Translational control of the expression of bacteriophage T7 gene 0.3

J. Mol.

Biol.

(1978)

125, ‘75-93

Translational

Control

of the Expression T7 Gene U-3

SUSAN STROMEAND ELTON Departments

of Bacteriophage

T. YOUNG

of Biochemistry and Genetics, University Seattle, Wash. 98195, U.S.A. (Received

8 May

of Washington

1978)

When Escherichia coli are infected at 43°C with a bacteriophage T7 mutant that produces a temperature-sensitive RNA polymerase (ts342), the rate of transcription of the T7 late genes is reduced threeto fourfold below the rate of transcription in cells infected with wild-type T7. The reduction in T7 late mRNA concentration in cells infected with ts342 is accompanied by the over-production at late times of at least one T7 early protein, the gene O-3 protein. Despite the difference in T7 late mRNA concentration in cells infected with wild-type T7 and ts342 at 43”C, T7 late proteins are synthesized at the same rate in the two infectedcell cultures. These findings support an hypothesis of discrimination against 0.3 mRNA translation in favor of translating T7 late messages when mRNA is in excess of the protein synthetic machinery of the cell.

1. Introduction The

of translational control has been recognized in systems in which stable messenger RNA species initiate protein synthesis at different rates. In reticulocyte lysates /3-globin mRNA initiates synthesis 40% more efficiently than cr-globin mRNA (McKeehan, 1974), and the rate of translational initiation on ovalbumin mRNA is about twofold higher than the rate of initiation on conalbumin mRNA (Palmiter, 1974). Other than the RNA bacteriophages, there are few well-documented examples of translational control in bacterial or DNA bacteriophage systems (Lodish, 1976; but see Russel et al. (1976) and Krisch et al. (1977) for an exception). Regulation of gene expression in bacteria and in DNA phage-infected bacteria occurs mainly at the level of transcription. Although this is also true of Escherichia coli infected with bacteriophage T7, the work presented here suggests that translational control exists as well. Upon infection of E. coli by T7, host RNA polymerase transcribes the early region of the T7 genome. The product of one early gene, 0.7, shuts off transcription of E. coli DNA and T7 early genes, probably by inactivating the host RNA polymerase. In the absence of the gene 0.7 protein, one or more late functions also serve to shut off transcription by E. coli RNA polymerase. A T7 early gene, gene 1, codes for T7 RNA polymerase which transcribes the late region of the T7 genome. Genes 0.7 and 1 function to switch transcription from host DNA and T7 early genes to T7 late genes. This sequence of events after T7 infection has been reviewed in detail (Studier, 1972, 1975). Transcription of E. coli DNA and the T7 early genes by host RNA polymerase is shut off by four to five minutes after infection at 30°C (Brunovskis & Summers, importance

75 0022-2836/78/290075-19

$02.00/O

0

1978 Academic

Press Inc.

(London)

Ltd.

S. STROME

76

AND

E. T. YOUNG

1971,1972 ; Rothman-Denes et al., 1973), and synthesis of host probeins and t,he Ti early proteins is shut off by about eight, minutes after infection (Studier, 1972). Since at least one E. coli mRNA decays with a half-life of two t,o three minutes hoth befort, and after T7 infection (Marrs & Yanofsky. 1971), the shut-off of host proteins can probably be attributed to transcriptional control. However, since T7 mRNA is chemically stable (Summers, 1970). t’ranscriptional control alone cannot’ account for the shut-off of expression of the T7 early gems. Several cxplanat,ions for the shut-off have been proposed. Herrlich et aE. (1974) suggested that a phagc-coded repressor. specifically inhibits translation of host and T7 early RNA. However. Yamada $ Nakada (1976) failed to find any evidence for a translational repressor. After infection of E. coli by T7 gene 2 amber mutants. shut-off of T7 early protein synthesis is delayed (Studier, 1972: Rothman-Denes et aZ.. 1973). Since gene I amber mutants do not, synthesize T7 late mRNA. the persistence of early protein synthesis was interpreted as indicating that a late function prevents t’ranslation of the earl) mRNAs, possibly by inactivating them (Studier. 1975). However, none of the mutants in T7 late genes presently identified is defective in shut-off of T7 early gene expression. Yamada et ~2. (1974a.6) found that the functional stability of T7 early mRNA. as measured by cell-fret translation, decayed after infection of E. coli t)y a TS gene I amber mutant. Based on evidence that late in infection some T7 early mRNA species appeared to migrate more rapidly in polvacrylamide gels. theg suggested that functional inactivation might occur by nuclease cleavage of the 5’ end of the message. Observations from this laboratory (Hopper of al.. 1975: Pachl & Young. 1976: Strome & Young, unpublished observation) suggest,. in apparent contradict’ion to tht results cited above, that functional T7 early mRNA coding for at least genes 0.3. I and 1.3 is present late in T7 infection and has the same size as it does earl,v in infectsion. In contrast to the rapid decrease in the rates of gene 0.3 and gene 1 protein synthesis as infection proceeds ilz vivo, cell-free translation of RNA extracted from E. coli at corresponding times after infection demonstrates that 60 to 70”; as much functional U-3 and I mRNA is present late in infection as is present ra,rly in infection. If T7 early mRNA is functionally stable. as our results suggest. competition tletween T7 early and late mRNA for a limiting amount of tra,nslationa,l machinery in the infected cell could explain the shut-off of T7 ear1.v protein synthesis. Translational discrimination against, T7 early mRNA might occur if ribosomes initiate translation on early mRNA less efficiently than on late RNA, and if some part’ of the translational apparatus, rather than the mRNA, is limiting in determining the rate of polypeptidc chain initiation. The persistence of T7 early protein synthesis in cells infected with T7 gene I amber mutants would be explained by the absence of late TS mRNA. rather than the absence of a specific late T7 protein. To test the hypothesis. we infected E. coli with a T7 gene I temperature-sensitive mutant (t&W) at a temperature that would reduce but not eliminate late mRNA trarlscript,ion. We conclude from analysis of this mutant that there is translational control of expression of at least one earl>, gene, 0.3.

