- Email: [email protected]
Translational discrimination against bacteriophage T7 gene 0·3 messenger RNA
J. Mol.
Biol.
(1980)
Translational
136, 433450
Discrimination Against Bacteriophage T7 Gene 0.3 Messenger RNA SUSAN STROME-~AND
ELTON T.YouNG’~
Department of Biochem,istry University of Washington Seattle, Wash. 98195, U.S.A. (Received
8 August
1979)
Based on genet’ic manipulation of T7 l&e messenger RNA levels in viva, we previously hypothesized that wild-type T7 infection of Escherichia coli develops in mRNA excess and that there is translational discrimination against T7 gene 0.3 mRNA (Strome & Young, 1978). The results presented here support our hypothesis. The discrimination against 0.3 mRN,4 translation observed in. viva can be mimicked in a cell-free system by increasing the concentration of T7 RNA beyond the level needed to saturate the translational machinery or by translating T7 RN,4 with a low concentration of ribosomes. This discrimination can be overcome by adding ribosomes to the cell-free system (increasing the ribosome to mRNA ratio) or by slowing the rate of polypeptide chain elongation. In addition 0.3 mRNA activity as well as a substantial fraction of T7 late mRNA activity is found to be shifted off of polysomes late in T7 infection, Our results are indicative of a low initiation rate constant for 0.3 mRNA compared to T7 late mRNAs.
1. Introduction As described in the accompanying paper (Strome & Young, 1980), T7 gene 0.3 protein synthesis is shut-off at lat’e times during T7 infection when chemical and functional 0.3 messenger RNA persists. The hypothesis that O-3 gene expression is at least partially controlled at the level of translation was initially tested by genetically manipulating T7 late mRNA levels ill vivo (Strome & Young, 1978). Reduction of T7 late mRNA levels in T7-infected Escherichia coli did not result in reduced T7 late protein synthesis, suggesting that wild-type T7-infected cells are normally in mRNA excess. In the presence of reduced levels of T7 late mRNA, O-3 mRNA was translated much more efficiently than in the presence of wild-type levels of late mRNA, which is consistent with discrimination against 0.3 mRh’A translation in the presence of T7 late mRNA levels that saturate the translational machinery of the cell. It is not necessary to invoke a specific mRNA-discriminatory factor to explain the translational discrimination against 0.3 mRNA. Rat’her, mRNAs differ in the inherent rates at which they initiate protein synthesis, as best illustrated by studies of ‘/’ Present of Colorado, $ Author
address: Boulder, to whom
Department of Molecular, CO 80309, U.S.A. reprint requests should
Cellular
and
Development.al
Biology,
University
be addressed. 433
0022-2836/80/040433-18~$02.00/0
Q 1980 Academic
Press Inc.
(London)
Ltd.
S.
434
RTROillll:
h&I)
E.
T.
YOUNG
lysates /I globin mRNA than CCglobin mRNA (Lodish & ,Jacobsen, 1972; McKeehan, 1974: Lodish, 197lh). A kinet’ic analysis of protein synthesis (Lodish, 1974,1976) predicts that if a component, required for initiation of translation were limit’ing, those mRNAs wibh the lowest rate constants for initiation would be discriminat’ed against. Non-specific reduction of polypeptide chain alongabion should prevent discrimination at the initiaCon stage of protein synthesis and result in protein synthesis more as a funct,ion of relative mRNA concentrations and less as a function of initiation rate constant,s. These predictions have been borne out nit,h GCand /3 globin synthesis (McKeehan, 1974; Lodish, 1971b,1974), as well as with the translation of ovalbumin and conalbumin mRNA, t,wo oviduct mRNAs that differ in their rates of translational initiation (Palmiter, 1974). In this paper, experiments are described that test the applicability of Lodish’s hypothesis of differential mRNA t,ranslation to control of 0.3 mRNA translation. The results demonstrate t,hat the t’ranslational discrimination against 0.3 mRNA observed in z&o can be mimicked in a cell-free translational system and that this discrimination can be overcome by increasing the ribosome to mRNA ratio or by slowing the rate of polypeptide chain elongation. The results are also consistent with T7 infection developing in mRNA excess. C( and
fl globin
initiates
synthesis.
protein
In
synthesis
ret,iculocytes
400/:,
rnort
2. Materials (a) E’. coli
BSt and
wild-type
Bacterial T71,
and
chemicals
are described
ret~iculocytc
and Methods
cmd bacteriophage
(wild-t,ype (b) Media
Media
and
rfficicntly
T7) and
wcrc
paper
(d) of S30 extracts for fractionation (1977). (e)
(Strome
F. W.
Studier.
& Young,
1980).
Fractionation
Strome & Young (1978), light irradiation of cells, cells, determination of of 35S-labelled proteins,
of 830
leas been described elsewhere (Hagen of S30 extract into SlOO and crude
Cell-free
Dr
procedures
The following methods are described by Hopper et al. (1975), Studier (1973), and Hagen & Young (1973,1978): ultraviolet labelling T7 proteins in viva, RNA extraction from T7-infected [35S]methionine incorporation, preparation and electrophoresis densitometric scanning of autoradiograms.
