical to 20-min CM-RNA and that when all IE sites on T4 DNA were blocked by 20-min CM-RNA, the level of competition was not increased by the addition of 5-min KD-RNA(Fig. IA).

In contrast, approximately 65% ofthe (0-12)-amRNAwascompetedoutby20-minKD-RNA

andblockage ofIE sites with 20-min CM-RNA did not alter the competitive power of 20-min KD-RNA. Therefore, 20-minK-RNAcontained all IE species of RNA andabout 40% oftheDE species of RNA (see legend to Table 1 for

calculation methods).

(ii) Limited aeration growth and limited aeration infection. Under these conditions competition of(0-12)-amRNAbyKD-RNAwas

greaterthan 50% (Fig. 1B). However, therewas a tendency for KD-RNA to compete out less

(0-12)-amRNA when used alone than when IE sites were blocked by the presence of 20-min

CM-RNA indicating the presence ofsome DE species ofRNAand the lackofsomeIE species of RNA. Specifically, 5-min KD-RNA con-tained approximately 90% of the IE species of RNA and 30% ofthe DE species ofRNA. The

20-min KD-RNA containedabout80% ofthe IE

species of RNA and 60% of the DE species of

RNA.

(iii) Aerobic growth and limited aeration

infection. Under these conditions, 5-min KD-RNA contained approximately 50% of the IE species of RNA and 40% of the DE species of RNA(Fig. 1C).The 20-minKD-RNA contained

about 75% ofthe IE species of RNA and 55% of

the DE species ofRNA.

(iv) Limited aeration growth and aerobic

infection. Under these conditions, 5-min KD-RNA contained approximately 60% of the IE RNA species and 40% of the DE RNA species (Fig. 1D). The20-min KD-RNA contained about

40% ofthe IE RNA species and 85% of the DE species of RNA.

These data are summarized in Table 1.

Accumulation of functional mRNA in the

absenceofprotein synthesis. Several laborato-ries have shown that IE RNA accumulated in vivo in thepresence of CM can serve as mRNA

for the synthesis of HMase, but not for the

synthesis ofdCTPase, a-gt, or deoxynucleotide kinase (kinase) (19, 28, 29, 35, 39).

Since all of these enzymes are synthesized

shortly after infection, these results suggested

that HMase mRNA is an IE species, whereas the mRNA's necessary for dCTPase, a-gt, and kinase synthesis are members of the DE class. To determine whether the T4 RNA that

accu-mulates in the absence of K+ contained func-tional IE and DE mRNA, the following

experi-ment was performed.

E. coliB207wasinfected with T4 either in the absence of K+orin thepresence of CM. Either 5

or 20 min later, rifampin was added to block further initiation of RNA transcripts (33) and 5 min later CMwas washedout orK+ wasadded back to allow protein synthesis to resume.

Twenty minutes later the cells were collected and assayed for the early enzymes HMase, dCTPase, kinase, a-gt, and the late enzyme, lysozyme. Again, these experiments were per-formed under four different conditions ofgrowth and infection.

(i) Aerobic growth-aerobic infection. As reported previously (19, 28, 35), of the enzymes tested, only HMase mRNA accumulates in T4-infected cells pretreated with CM.However, in K+-depleted cells, although all IE and as

muchas40% of the DE mRNAspeciesappeared to be transcribed (Fig. 1A, Table 1), they did

not direct the synthesis of any of the enzymes

wetested (Table 2).

(ii) Limited aerationgrowth-limited aer-ation infection. Again, of the enzymestested, only HMase and mRNA accumulated in CM pretreated cells. However, in K+-depletedcells, not only did the HMase mRNA accumulate (IE), but dCTPasemRNA (DE), and toalesser extent a-gt (DE) mRNA, were transcribed. Neither kinase mRNA nor lysozyme mRNA appeared to accumulate under these conditions (Table 3). These results areconsistent with the fact that almost all the IE and as muchas 60% of the DE mRNA species accumulate under theseconditions (Fig. 1B, Table1).