2. Materials (TV) Bacteriophage

and Methods and

bacterial

strai,cs

coli BSt and wild-type T7L were obtained from Dr F. \I’. Studier. ‘l’l~r t(~~npwatw(~sensitive gene 1 mutant,, ts342-15, picked up as a te~rlpc~~~~trl~c~-s~~~lsiti~~~wvcrtarlt of E.

gene I am342 (Summers wild-type-size plaques ts342 in this paper.

TRANSLATIONAL

CONTROL

& Siegel, 1970), was at 30°C and pinpoint

obtained plaques

(b) The method Polyethylene to concentrate

Prepara$ion

of phage stock glycol precipitation, the phage.

IN

stocks

is essentially that by Yamamoto

(c) Media

and

77

from Dr W. C. Summers. It gives at 43°C. ts342-15 is referred to as

of bacteriophage

preparation described

T7

described & Alberta

by

Studier (1969). (1970), was used

chemicals

One liter of M9 medium is made by mixing the following sterile solutions: 900 ml of a solution containing 7 g Na,HPO,, 3 g KH,PO,, O-5 g NaCl, 1 g NH,Cl, 0.1 ml of 1 M-CaCl,, and 1 ml of 1 M-MgSG,, and 100 ml of a solution containing 4 g glucose. M9 amino acid medium (MSAA) contains additionally 2 g Casamino acids/l. Radioactive methionine was (from New England Nuclear) following isolated from cells grown in the presence of e5SGithe procedure of Crawford & Gesteland (1973). Electrophoretically purified DNase (RNase-free) was purchased from Worthington Biochemicals. Contaminating RNase was inactivated with iodoacetate according to the procedure of Zimmerman & Sandeen (1966). (d) Cells G15T8 30 min

Ultraviolet

light

irradiation

of cells

were irradiated with U.V. light at a distance of 40 cm from twin General Electric 15W lamps for 8 min. After irradiation the culture was aerated at 30°C for at least in the dark prior to infection. (e) Labeling

T7 proteins

in vivo

The procedure for pulse labeling T7 proteins in. vivo has been given in detail by Hopper et al. (1975). Fo: continuous labeling of T7 proteins in vivo a 4-ml portion of the cell culture was transferred to a small bubbler tube containing O-2 ml of M9 medium, 40 PCi of [35S]methionine, and cold methionine to give a final concentration of 1 rg/ml. At 2-min intervals a O-5-ml portion was transferred to a tube containing 05 ml of 10% (W/V) C&amino acids, and the incubation was continued for 2 min. Samples were processed as pulse labeled samples. (f) Labeling

T7

RNA

in vivo

At 4-min intervals throughout infection a 1 ml portion of the culture was transferred to a tube containing 0.1 ml of M9 medium and 1 &i of [3H]uridine and incubated for 2 min. The labeling was stopped by adding 1 ml of cold 10% (w/v) trichloroacetic acid. After at least 30 min on ice the precipitates were collected on filters for counting. (g) RNA

extraction

from

T74nfected

cells

RNA was extracted from cells using the hot sodium dodeoyl sulfate extraction procedure detailed by Hagen & Young (1978) or by the standard cold extraction procedure previously described by Hagen & Young (1973) with the following changes: 2 M-sodium acetate (pH 5.2) was replaced with 2 M-ammonium acetate (pH 5.2). Instead of treating the crude lysate with DNase, the extracted RNA in a solution of O-1 M-Tris (pH 7-O), 5 mM-MgSG,, and 10e4 M-CaCl, was incubated with DNase at a concentration of 5 to 10 pg/ml for 15 min at 37°C. RNA extraction with phenol and precipitation with ethanol of RNA were repeated to remove DNase. Radioactive RNA was isolated in the same manner after incubating T7-infected cells in the presence of [3H]uridine (1 &/ml culture) for the desired length of time. Incorporation was stopped by the hot sodium dodecyl sulfate method of lysing cells. (h) Separation

of T7

DNA

strands

The most effective strand separation occurred when the ratio of absorbance at 260 nm for T7:poly(U.G) was 10:7*5. A solution containing 1.7 ml of 0.03% (w/v) Sarkosyl, 3 m&r-NaGH, 1 m&r-EDTA, 3 mM-NaCl, 10 A,,, units of DNA, and 7.5 Azeo units of

S. STHOME

7s

ANI)

E.

‘1’. YOVNC:

was boiled for 5 min and cooled on icca. Six ml of C&lphenol-extracted poly(U.G) saturated, 0.01 M-EDTA, 0.5 M-Tris (pH 7.5) was added t,o t)be DNA-poly(U*G) solut,ion. The mixture was centrifuged in cellulose nitrate tubes at, 40.000 revs/min and 5°C for 40 11. Fractions were collected, and the absorbance at 260 nm was determined for each. The, tieavv strand fractions were pooled, made 0.5 by in NaOH, and incuba,trd at 45°C f~lr 30 iin to remove and hydrolyze the poly(U. G). Ttlcx rcasultinp soltltioll \vax tlialyzetl against0.01 M-T& (pH 7.5). 0.01 &I-NaCI.

Tris/KCl buffer (0.01 31.Tris (pH 7.2), 0.5 X-Kc‘l) \vas used. Since ‘1’5 mlCN:X is trarlacribed exclusively from r-strand TS DNA (Summers Oz Szybalski, 1968). and in order to prevent DNA-DNA reannealing from competing wittL RNA -DNA hybridization. r-strand it1 all competition hybridizations. After T7 DNA prepared using poly (U . G) was used incubation for 5 h at 65°C in a volume of 0.2 ml, t,hrh DNA-RNA llybrids were treat,cd wit,11 a mixture of previously boiled pancreatic HNasc (2.5 gg) and T, RNasr (5 units) fol 15 min at 37°C. Three ml of cold Tris/KCl buffer was added AtId the llybrids were collect’etl on Millipore HA filters and wasbed with Tris/K(:l buffer. The data were analyzed by plotting c,/cX ??er.so.s J./U according to tilt, cLc{uatir)rl

(‘0 -=(.L& ” cx ’ .f, 0 !/ ’ where fX = fraction of competitor RNA that is T7 late 1nKNA; .L ~~: (~l)tlcetltritt,iolr ot competitor RNA in mg/ml; f, = fraction of 13H]KNA that is ‘I’7 lat,e mRNA: y _ tollcentration of [3H]RNA in mg/ml; c0 = cts/min of L3H]RNA hybridized ill the absence of compet,itor RNA ; c, : ct,s/min of [3H]RNA hybridized in the presence of competitor RNA.