The prepasation and the procedure Belin & Epstein
from
chemicals
in the accompanying (c) General
strains
obtained
protein
& Young, ribosomes
1973) is from
synthesis
The details of cell-free protein synthesis are essentially those described by Hagen & Young (1973) with the following changes. Instead of 48 mm-NH,Cl in t,he cell-free synthesis reaction, 110 mM-NH,Ac was used. DNase at a final concentration of 2.5 pg/ml was added to inhibit synthesis of endogenous E. coli RNA in t,he S30 extract. The concentration of 530 (or SlOO plus ribosomes) used in the protein synthesis reactions varied as indicated in the Figure legends. Synthesis of labelled polypeptides was achieved by adding 2 to 10 &i [35S]metliionine to 100 ~1 total reaction mixture. No unlabelled
0.3
mRNA
435
DIRCKIMIKATION
methionine was added to the reactions. At the end of tlio synthesis, the reaction mixtures were immediately chilled and processed for olectrophoresis. Tlre duration of linear [35S]mrthionine incorporation varied somewhat between S30 preparations. Wlren necessary the length of incubation was reduced below the usual 20 min to maintain the linear relationship between time and incorporation. (f) The
procedure
for
Extraction
of polysomes
extracting
polysomes is essentially t)hat of Linda111 & Forchhammer wit11 aeration in MQ medium, at 37”(.‘, to a cell density of 6 x 10E/ml. The cells were infect,ed with T7 with a multiplicity of infection of 10. Before and at irltervals during infection l&ml samples were removed to tubes wit,h IO g ice and 1.5 ml I M-NaN,. The cells were harvested by centrifugat)ion at, 10,000 revs/min for 5 min at 5°C. x.\-aslled wit11 1 ml lysis buffer (5 mM-Tris (pH 7.5), 5 m>l-MpSO,, GO m&l-KCl, ISIS, (w/w) sucrose, 0.01 M-NaN,, and 300 pg lysozyme/ml), and collected again by centrifuRation. The pellet was frozen in solid COz/acetone and could be st’ored at -70°C. The pellet, was thawed at O”C, resuspended in 50 ~1 lysis buffer, incubated 10 min at O”C, frozen ill solid CO,/acetone, and thawed at 0°C. The cells were lysed by adding 0.6 ml TMK hufk (5 mM-Tris (pH 7.5), 5 m&f-MgSO,. 60 mnr-K(J). 60 ~1 5:/, Rrij 58, 60 ~1 59, deoxycholate, and 15 ~1 DNase (1 mg/ml). The lysatc was incubated for 10 min at 0°C and cerltrlfilged at 10.000 revs/mill for 10 min at 5’C. The supernatant was layered ont)o an II-ml 15”‘, t,o 4i0t ,;, (w/w) sucrose gradient in TMK buffer. Tile gradient was cent.rifllped for 90 min at 4O:OOO revs/min at 5°C and collected using an Isco gradient fractionator. The absorbance at 254 nm was monit,ored continuously as tile gradient, was collected. Tllr fractiorls were made 0.5% in sodium dodecyl sulplmte and 0.2 ~1 in NH,Ac and tllcrr exkacted wit’h an equal volume of phenol saturated in 50 mM-NH,Ac and an equal volume of chloroform. Phases were separated by cenkifugation at 2000 revs/min for 5 min. The aqrleous phase was re-extracted with 2 l-01. chloroform. and the RNA was precipitated with 2.5 1.01. cold 95% ethanol. Tile precipitate was collected by centrifugal&r either at 3000 Ir>vs/min for 90 min or at 10.000 revs/min for 20 to 30 min, washed with 2 vol. cold 95:/o ettlanol, lyophilized, and resuspended in an appropriato volume of water for cell-free translat~i~)t1.
(1969). E. coli RSt was grown
3. Results (a) RNA
saturation
of the translational
machinery
in vitro
In an att,empt to mimic in vitro the situation late in T7 infection, when mRNA is apparently in excess of the translational machinery, increasing concentrations of T7 were translated in a cell-free translational system with a constant amount of E. coli 530 extract. The RNA has been extractled from cells 14 minutes after T7 infection, a time at which the majority of T7 late gene transcription is complete (Hopper et al., 1975). Since O-3 mRNA is relatively stable, both chemically and functionally (see accompanying paper, Strome & Young, 1980), 14 minute RNA contained a substantial amount of functional 0.3 mRNA. The [35S]methionine-labelled proteins that were sYynthesized in vitro were analyzed by SDSt/polyacrylamide gel electrophoresis followed by autoradiography (Fig. 1 (a)) ; q uantitation of individual mRNA activities is shown in Figure I(b). As the concentration of T7 mRNA was increased beyond the level needed to saturate the translational apparatus, [35S]methionine incorporation into total protein and individual late proteins reached a maximum and then remained t Abbreviations prokin. 21
used:
SDS,
sodium
rtodecyl
sulphate,
15K
protein,
15,000
molecular
weight
4x
S. STROME
ANI)
E.