(iii) Aerobic growth-limited aeration in-fection. Under these conditions, very little functional HMase mRNA accumulated in CM-pretreated cells, but this mRNA was again the only one of those tested which was detectable. However, in K+-depleted cells, significant

amounts of HMase mRNA (IE) and dCTPase mRNA(DE) accumulated andtoalesser extent a-gt mRNA (DE) accumulated. Neither kinase mRNA nor lysozyme mRNA were detectable

(Table 4). These results are consistent with the DNA-RNA hybridization data (Fig. 1C, Table 1) which show that most of the IE mRNA's and as much as 60% of the DE mRNA's accumulateinK+-depleted cells under these conditions.

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mRNASYNTHESIS IN T4-INFECTED CELLS

_100

I

< 75

z

I

to

- 50

c

25 0

Ratio Unlabeled/ Labeled RNA

I

z

I

E >

x

0 50 100 150 200

Rotio Unlabeled/Labeled RNA

I

z

c-0

z

0

E

a

0 50 100 150 200

Ratio Unlabeled/ Labeled RNA Ratio Unlabeled/Labeled RNA

FIG. 1. Hybridization properties of T4 RNA accumulated in K-depleted E. coli B207. Radioactive IE and DE

T4 RNA was isolated after infecting 50-ml cultures of E. coli B207 for 12minat 30 C with T4 amBL292

(multiplicity of infection of 5) in the presence of [6-3H]uracil (0.5 ,uCi/ml, 0.25 ug/ml). This RNA was

designated 3H-labeled (0-12)-amRNA. Competing RNAwasprepared asfollows: 20-minCM-RNA: CM (100

i.g/ml) wasadded 5minbefore infection, the cellswereinfectedwithwild-typeT4, and 20 min later the cells were collected and the RNA waspurified aspreviously described (28). 5-min KD-RNA: E. coli B207was

depleted ofK+5minbefore infection and infected, and 5minlater the cellswerecollected and the RNAwas

purified. 20-min KD-RNA: as5-minKD-RNA exceptthatinfectionwasallowedtoproceed for20minofK+

depletion before collecting the cells and purifying the RNA. Filters containing5 jigof denatured T4 DNAwere

incubatedinthepresenceof2ggof 3H-labeled (0-12)-amRNAat64 Cinatotal volumeof 0.5mlasdescribed

previously (28). Competing nonradioactive RNA wasaddedatthe indicatedconcentrationsatthesametimeas

the radioactive RNA. When20-min CM-RNAwasusedtoblock all IE sites in mixedcompetitor experiments,it

was used at the saturating RNA/DNA ratio of 200 and designated 20-min CMS-RNA. The competing

nonradioactiveRNAswere:0,5-minKD-RNA;0,20-min KD-RNA; V, 20-min CM-RNA;*, 5-minKD-RNA

+20-min CMS-RNA;E, 20-minKD-RNA +20-min CMS-RNA; A,nonradioactive(0-12)-amRNA.Cellswere

growneitheraerobicallyorwith limited aeration(seeMaterials andMethods)andinfectedinthe absenceofK+ eitheraerobicallyorwithlimited aerationin thefollowingcombinations:(A)aerobicgrowth-aerobic infection;

(B) limited aerationgrowth-limited aeration infection; (C)aerobicgrowth-limited aerationinfection; (D)

limited aerationgrowth-aerobic infection.In the absenceof competing RNA, about 15%oftheradioactivity

washybridized toT4DNA (2,800 counts/min).