The slopes of ttlta lines c,ach competitor R.NA

yield t)bat

fxif,, is T7

and late

the ratio mRNA.

of ttrcb slopf~

is ttlc, r&i0

of the fractiofls

The procedllres for crll-free synthesis, preparation and electrophorcsis determination of [35S]mettiiot~inr incorporation. dcnsitometric scanning grams, and tile assay for lysozyme activity Ilave been described (Hopper Studier, 1973; Young & Menard, 1975; Hagen & Young, 1973).

of

of

nf

samples, aut,oradioef al., 1975:

3. Results (a)

Comparison

r$ T7 late mRNA and

The

hypothesis

of’ translational

a gene

synthesis

1 mutad discrimination

in cells

Of T7

injected

with

wild-i!yprl

T7

early

mHNAs

late

(ts342) against

T7

in

infection has been tested using a T7 mutant that, has a temperature-sensitive mutation in gene 1, ts342. Temperatures intermediat’e between the permissive and restrict’ive temperatures should reduce the activity of the mutant T7 RNA polymerase and result in reduced synthesis of T7 late mRNAs. If T7 early mRNAs are excluded from the translational machinery in the presence of normal levels of late mRNA, tlrch synthesis of early proteins might persist late in infection in the presence of reduced levels of late mRNA. After observing that at least one early prot,ein, t’he gene 0.3

TRANSLATIONAL

CONTROL

IN

T7

79

protein, was dramatically overproduced late in infection of E. co.5 by ts342 at 43°C (next section), we measured the relative concentrations of late mRNA in cells infected with this mutant at several temperatures. (i) [3H]uridine

incorporation

Figure 1 shows the rates of [3H]uridine incorporation into RNA in E. coli infected with wild-type T7 and the gene I temperature-sensitive mutant at 30, 37, 43 and 46°C. Since E. co.% were u.v.-irradiated prior to infection, there was no host RNA synthesis. In cells infected with ts342 at different temperatures, rates of [3H]uridine incorporation at late times were progressively reduced below wild-type rates as the temperature was elevated. Assuming that T7 mRNA is stable (Summers, 1970), integration of the curves of [3H]uridine incorporation into RNA at 43°C indicated a three- to fourfold reduction in T7 late mRNA in cells infected with ts342 relative to cells infected with wild-type T7. (ii) Competition

hybridization

Since the specific activity of the uridine pool in cells infected with wild-type T7 and ts342 is unknown, competition hybridization was used as an alternate measure of late mRNA levels. Since total RNA per cell does not change significantly during infection, the amount of T7 RNA in two different preparations of total RNA extracted from either wild-type or mutant-infected cells can be compared by competing radioactive T7 mRNA by the two RNA preparations. In order to label only T7 late messages, RNA was labeled 13 to 21 minutes after infection of E. coli with wild-type T7 at 30°C. Non-radioactive competitor RNA was extracted 13 minutes after infection of cells with wild-type T7 or ts342 at 43°C. In order to compare the concentration of T7 late mRNA in E. coli infected at 43°C with either wild-type T7 or ts342, radioactive RNA was hybridized to r-strand T7 DNA in the presence of increasing amounts of non-radioactive competitor RNAs (Fig. 2(a)). As expected, high concentrations of both preparations of RNA competed more than 95% of the radioactive RNA, indicating that the sequence complexity of late mRNA extracted either from wild-type T7-infected or mutant-infected cells was the same. However, the initial slope of the competition curve was steeper when wild-type T7 RNA was used, reflecting a higher concentration of T7 late mRNA in cells infected by wild-type T7 compared to cells infected by ts342. The ratio of the slopes of the lines in Figure 2(b) is the ratio of concentrations of T7 late mRNA in wild-type versus ts342-infected cells (see Materials and Methods). This ratio was 2.9, consistent with the three- to fourfold reduction in late gene transcription measured in ts342-infected cells by [“Hluridine pulse labeling. (iii)

Cell-free

translation

Both [3H]uridine labeling and hybridization are chemical assays of RNA. As an independent assay of T7 late gene activity and one that would measure functionally intact mRNA, RNA extracted from infected cells was translated in a cell-free translational system. RNA was extracted from u.v.-irradiated cells infected with wild-type T7 or ts342 at 1, 5, 13, and 21 minutes after infection at 43°C. This RNA was translated in an E. coli cell-free translational system in the linear range of [35S]methionine incorporation, and the proteins synthesized were analyzed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (Fig. 3). The microdensitometric measurements

S.

STROME

AND

E.

‘I’.

YOUNG

,,-

(dl

Time

after

infectIon

(rmn)

FIG. 1. L3H]uridine incorporation iu viva during infwtion of ti. wli BSL b)- willi-tylw Ti itntl ts342 at 30”, 37”, 43” and 46°C’. E. coli Bst was grown with aeration in M9 medium at 37°C to a cell density of 4 x 108/ml. The cult,we was u.v.-irradiated and aerat,ed at 30°C for 30 min. .Iftrt dividing the culture into IO-ml subcultures, the cult,ures mew placed at (a) 3O”C, (b) Bi”(‘. (c) 43Y’. and (d) 46°C and infected wit,h wild-type T7 or ts342 with a multiplicity of infection of 10. At 4-min intervals, 1 ml samples were removed for pulse labeling of RNA wit,h [3H]uridinc i,z virv at the temperature of infection. The details of labeling in viva and precipitation with t.richloroacetic acid are described in Materials and Methods. A background radioactivity of 5716 cts/min was obtained by pulse labeling uninfected E. co& from the irradiated culture at 37°C’. Hecausc RNA synthesis is inhibited by T7 infection, the background was not subtracted from the data. --@-e--, Wild-type T7; -~ ( ~--~r> mm-m.ts.742.

et al.. 1975) of individual mRNA activities are shown in Figure 4. Total trichloroacetic acid-precipitable radioactivity (cts/min) incorporated into protein and the intensities of individual T7 late protein bands on autoradiograms were all threeto fourfold lower upon translation of RNA extracted from cells infected with ts342 than when RNA extracted from cells infected with wild-type T7 was translated. The (Hopper

TRANSLATIONAL

LO)

CONTROL

20

0.2

IN

81

T7

(b)