333

'It

z

E

-'I

cr

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TABLE 1. IEand DE RNAcontentof T4-infected K+-depletedcells: effect of culture conditions

% of % of Data

sum-Conditionsof: total total

Dataisum

T TypeofRNA IEtran- DEtran-

marized

Growth Infection scriptsa scriptsa from:

Aerobic Aerobic 5-min KD 100 0 Fig. 1A

20-min KD 100 40

Limitedaer- Limitedaer- 5-minKD 90 30 Fig. 1B

ation ation 20-min KD 80 60

Aeration Limitedaer- 5-minKD 50 40 Fig. 1C

ation 20-min KD 75 55

Limited aer- Aerobic 5-minKD 60 40 Fig.1D

ation 20-minKD 40 85

aTotal IE transcripts, designated CM, are defined as the percent of 3H-labeled (0-12)-am transcripts competedoutof T4DNA-RNAhybridsby20-minCM-RNAat aratioof unlabeledtolabeled RNA of 200.Total DEtranscripts, designated (0-12)-am - CM, aredefinedasthepercentage of 3H-labeled (0-12)-amtranscripts

competedout ofT4 DNA-RNAhybrids by(0-12)-am RNAat aratio ofunlabeledtolabeled RNA of 200(i.e.,

total IE and DEtranscripts)minustotal IEtranscripts(CM). Total IE and DE transcripts presentinKD-RNA, designated KD, aredefined asthe percentage of 3H-labeled (0-12)-amtranscripts competed out of T4 DNA-RNA hybrids by KD-RNA at a ratio ofunlabeled to labeled RNA of 200. Total DE transcripts present in KD-RNA, designated (KD + CM) - CM, aredefined asthe percentage of 3H-labeled (0-12)-amtranscripts

competed out of T4DNA-RNAhybrids byKD-RNA whenIE sitesinT4DNAarefilledby20-minCM-RNA transcripts, (KD+CM), minustotal IE transcripts(CM). Accordingtothese definitions:%of the totalIE

tran-scripts present inKD-RNA = gKD - [(KD + CM) - CM]I/CM x 100; %of total DEtranscripts present in KD-RNA= [(KD+CM) -CM

I/[0-12)-am

-CM] x 100.

TABLE 2. Synthesisofprereplicativeenzymesduring recovery from CMtreatmentandpotassium depletion in aerobic cellsa

Minutes after infection Enzyme activityb

Cul-tr5 0 i-S +10 ±20 +25 +30 ±40 +45 HMase

dT-a-gt

Ki Lyo

ase nase zvme

1 CM Infect Harvest 0 0 0 0 0

2 CM Infect Wash Harvest 1,397 5,143 15,500 1,132 13,811

3 CM Infect +Rif Wash 295 167 0 0 0

+Rif Harvest

4 CM Infect +Rif Wash Harvest 371 0 0 58 17

+Rif

5 KD Infect Harvest 0 0 0 0 0

6 KD Infect +K+ Harvest 2,098 4,060 10,052 3,918 5,921

7 KD Infect +Rif +K+ Harvest 50 45 0 250 0

8 KD Infect Rif +K+ Harvest 15 145 0 148 0

aFour 50-ml cultures ofaerobically grownE. coli B207 wereincubatedfor 5minin thepresenceof CM (100

,ug/ml)

andthen infected under areobicconditionsat amultiplicityof 5.After20min furtherincubation, culture 1 was harvested. Another culture (2) was washed free ofCMat 20 min andincubated foranadditional20minin growth mediumlackingCM andthen harvested. Rifampicin(Rif), at aconcentration of 200gg/ml,wasaddedtoculture3, 5min after infection.Theculturewas thenwashed free ofCMat10minandincubatedfor anadditional20min infresh growth medium lackingCM butcontaining Rif.Rifwasaddedtoculture4, 20 minafter infection.The culturewasthenwashedfree ofCM at 25 min and incubated for an additional20min infreshgrowth medium lacking CMbut containing Rif. Thecellswereharvested,extracts were made, and enzymes wereassayedasdescribed previously (28).Assays 5 to 8 weresimilarto 1 to 4except thatK+depletion(KD) wasused toinhibitproteinsynthesis. Significant synthesisofenzyme (at least 8% control values) was observed only in thesituations indicatedby underlinednumbers.

bEnzymeactivitiesareexpressed ascounts per minuteinproductper108 infectedcells minus counts per minute inproduct per 10' uninfected cells. Average counts per minute per 10' uninfected cells was: HMase 106counts/min: dCTPase 623

counts/min,a-gt,1,065counts/min; kinase,602counts/min; lysozyme,380counts/min.

(iv) Limited aeration growth-aerobic in- pretreated cells, but of the other enzymes fection. Under these conditions, a significant tested, no mRNA accumulation was observed. amount ofHMase mRNAaccumulated in CM- However, in K+-depleted cells, significant

334 AND COHEN

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TABLE 3. Synthesisofprereplicative enzymes during recovery from CM treatment and potassium depletion of limited aeration grown cellsa

Minutes after infection Enzyme activity"

Cul-ture -5 0 +5 +10 +20 +25 +30 +40 +45 HMase dT a-gt Ki-

Lyso-ase nase zyme

+Rif

6 KD Infect +K+ Harvest 2,40 5.058 7,009 4,643 4,592

aSame asTable2, exceptthat cells were grown with limited aeration and infected with limited aeration.

"As inTable2, average counts per minute per106uninfected cells subtracted was: HMase, 96 counts/min;dCTPase, 793

counts/min;a-gt, 1,221counts/min;kinase, 709counts/min;lysozyme,504counts/min.

TABLE 4. Synthesis of prereplicative enzymes during recovery from CM treatment and potassium depletion after aerobic growth and limited aeration infectiona

Cul-ture 5 0 +5 +10 +20 +25 +30

+40

+45

HMase dCTP- | Ki-

Lyso-ase nase zyme

1 CM Infect Harvest 0 0 0 0

+Rif

7 KD Infect +Rif +K+ Harvest 41 1,377 813 0 0

8 KD Infect +Rif +K+ Harvest 451 1,117 1,667 0 0

aSame as Table 2, except that cellsweregrownaerobicallyand infected withlimited aeration.

b As inTable2, average counts per minute per 108 uninfectedcells subtracted was: HMase, 75 counts/min; dCTPase, 742

counts/min;a-gt, 1,166counts/min; kinase,779counts/min; lysozyme,743counts/min.

TABLE 5. Synthesisofprereplicativeenzymesduringrecoveryfrom CMtreatmentandpotassium depletion after limited aerationgrowthand aerobicinfectiona

Cul-

-I

ture 5 0 +5

+10

+20 +25 +30 +40 +45 HMase dCTP- a-gt Ki-

Lyso-ase nase zyme

3 CM Infect +Rif Wash Harvest

|

10|

54 0

1

0 186

+Rif

7 KD Infect +Rif +K+ Harvest

|I1T

2,026

1

28

0

8 KD Infect +Rif +K+ Harvest 484 2,818 5,802 32 209

aSameasTable2, except thatcells weregrownwithlimitedaerationand infectedaerobically.

',AsinTable 2, averagecountsperminuteper 108 uninfected cells subtractedwas:HMase,60counts/min; dCTPase,641

counts/min;a-gt,1,008counts/min;kinase,802counts/min;lysozyme,467counts/min.

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336 MORSE AND COHEN

amounts of HMase mRNA (IE), dCTPase

mRNA (DE), and a-gt mRNA (DE)

accumu-lated. No kinase mRNA or lysozyme mRNA were observed (Table 5). These results are

con-sistent with the fact that about half the IE mRNA's andasmuchas85% of the DE mRNA's

accumulate under these conditions (Fig. 1D, Table1).

It is important to emphasize that lysozyme mRNA did not accumulate under any of the

growth and infection conditions used in the

present studies (Tables 2-5). Functional

tran-scription of the lysozyme gene requires the

synthesis of IE and DE early enzymes and T4

DNA synthesis (22, 32). The lack of

accumula-tion of lysozyme mRNA in these experiments therefore serves as anadditional control,

ensur-ing the validity of each experiment. It is also important to note that the enzyme activities

observed in the control cultures (rows 2 and 6,

Tables 2-5) were comparable to those found

after 20 minin normallyinfected cells. To clarify the relationships between growth

and infection conditions and the accumulation

of IE and DE mRNA's, the data presented in

Tables 2-5aresummarized in Tables 6 and 7.