0.3

0.4

Competitor

0.5 RNA

0.6

0.7

0.0

(mg/mll

FIG. 2. Competition of T7 RNA labeled late in infection by late RNA extrected from E. coZi Bat infected with wild-type T7 or ts342 at 43°C. (a) The concentration of r-strand T7 DNA was 5 pg/ml. RNA was labeled 13 to 21 min after infection of u&radiated E. coli with wild-type T7 at 30°C. The concentration of [sH]RNA w&s in slight excess of that needed to saturate the DNA in the absence of competitor RNA. Competitor RNA was extracted 13 min after infection of u.v.irradiated E. coli with wild-type T7 or ts342 at 43°C. Hybridization reactions were carried out in a volume of 0.2 ml at 66°C for 6 h. Samples were treated with RNase before filtering &B described in Materials and Methods. The background radioactivity (47 cts/min) detected in the absence of DNA was subtracted from the data. The competition values at the 3 lowest competitor RNA concentrations represent the average velues from duplicate reactions. Repeat experiments with different DNA and competitor RNA preparations contimed the results shown here. (b) The data from (a) were analyzed as described in Materials and Methods. -e--•--, Wild-type T7; -- 0 -- 0 --, ts342.

three independent methods of determining T7 late mRNA levels in cells infected with ts342 at 43°C were consistent in revealing a three- to fourfold reduction compared to the amount of late mRNA present after infection with wild-type T7 at 43°C. (b) Comparison

qf T7

early protein synthesis in vivo during with wild-type T7 and ts342

infections

The reduced synthesis of T7 late RNA in E. coli infected at an elevated temperature with ts342 enabled us to ask what effect, if any, the reduced levels of T7 late mRNA would have on the expression of the T7 early genes in vivo. A comparison of acidprecipitable [35S]methionine incorporation during infection of u.v.-irradiated E. wli by wild-type T7 and ts342 at 30, 37,43 and 46°C is shown in Figure 5. At 30,37 and 43”C, [35S]methionine incorporation reached the same maximum rate in ts342-infected cells as in cells infected with wild-type T7, although there was a slight lag in reaching the maximum rate in the cells infected with the mutant. Only at 46°C was there a depression in the rate of [35S]methionine incorporation late in infection in mutantinfected cells. The radioactive proteins, analyzed by sodium dodecyl sulfat,e/polyacrylamide gel electrophoresis, revealed a very similar pattern of protein synthesis in cells infected with wild-type T7 and ts342 at 30 and 37°C. A lag in the appearance of 4

82

Y.

HTROME

wt

AND

E.

T.

ts 342

YOUNG

Gene products

PI6 PI

PlOh P9 PO-7,

PIa ,3,

PI0 P(D’JP),

P6

P3.5

14*4

PO-3

synthesis directed by RKA extracted from E. coli R St infected by wild-type T7 (w-t) anti ts342. K. coli Bst was grown with aeration in MS medium at 37”(‘ to a cell density of 4x 108/ml. Thr culture was u.v.-irradiated, aerated at, 30°C for 30 min, and divided into 100.ml subcultures. The cultures were transferred to 43°C and infected with wiltl-type T7 or ts342 with a multiplicity of 20.1111 portions of the culture wtve poured infection of 10. At, 1, 5, 13 and 21 min after infection, over 7 g of ice with NaN, added to a final concentration of 0.01 M. The cells were concentrated 4-fold by pelleting, and RR’A was extracted as described in Materials and Methods. The RNA samples were translated in a cell-free system as described in Materials and Methods. Equal volumes of the [35S]methionine-labeled proteins synthesized in vitro were run on a sodium dodecyl nulfate/16~/0 polyacrylamide gel for 1.75 h at 9 mA and then 3.25 h at 25 mA.

late proteins was observed after infection of cells by the mutant, but individual protein bands reached the same intensities on autoradiograms displaying proteins synthesized in ts342-infected cells as on autoradiograms showing proteins synthesized in cells infected with wild-type T7 (data not shown). At 43 and 46°C. however, 0.3 protein was dramatically overproduced at late times in cells infected with t’s342 (Fig. 6). Figure 7(a) shows the time-course of synthesis of 0.3 protein in wild-type T7 and mutant-infected cells at 43°C. The maximum amount of 0.3 protein synthesized

TRANSLATIONAL

CONTRO-I,

*-a

(a)

l

IN

T7

83

(d)

f

/-.

4

/

7 ,o---,---o ( i:

,

1 ,

,

,

,

,

,

(

,

2

-- -o---

, I

2 c

.z

llllllll1I

0

(e)

g = E

6=

0

9 2

-A

l

-4

-2

/

0

0

a---

---

1111,1111111

(c1 /‘\. i

b

l@L

/

x)

/

’

0.

e---O

,I,

6

12

I6

-2

\ \

/I

,IIl,,,l,l

4

og

If)

24

Time

after

4 InfectIan

38 I

-o----o

,I,I,IIIIIII

20

2 Et ‘Z 2 E 2 .c 2

o

6

I2

I6

20

24

!m!r,j

FIG. 4. Patterns of [3sS]methionine incorporation in vitro directed by RNA extracted from E. coli Bat infected with wild-type T7 and ts342 at 43°C. (a) A portion of each in vitro translation reaction was precipitated with trichloroacetic acid to determine total [35S]methionine incorporation as described in Materials and Methods. The background radioactivity of 3231 cts/min (no RNA) was subtracted from the data. (b) to (f) Data were obtained from microdensitometric scans of autoradiograms equivalent to Fig. 3. The same amount of total RNA was translated in the linear range of [35S]methionine incorporation, presumably reflecting the concentrations of corresponding mRNAs in the RNA extracts. (b) PO.3; (c) P8; (d) P9; (e) PIO; (f) T7 late protein above PO.3 (M, = 16,000 to 16,000). -e-e--, Wild-type T7; --O--O--, ts342.

in a two-minute pulse after infection by ts342 was 2.2 times greater than the maximum amount of O-3 protein synthesized after wild-type T7 infection. At later times the difference increased to tenfold. The synthesis of another early protein, the product of gene 1, was shut off normally in mutant-infected cells at 43°C. We also observed persistence of O-3 protein synthesis late in infection of unirradiated E. coli infected with ts342 at 43°C. Thus, this phenomenon is not a peculiarity of infection of u.v.irradiated cells.

84

8.

STROME

AND

E.

T.

YOUNG

1

I

I

(b)

I

III

I

,,I

Cdl o-0 /

i

/ 8” I 2

/

/ p-,

‘O- _ - -J-O---_o

I 4

I 6 Time

I 6

I IO after

I 12

I I 14 16

infectIon

, I 18 20

I 22

(mln)

FIG. 5. [35S]methionine incorporation in viva during infection of E. col% 13”’ by wiltl-type ‘J’i and ts342 at 30”, 37”, 43” and 46°C. E. coli Bstwas grown with aeration in MS medium at 37°C to a cell density of 4 x 108/ml. The culture was u.v.-irradiated and aerated at 30°C for 30 min. After dividing the culture into 20.ml subcultures, the cultures were placed at (a) WC, (b) 37”C’, (c) 43°C. and (d) 46°C and infected with wild-type T7 or ts342 with a multiplicity of infection of 10. At, 4.min intervals 2.ml samples were removed for pulse labeling irr GXI of protein with [“%Imethionine at the temperature of infection. The details of labeling in rtivo and precipit,at,ion with trichloroacetic acid are described in Materials and Methods. The 46°C values were obtained from a separate experiment, so wild-type and ts342 values are comparable, but they canuot btx rlirwtl5 compared to 3O”C, 37”C, and 43°C values. -e-a--. Wild-type T7: lI_ t,s342.

(c) Comparison

of T7 early mRNA

transcription and ts342

in infections

with wild-type

T7

(i) Effect of rifampicin One explanation for the overproduction of (1.3 protein after infection by ts342 at 43°C is failure to stop early gene transcription by the host RNA polymerase. Since the

TRANSLATIONAL

CONTROL

IN

T7

85

Gene products PI6 PI

PlOh P9 PO-7, PI,*3 PI0 P (DUP), P6

. P3.5 - PO.3

Fro. 6. Autoradiogram of sodium dodecyl sulfate/12G”h polyacrylamide gel of protein synthesis in viwo after infection of E. coli Bst by wild-type (wt) T7 and ts342 at 43°C. The details of u.v.irradiation, i&e&ion, and pulse labeling were as described in the legend to Fig. 6. Equal volumes of the proteins synthesized in viva were run on a sodium dodecyl sulfate/l2.5% polyacrylamide gel at 25 mA for 3.6 h.

product ofgene 0.7, which is responsible for shutting off T7 early transcription, should be synthesized at the same rate in cells infected at 43°C with wild-type T7 and ts342, prolonged synthesis of T7 early RNA by host RNA polymerase in mutant-infected cells did not seem like a plausible explanation of the overproduction of 0.3 protein. This possibility was tested by adding rifampicin at early times after infection of u.v.-irradiated E. c&i. Rifampicin inhibits E. c&i RNA polymerase, but not T7 RNA polymerase (Chamberlin et al., 1970). The concentration of rifampicin used was sufficient to prevent T7 proteins from appearing when it was added prior to infection. Rifampicin addition at 3, 5 or 7 minutes after infection did not prevent the overproduction of 0.3 protein late in infection by ts342. Figure 8 shows the time-course of O-3 synthesis in cells infected with wild-type T7 or ts342 at 43°C and treated with rifampicin at seven minutes after infection. The overproduction of O-3 protein in mutant-infected cells is not dependent on continued transcription by E. coli RNA polymerase.

86

X.

STROME

0

\

4

42

0

/

16 -

/

8

,Q.

/ /

8-

ANJI

E.

12

I’.

16

YOUNC:

20

\

/

b

24

l\

l \.

,;x----0

l

-8

‘.

-4

d’

d’ 1 0

I 4

I

I 8

I

I 12

I

I 16

I

I 20 Time

I

I 24 after

1111,11l,1,1, 4 8 infectlon

12

16

20

24

’

Cmtnl

FIG. 7. Patterns of [35S]methionine incorporation i/t r,i~o into protein during infectlou of E. ~st at 43°C with wild-type T7 and ts342. Data were obtained from microdensitomet’ric JCBIIX of autoradiogram shown in Fig. 6 and are expressed as rrlat,ive densitometric units. (a) PO.:3 ; (h) (c) 1’8; (d) P11: (e) Plfi. -a-~a, C!‘ild-t,vpcx late protein (M, =- 26,000 t.o 48,000); -- _ 0 -- ()---, ts34”.

(ii)

Competition

coli thri T7 T7:

hybridization

The amounts of T7 early mRNA present car1.v and late in infection of cells by wild-type T7 and ts342 at 43°C were also compa’red directlp by competition hybridization. These experiments were done in order to examine the possibilities of enhanced synthesis of T7 early mRNA by host RNA polymerase or transcription of early mRNA by T7 RNA polymerase Iate in infection as causes of O-3 protein synthesis at late times in mutant-infected cells. T7 early mRNA wxs labeled and extracted as described in the legend to Pigure 9. Competitor RNA was extracted from E. coEi 5 minutes and 13 minutes after infection by wild-type T7 or ts342 at 43°C. Radioactive RNA was hybridized to r-strand T7 DNA in the presence of increasing amounts of non-radioactive competitor RNA (Fig. 9(a) and (b)). RNA extracted from cells

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: .-. /,,,\TIF,, 4

IN

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FIG. 8. Patterns of [%]methionine incorporation in wiwo into PO.3 during infection of E. coli Bst with wild-type T7 and ts342 at 43°C in the presence of rifampicin. Procedures were the same as described in the legend to Fig. 5. Rifampicin was added to a concentration of 50 pg/ml at 7 min after infection. Addition of rifampicin at this concentration 1 min prior to infection prevented T7 proteins from appearing. Other details of polyecrylamide gel electrophoresis and microdensitometric scanning are described in Materials and Methods. The data were obtained from sodium dodecyl sulfate/l’/.S% polyacrylamide gels run at 25 mA for 4 h. -a-@-, Wild-type T7; -- 0 -- 0 --, ts342.