DISCUSSION

Protein synthesisisnotrequiredfor

trans-cription of some DE RNAs. When E. coli is

treated with the amino acid analogue 5-methyl-tryptophan (19) or is starved for a required

amino acid (A. Baros and H. J. Witmer, Arch. Biochem. Biophys., inpress) and then infected withphage T4, transcription ofmanyDEgenes

occurs. However, since significant amounts of protein are synthesized under these conditions

(19;A. Baros and H. J.Witmer, Arch. Biochem. Biophys., in press), such experiments havenot

resolved the question as to whether protein synthesis is required for DEgenetranscription.

Inthe present investigation we have

unequivo-callyshown thatprotein synthesisis not neces-sary for the transcription of substantial

amounts ofdCTPase and a-gtmRNA's bothof

which have been characterized as belongingto

the DE class (19, 28, 29, 39). This conclusion

requires that K+ depletion of E. coli B207

prevents protein synthesis at least as well as

CMtreatmentof E. coli. That such is thecaseis

shown by the following facts: (i) both CM

treatment (100

Ag/ml)

and K+ depletionof E.

coliB207 result invirtuallythecomplete

inhibi-tion of protein synthesis as measured

chemi-cally or by incorporation of radioactive amino

acids into protein (12, 13); (ii) whereas treat-ment ofE. coliwithCM (100 gg/ml) allows the

synthesis of those proteins necessary for

in-fected cells to acquire resistance to lysis from

without, K+ depletion blocks the synthesis of those proteins (28); (iii) whereas CMtreatment allows the accumulation of small polysomes (9, 15) and the synthesis ofsmallpeptides (9)inE. coli,K+depletionresults in thecomplete break-down ofpolysomesto monosomes (10, 11).

Not all DE genes are transcribed in the absence of protein synthesis. When infected cellsare eitherpretreated with

5-methyltrypto-phan (19), starvedforarequiredaminoacid (A. Baros, and H. J. Witmer, Arch. Biochem.

Biophys., inpress), or allowed 1.5minofRNA

and protein synthesis prior to the addition of

rifampin (25, 28), someDE genetranscriptsdo not accumulate. Consequently, it has been suggested that the presence of significant

amounts of a phage-specific protein(s) in the

cell is necessary for the transcription of these

genes. Deoxynucleotide kinase has been

desig-natedas amember of this "quasi-late" class (8).

The resultspresented heresupport this view in that conditions have been found in which both

dCTPase mRNA and a-gt mRNA accumulate

in the complete absence of protein synthesis,

but in no case does kinase mRNA accumulate. It is important to note that 85% ofthe DE transcripts can accumulate to a significant

extent under conditions of limited aeration

growth followed by aerobic infection in the

absence of K+ (Fig. 1D). These transcripts are

functional (Tables 5 and 7). Since kinase

TABLE 6. Accumulation ofT4enzyme-specific mRNA's during CM treatment

Conditions of: mRNA's accumulated Data

sum-Datarized

Growth Infection HMase dCTP- a-gt Kinase Lyso- from:

ase zyme

Aerobic Aerobic ++ a - _ Table 2

Limited aeration Limited aeration + - - _ _ Table 3

Aerobic Limited aeration + - - - - Table 4

Limitedaeration Aerobic + + - - - - Table 5

aSymbols: -, <8% of control enzyme activity (that in row 2); +, 8 to 10% of control enzyme activity; +,10 to

20% of controlenzyme activity; ++,20to 40% of control enzyme activity.