five minutes after infection by either wild-type T7 or ts342 competed radioactive RNA to a similar extent at every concentration of competitor RNA used (Fig.S(a)). This demonstrates that there is no substantial difference in the amount of T7 early mRNA transcribed by host RNA polymerase by five minutes after infection of cells with wild-type T7 and ts342 at 43°C. The competition curves obtained using RNA extracted 13 minutes after infection by either wild-type T7 or ts342 were also very similar (Fig. 9(b)), confirming that similar concentrations of T7 early mRNA are present late during infection of cells by wild-type T7 and ts342 at 43°C. (iii) Cell-free

translation

The amounts of functional 0.3 mRNA present throughout infection by wild-type T7 and ts342 at 43°C were also measured by cell-free translation. RNA was extracted from cells infected by either wild-type T7 or ts342 at 43°C as described for the analysis of late RNA (section (a),(iii)). Measurements of 0.3 mRNA activity are shown in Figure 4(b). The same amount of 0.3 mRNA activity was present at 13 and 21 minutes after infection by either wild-type T7 or ts342. At one and five minutes after infection there was about twice as much 0.3 mRNA activity detectable in RNA prepared from ts342-infected cells as in RNA prepared from cells infected with wild-type T7. This result could be due to either the presence of twice as much functional 0.3 mRNA in cells infected with ts342 at 43°C relative to wild-type T7-infected cells, in apparent contrast to the equivalent amount of total early T7 RNA measured by competition hybridization, or to an in vitro competition phenomenon (see Discussion).

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RNA img /ml 1

FIG. 9. Competition of labeled T7 early RNA by early and late HS.\ c~strwtvd fr~un K. r,o[i FP’ infected with wild-type T7 and ts342 at 43°C. The concentration of r-strand 1’7 I)N.\ was 5 &ml. 3H-labeled T7 early RNA was extracted from u.v.-irradiated E. coli that \vwr treated with chloramphenicol (100 pg/ml) 5 min before infection, infected with wild-type T7 at 3O”C, and labeled 0 to 20 min. Chloramphenicol prevent,s synt.hesis of the gene 0.7 and gene I products (Siegel & Summers, 1970), thus allowing cont,inued synthesis of 1’7 early RNA in thp absence of T7 late mRNA transcription. The concentration of [ 3H]HNLI was 16X pg/ml, a roncrntrat~ion just sufficient to saturate the DNA in t,hr absence of comprtitor. A total of 26?<, OI 2458 cts/min wew RNase-resistant, after hybridization in the absence of competitor RN;\. (‘ompetitor RX.4 was extracted at (a) 5 min and (b) 13 min from u.\,.-irradiat,ptl .%‘. di infected with wiltl-type ‘I’7 or ts342 at 43°C. Hybridization reactions were carried oat, in a volume of 0.2 ml at 65”(‘for 5 h. Samples were treated with RNase before filtering. The background radioactivit,y (45 cts/rnin) (Letected in the absence of DNA was subtracted from the data. .III values wpws+~nt thea average of at leant 2 reactions. -e--e-, Wild-type T7; --i - :. ts342.

(d) C’wnxparison

of in vivo T7 late protei)r synthesis with wild-type T7 ad ts342

in itl,fk:tims

The overproduction of 0.3 protein was seen in cells infected with ts342 at 43°C in spite of apparent wild-type rates of T7 late probein synthesis (Fig. 6). Densitometric quantitation of several T7 late proteins synthesized during infection of cells by wildtype T7 and ts342 at 43°C is shown in Figure 7(b). (c) and (d). Even though the rate of RNA synthesis was depressed in ts342-infected cells late in infection, late protein synthesis appeared to reach the same rate in ts342-infected cells as in cells infected with wild-type T7. This is based on [35S]methionine-labeled protein band intensities on autoradiograms. If the specific activity of the utilizable methionine pool in the

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cultures infected with wild-type T7 and ts342 were the same, comparison of [35S]methionine pulse labeling of proteins could be used directly to compare rates of protein synthesis. It seemed unlikely that infection of E. coli by wild-type T7 and ts342 would have a differential effect on the utilizable methionine pool. However, in order to show that protein band intensities on autoradiograms do indeed reflect rates of protein synthesis, the accumulation of a T7 late protein, from gene 3.5 (lysozyme), was analyzed by densitometric scanning of autoradiograms and by an enzyme assay. If the specific activity of the methionine pool were the same in wild-type T7 and mutantinfected cells, the ratio of enzyme activity to total [35S]methionine incorporation into lysozyme should be the same for protein extracted from the two infected cell cultures. Portions of cultures infected at 42°C with wild-type T7 or ts342 were continuously labeled with [35S]methionine in vivo, and the labeled samples were electrophoresed on sodium dodecyl sulfate/polyacrylamide gels. Portions of the infected cell cultures were also removed at intervals during infection, sonicated, and assayed for lysozyme activity. The results are shown in Figure 10. Figure 10(a) represents the densitometric quantitation of lysozyme after continuous labeling. [35S]methionine incorporation was approximately linear for the duration of the labeling period. (The kinetics of lysozyme accumulation were the same when measured by pulse labeling proteins throughout infection by wild-type T7 or ts342 and summing lysozyme band intensities on autoradiograms ; data not shown.) Figure 10(b) shows the accumulation of lysozyme

,d

4 Time

after

,

1

I

8

12

16

infection

(min)

(0) 0

4 Time

8 after

12 infectlon

16 (mid

(bl

FIG.

10. Comparison of lysozyme activity with [35S]methionine incorporation in viva into lysozyme during infection of E. coli Bat at 42°C with wild-type T7 and ts342. E. coli Bat was grown with aeration in M9 medium at 37% to a cell density of 4 x lO*/ml. The culture was u.v.-irradiated and aerated at 30°C for 30 min. After dividing the culture into 60.ml portions, the cultures were placed at 42°C and infected with wild-type T7 or ts342 with a multiplicity of infection of 10: (a) 4 ml of each culture were removed for continuous labeling in wivo and portions of this continuously labeled culture were chased with 10% Casamino acids at 2.min intervals as described in Materials and Methods. Proteins labeled in viva were run on 13.75% polyacrylamide gels for 2 h at 16 mA and then 3.76 h at 45 mA. Data were obtained from miorodensitometric soam of the lysozyme bands on the autoradiograms and are expressed as relative densitometric units. -@-a--, Wild-type T7; -~ O-O--, ts342. (b) At 4.min intervals, 6-ml portions of the cultures were removed and assayed for lysozyme activity as described in Materials and Methods. -A-A--, wild-type T7; --n--a--, ts342.