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T'ABLE 7. Accumulation ofT4enzyme-specific mRNA's during K+ depletion

Conditions of: mRNA's accumulated Data

sum-1~~~~~~~~~~~~~~

marized

Growth Infection HMase dCTPase a-gt Kinase

Lzysome

frm

Aerobic Aerobic _a I Table2

Limited aeration Limited aeration + + + + + _ - Table 3

Aerobic Limited aeration + +++ 4 --_ Table 4

Limited aeration Aerobic + + + + + + + - Table 5

aSymbols: -, <8% of control enzyme activity (that in row 6);

±,

8 to 10% of control enzymeactivity; +, 10 to 20% of control enzyme activity; ++, 20 to 40% of control enzyme activity; +++, >40% of control enzyme activity.

mRNAdoes not accumulate under these condi-tions and the kinase gene is a quasi-late func-tion (8), it seems reasonable to assume that if

all quasi-late genes act like the kinase gene,

they direct the synthesisof no morethan 15% of

theDE transcripts.

DE gene transcription may prevent the transcription of certain IE genes. When

pro-tein synthesis is blocked by starving E. coli

B207 for K+, the transcription of DE genes is

accompanied

by

a decrease in the percentage ofIE genes transcribed (Table 1). This

observa-tionisparticularly strikinginthecaseoflimited

aerationgrowth followed by aerobicinfection in

the absence of K +. Under these conditions, 20-min KD-RNAcontains 85% oftheDE tran-scripts, but only 40% of the IE transcripts

(Table 1). The DE transcripts accumulated

under these conditions are in relatively high

concentration as evidenced by thefactthatthey

can serve as a template for thesynthesis of53

and 37% ofthe controlamountofdCTPaseand

a-gt, respectively (Tables 5 and 7). The expla-nation that some IE genes require K+ for transcription is ruled out by the fact that cells

grown aerobically and infected aerobically in

the absence ofK+ accumulate 100% ofthe IE

transcripts (Fig. 1A, Table 1). However, it is

possible that extensive transcription of DE genes limits the free pool ofRNA polymerase and consequently theonly IEgenestranscribed

arethose with astrongaffinityforthisenzyme. Ifso, the shutoffofgroup AIE gene

transcrip-tion inthefirst halfofthelatentperiod during normal infection (1, 35) would not require the synthesis of any phage-specific protein but

might result as a consequence of DE gene transcription.

The physiological state of E. coli deter-mines which transcripts accumulate in

K+-depletedcells. Inapreviouspaper, we

reported

that K+-depleted cells accumulate IE

tran-scripts exclusively under aerobic conditions at

30C (28). In the present investigation, all

experiments were performed at 37 C. When

cells were grown and infected under aerobic

conditions, K+-depleted cells accumulated IE

transcriptsexclusivelyinthe first 5min, but by 20 min after infection significant transcription ofDE genes had occurred (Fig. 1A, Table 1). However, as reported previously (28), under

aerobic conditions we were unable to detect

accumulation offunctional mRNA's in K+-de-pleted cells asdefined by their ability to direct

the synthesisofenzymes in vivo(Tables 2, 7). In contrast, cells grown under limited aeration

conditions and infected under limited aeration

conditions in the absence of K+ accumulated

most ofthe IE transcripts and approximately

60%ofDE transcriptsby 20 min after infection

(Fig. 1B, Table1).TheRNAthusaccumulated was able to direct the synthesis of small amounts of the IE enzyme HMase, small

amounts of the DE enzyme a-gt, and large

amounts oftheDE enzymedCTPase (Tables3

and 7). It therefore seems possible that the IE and DEtranscripts accumulated underlimited

aeration conditions areeithermorestable than the same transcripts accumulated under strict

aerobic conditions or that IE and DE

tran-scripts terminate prematurely under aerobic but not limited aeration conditions in infected cellsdepleted ofK+.

Itis important to note that under all

condi-tions of

growth

and infection

only

HMase mRNA, of the five mRNA's

tested,

accumu-lated in CM-treated cells (Tables 2-6). This result suggests that transcription is limited to

IE genes in CM-treated cells even when the

physiology of those cells favors accumulation of

functional DE transcripts and argues against

the hypothesis (2) that rapid breakdown of distal DEportionsofT4transcriptsaccountsfor the exclusive accumulation ofIEtranscripts in

CM-pretreated cells. This view is

supported

by

the results of experiments in which IE genes were transcribed exclusively in both

CM-treated SuA+ and SuA- strains of E. coli

(A.

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338 MORSE AND COHEN

Baros and H. J. Witmer, Arch. Biochem. Bio-phys., in press).

RNA is far more stable when bacterial cells are grown anaerobically than aerobically, but the reason is not as yetfullyunderstood (16, 20). Fromthe results of the presentstudy, itisclear that functional T4 mRNA can accumulate in K+-depleted E. coli as long as the cells are either grown orinfected under limited aeration conditions. In fact, it appears that aerobic infection ofcells grown under limited aeration conditions specificallyallows the accumulation of significant amounts of a-gt mRNA in the absence of K+. That is, the ca-gt mRNA

ac-cumulated under these conditions results in the synthesis ofabout 35% as much a-gt as in the control culture (Tables 5 and 7). Infection underanyother set ofconditionsresulted in the accumulation offunctional a-gt mRNA which yieldedat mostabout 10% of the control level of a-gt synthesized (Compare Tables 2, 3, and 4

with Table 5). In contrast, significant amounts ofdCTPasemRNA were accumulated under all conditions ofgrowth and infection except

aero-bic growthand infection (Tables2-5and 7). At present, the reason for these differences is

unclear, but it is interesting to note that a-gt mRNA accumulated significantly only when 85%ofthe DE mRNA species were transcribed (Fig. 1D, Tables 5 and 7), whereas significant

amountsofdCTPasemRNAaccumulated when

asfew as40% ofthe DE genes weretranscribed

(see Tables 4 and 5, rifampin added at 5 min and Fig. 1C and D, hybridization of 5-min KD-RNA). Whether these results point to dif-ferential rates of transcription of the two DE genesordifferential rates of turnover of the two transcripts under the different experimental

conditions remains unclear. It has been shown previously, however, that when T4-infected cells are treated withCMshortlyafter infection,

theturnover ratesofa-gtmRNA, T4 endonucle-asemRNA, and deoxynucleotide kinase mRNA arevastly different (29, 38).

The reason chloramphenicol restricts T4 transcription to IE genes, whereas K+ depletion allows transcription of most DE genes, remains obscure. However, it should be emphasized that CM and K+ depletion act in entirely different ways. For example, it is known that CM does not prevent initiation of protein synthesis nor does it prevent polysome formation (9, 15, 27), but it does inhibit elongation of the peptide chain by inhibiting the peptidyl transferase reaction (27). In contrast, K+ depletion results

in the complete breakdown of polysomes (10, 11). Therefore, iftranscription of DE genes like a-gtordCTPaserequires the action of a normal

E. colipolysomal protein at specific sites imme-diately proximal to such genes, it is possible that transcription ofthese genes ispreventedin CM-treated cells because ribosomes are con-fined to IE promoter sites; however, in K+-de-pleted cells, it ispossiblethat the dissociation of polysomes to monosomes (10, 11) releases

poly-somaltranscription proteins which then bindto

the aformentioned DE sites on the T4 genome and stimulate transcription of these genes. In this context, it has recently been shown that

ribosomal proteins can stimulate T4 transcrip-tion at the initiation step (18).Alternatively, it ispossible thatanE. coliproteinresponsiblefor restricting transcription to IE genes in CM-pre-treated cells requires K+ for activity.

ACKNOWLEDGMENTS

This investigationwassupportedby Public Health Service grantA111518-01fromthe NationalInstitute ofAllergy and Infectious Diseases and the National Science Foundation grantGB-37942. Thispaper is from adissertationbyJ. W. Morse inpartial fulfillment oftherequirement for aPh.D. degreeinBiologicalSciences, UniversityofRhodeIsland.

We express our appreciation to Herbert L. Ennis for helpfulcriticism of ouroriginalmanuscript.

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