20

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YOUSG

activity in sonicates of cells infected with wild-type T7 and ts342. There was a close correlation between lysozyme activity and the accumulation of labeled lysozyme at corresponding times in the two infected cell cultures. Therefore, rates of protein synthesis can be compared using [35S]methionine pulse labeling of proteins throughout, infection. Figure 10(b) also shows that, after a lag. cells infected with ts342 accumulated at least 80% as much lysozyme as cells infected with wild-type T7. From the experiments discussed in this section, it is apparent that the reduction in late mRNA levels in ts342-infected cells is not reflected in late protein concentrations.

4. Discussion At least one T7 early protein, the gene 0.3 protein, is overproduced late in infection of E. coli at 43°C with a T7 mutant that produces a temperature-sensitive RNA polymerase. At this temperature there is three- to fourfold less T7 late mRNA in cells infected with the mutant than in wild-type T7-iufected cells. Surprisingly, T7 late proteins are synthesized at about the same rate in the tbvo infect’ed cell cultures. These findings support an hypothesis of t’ranslational discrimination against 0.3 mRNA at late times during T7 infection when mRNA is saturat,ing the protein synthetic machinery of the cell. (a) RNA qu,antitatimc The reduction in T7 late mRNA transcription in cells infected a-it’h ts342 at 43C’ was demonstrated by [3H]uridine pulse labeling (Fig. I ). competition hybridization (Fig. 2), and cell-free translation (Fig. 4). These methods assay RNA according to different criteria. Taken together, they provide strong evidence for the presence of about one-quarter to one-third of the wild-type level of T7 late mRNA in cells infected with ts342 at 43°C. The results of other hybridization experiment’s (Fig. 9) and the, rifampicin experiment (Fig. 8) demonst,ratc that, similar, and perhaps identical. amounts of T7 early RNA are present in wild-type T7 and ts342-inf%cd cells at

43°C. (h)

0.3 protein

synthesis

in viva a&

in vit,ro

Even though 0.3 mRNA wits present at about the same concentrat’ion late in infections by wild-type T7 and ts342, as judged by competition hybridization (Fig. 9(b)) and cell-free translation (Fig. 4(b)), th erc \vas about ten times more 0.3 protein synthesized late in infection by ts342 than late in infection by wild-t,\pe T7 (Fig. 7(a)). It, is this finding that supports the hypothesis of t,ranslational discrimination against 0.3 mRNA in the presence of saturating levels of T7 late mRNA. The high ratr of synthesis of 0.3 protein at 5 to 7 and 9 to 11 minutes after infection hy ts342 at 43°C’ (Fig. 7(a)) may be due to the delay in synthesis of T7 late mRNAs. thereby allowing efficient translation of 0.3 mRNA. Alterna,tively. 0.3 protein synthesis may be repressed by a late function whose synthesis is delayed in cells infected wibh ts342, allowing the early rate of 0.3 protein synthesis to increase beyond the maximum early rate in wild-type T7 infections. This possibility does not negate t’he model of translational discrimination late in infection. When RNA extracted from cells infected at 43°C with wild-type T7 or ts342 was translated in a cell-free system, the maximum amount of 0.3 mRNA activity was

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detected using RNA extracted at five minutes (Fig. 4(b)). There was about twice the 0.3 activity in RNA extracted after infection with ts342 at 43°C than after infection with wild-type T7 at the same temperature. This result was seen in several experiments. Although T7 RNA was translated within the linear range of total [35S]methionine incorporation, the presence of T7 late mRNAs in RNA extracted from wild-type T7-infected cells at five minutes could have resulted in discrimination against 0.3 mRNA translation in vitro (see last section of the Discussion). It is striking that the kinetics of O-3 protein synthesis in viwo (Fig. 7(a)) and 0.3 mRNA activity in vitro (Fig. 4(b)) were more similar after infection of cells at 43°C by ts342 than by wild-type T7. The kinetics of O-3 protein synthesis in vivo and 0.3 mRNA activity in vitro after infection of cells by wild-type T7 at 43°C resembled that seen by Hopper et al. (1975) in a wild-type infection at 30°C. Namely, there was little 0.3 protein synthesized in viva at late times in infection, whereas the 0.3 mRNA activity in vitro had declined only about 40%. However, after infection by ts342 at 43”C, 0.3 protein synthesis declined in parallel with 0.3 mRNA activity in vitro and persisted at a level comparable to the maximum level observed in cells infected with wild-type T7. Thus 0.3 mRNA was expressed more as a function of its concentration in vivo in ts342-infected cells than in cells infected with wild-type T7 at 43°C. Persistence of 0.3 protein synthesis late in T7 infection is not limited to E. coli infected with mutant T7. Reduction of T7 late mRNA synthesis also occurs during abortive infection of male E. coli cells by wild-type T7 (Young & Menard, 1975): 0.3 protein is synthesized at a much higher rate late in infection of male cells than at corresponding times in infection of female cells. Thus the overproduction of 0.3 protein observed at late times in cells infected with the temperature-sensitive gene 1 mutant also occurs in male cells infected with wild-type T7.

(c) mRNA

excess

The hypothesis that T7 infection develops in mRNA excess is based on [35S]methionine incorporation into T7 late proteins in viva (Figs 6 and 7) and the amount of of rates of protein synthesis lysozyme activity in vivo (Fig. 10). S ince comparisons based on [35S]methionine incorporation require knowledge of the specific activities of the methionine pools, which were unknown, we measured the accumulation of a specific late protein, lysozyme, and found that its activity correlated closely with quantitation of that same protein by pulse labeling and continuous labeling. In this way we confirmed indirectly that band intensities of proteins labeled in cells infected with wild-type T7 and ts342 reflect rates of protein synthesis. Both [35S]methionine incorporation and enzyme activity demonstrated similar rates of late protein synthesis in cells infected with wild-type T7 and ts342 at 43”C, despite the three- to fourfold difference in concentrations of T7 late mRNA. Since T7 late protein bands on sodium dodecyl sulfate/polyacrylamide gels reached the same intensities and, since there was at least SOo/o as much lysozyme activity in cells infected with ts342 as with wild-type T7-infected cells, it seems probable that the putative repressor of early protein synthesis hypothesized by Studier (1972,1975) would be present at similar concentrations in ts342 and wild-type T7-infected cells. Therefore, the overproduction of 0.3 protein late in infection of cells by t,s342 is probably not attributable to the underproduction or absence of the hypothesized repressor. It is not possible at this time to extend the findings on 0.3 protein synthesis to

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synthesis of all of the T7 early proteins. In contrast to the sensitivity of 0.3 mRNA translation to the level of T7 late mRNA, translation of gene I mRNA was unaffected by reduction of T7 late mRNA levels. Since gene 1.1 polypeptide is not seen on OUI gels, and the products of genes 0.7 and 1.3 comigratr with T7 late proteins, it has not been possible to study their synthesis late in infection when T7 late mRNA levels have been reduced. Also, since the leftmost promoter for T7 RNA polymerase is in the right end of gene I (Oakley & Coleman, 1977). t ranscription of genes 1.1 and 1.3 continues after transcription of the early region by E. coli polymerase ha,s been shut off. Yamada et al. (1974a.6) hypothesized functional inactivation of TT early mRNA and detected a shift in migration rate on polyacrylamide gels of a large early radioactive mRNA, perhaps gene 2 mRNA, extracted late in infection. We found that two functional early mRNAs, from genes 0.3 and 7. did not differ detectably in mobility when they were isolated at 21 minutes compared bo 6 minutes a,fter infect’ion (Strome & Young, unpublished result; Pachl & Young, 1978). In a recent review on translational control of protein synthesis Lodish (1976) discusses the effect of different rate constant,s for polypeptide chain initiation on differential translation of mRNAs. If a component required for initia,tion of translation is limiting, the mRNAs with the lowest initiation rate constants are discriminated against, while the mRNAs with the highest rate constants are translated preferentially. In addition to indirect evidence indicating translational discrimination against, 0.3 mRNA in viva, we have direct evidence for inefficient translation of 0.3 mRNA compared to T7 late mRNAs (Strome $ Young. unpublished observation): 0.3 protein synthesis decreases Gn vitro as the T7 lat,e mRNA concentration is increased beyond the level needed to saturate the translational machinery, whereas synthesis of individual T7 late proteins remains constant. This supports the hypothesis of mRNA competition for a rate-limiting component of the translational machinery. anti Milt (:ordo~i for critical We wish to thank Drs Richard Palmiter, Davc~ Morris, evaluation of this manuscript and Drs W. C. Summers and F. W. Studier for supplying tlrc* bacteria and phage. We are also grateful to Rose Mnnard for technical assistance with some of the experiments and to Carole McCutcheon for help in preparation of the manuscript. This research was supported by National Institutes of Health grant AI-09456. One of IIS (S. S.) was supported by National Institutes of Healt,h training grant 5T32 GM07270. .KEFERENCKS Brunovskis, I. & Summers, W. C. (1971). l’irology, 45, 224- 231. Brunovskis, I. & Summers, W. C. (1972). I’irology. 50. 322-327. Chamberlin, M., McGrat,h, .I. & Waskell, 1,. ( 1970). .vatrcre (/,on,rlo,r,). 228. 227 23 I. Crawford, L. V. & Gesteland, R. F. (1973). ,/. 11101. Bid. 74. 627 -634. Hagen, F. & Young, E. T. (1973). l’irology, 55, 23~241. Hagen, F. & Young, E. T. (1978). J. r’irol. 26, 7!)3-804. Herrlich, I’., Rahmsdorf, H. J., Pai, S. H. & Sctiweigrr. M. ( 1974). I’roc,. LVat. ,dcarl. Sci.. U.S.A. 71, 1088-1092. Hopper, J. E., Ko, G. & Young, E. T. (1975). .I. .IIol. Hiol. 94. 5399554. Krisch, H. M., Van Houwe, G., Belin, D., Gibbs, W. & Epst)ein, R. H. (1977). I~irology. 78, 87-98. Lodish, H. F. (1976). Annu. Rev. Rio&em. 45, 3972. Marrs, B. L. 8: Yanofsky, C. (1971). Nature New Riol. 234, 168-170. McKeehan, W. L. (1974). J. Biol. Chem. 249, 6517-6526. Oakley, J. L. 8: Coleman, J. E. (1977). Proc. Nat. =Lcad. Sci., Cy.S.A. 74. 4266 4250. Pachl, C. A. & Young, E. T. (1976). Proc. XTat. Acad. Ax’., Ii.S..3. 73. 312 316. Pachl, C. A. & Young, E. T. (1978). .J. Mol. Sol. 122, fX-102.

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Palmiter, R. D. (1974). J. Biol. Chem. 249, 6779-6787. Rothman-Denes, L. B., Muthukrishnan, S., Haselkorn, R. & Studier, F. W. (1973). In Virus Research (Fox, C. F. & Robinson, W. S., eds), pp. 227-239, Academic Press, New York. Russel, M., Gold, L., Morrissett, H. & O’Farrell, P. Z. (1976). J. Biol. Chem. 251, 7263-7270. Siegel, R. B. & Summers, W. C. (197O).J. Mol. Biol. 49, 115123. Studier, F. W. (1969). Virology, 39, 562-574. Studier, F. W. (1972). Science, 176, 367-376. Studier, F. W. (1973). J. Mol. BioZ. 79, 237-248. Studier, F. W. (1975). Proc. Tenth FEBS Meeting, 45-53. Summers, W. C. (1970). J. Mol. BioZ. 51, 671-678. Summers, W. C. & Siegel, R. B. (1970). CoZd Spring Harbor Symp. Quant. BioZ. 35, 253-257. Summers, W. C. & Szybalski, W. (1968). Virology, 34, 9-16. Yamada, Y. & Nakada, D. (1976). J. Mol. BioZ. 100, 3545. Yamada, Y., Whitaker, P. A&. Nakada, D. (1974a). Nature (London), 248, 335-338. Yamada, Y., Whitaker, P. A. & Nakada, D. (19745). J. Mol. BioZ. 89, 293-303. Yamamoto, K. R. & Alberts, B. M. (1970). Virology, 40, 734-744. Young, E. T. & Menard, R. C. (1975). J. Mol. BioZ. 99, 167-184. Zimmerman, S. B. & Sandeen, G. (1966). Anal. Biochem. 14, 269-277.