T.
YOCNG
too _
Gene products
41
”
-
-P9 -
PlOh
PO.7, PI0
‘-
P (DUP),
-
PII
-
P3.5
-
Pl5K PO-3
PI.3,
P6
0
‘, ;
14.4
-
-
FIG. 1. Cell-free translation of increasing concentrations of T7 RNA with a constant concentration of S30. The RNA used in this experiment was extracted from u.v.-irradiated E. coli Bat 14 min after T7 infection at 30°C. The cell-free synthesis reactions (100 ~1) wore carried out at
0.3
6
mRNA
16 24
DISCRTMINRTION
32
40
56
48
RNA translated
FIG.
ii, vitro
437
64
72
80
80
(pg)
l(b).
constant. However, synthesis of 0.3 protein reached a maximum and then declined as the translational machinery became saturated. In several experiments the rate of 0.3 protein synthesis using high RNA concentrations ranged from 35 to 50% of the maximal O-3 protein synthetic rate. Based on the experiments described in the accompanying paper (Strome & Young, 1980), 0.3 mRPu’A is translated at 25 t,o 300/b efficiency in vivo late in T7 infection. Thus the discrimination against 0.3 mRNA translation in vitro is similar t’o that seen in vivo at late times during T7 infection. (h)
Sensitivity
of 0.3
mRNA
translation
to rihosome
concentration
The sensitivity of protein synthesis, especially 0.3 protein synthesis, to the concentration of translational component’s was tested in two ways. In the first experiment, 14 minute T7 RNA was translated using half the normal concentration of X30 extract, with either additional ribosome-free supernatant (SlOO) or additional crude ribosomes. In the second experiment, T7 RNA was translated using a constant amount of ribosome-free SlOO and various concentrations of crude ribosomes. The labelled proteins synthesized in the reconstituted systems were electrophoresed on SDS/ polyacrylamide gels, an autoradiogram of which is shown in Figure 2. The results are quantitated in Table 1.
37°C’ for 20 min using the RNA concentrations indicated and an 630 concentration of 2.8 mg/ml. A portion (10 ~1) of each reaction was trichloroncetic acid-precipitated to determine [?!J]methionine incorporation into total protein, and the remainder was acetone-precipitated and processed for electrophoresis as described in Materials and Methods. (a) Equal volumes of the 9. labelled proteins synthesized in vitro were run on an SDS/13.61& polyacrylamidc gel at 10 mA for 1 h and at 25 rnA for 3 h. (b) The intensities of individual protein bands from an autoradiogram equivalent to the one shown in (a) were quantitated by microdensitometric scanning as described in Materials and Methods. (0) [35S]methionine incorporation into total protein; (O), PO.3; (A) , 1’3.5. 3 (C), I’(Dvl’).
-.
(a I (b)
.*,r
Cd)
-Pl5K -PO.3
0.3
mRNA
DISCRIMINATTON
TABLE In vitro
Crude ribosomes
synthesis of T7 proteins [%]methionine incorporation total proteint LOWS
s30
1
at different
into
0.3
Highs
439
concentrations
of crude ribosomes
proteinj!
LOWS
15K High
5
Low
proteinf $
Hi&S
35,000
94,000
2.3
2.3
6.7
12.2
2 mg/ml
76,000
117,000
21.4
10.6
18.0
26.2
0.5 mg/ml
41,000
94,000
11.0
16.3
2 mg/ml
72,000
116,000
22.4
15.0
23.3
28.2
5 mg/ml
76,000
130,000
29.6
24.6
21.6
47.2
SlOO
3000
6000 3.3
t Trichloroacetic acid-precipitable [Wlmethionine $ Relative densitometric units. 5 Low RNA concentration, 92 pg/ml; high RNA The data are from the experiment) shown in Fig.
3.1
incorporation concentration, 2.
(cts/min). 650
pg/ml.
The addition of SlOO to the S30 extract did not significantly stimulate total incorporation into protein or specific incorporation into 0.3 protein (Fig. Z(a) and (b)). In contrast, synthesis of 0.3 protein was very sensitive to the concentration of ribosomes (Fig. Z(a) to (d)). As previously observed by Belin & Epstein (1977), total [35S]methionine incorporation into protein did not vary proport!ionally to ribosome concentration, and most of the change in protein synthesis was seen at low ribosome concentrations. Total protein synthesis only increased by a factor of about two between 0.5 and 2 mg/ml ribosome concentration, and most of the T7 proteins analyzed by gel electrophoresis were stimulated only slightly by higher concentrations of crude ribosomes (Fig. 2(a) to (d)). A benfold increase in the concentration of ribosomes caused a ninefold increase in 0.3 protein synthesis (Fig. 2(c)). A slightly lower (eightfold) stimulation was observed at the higher of the two RNA concentrations tested (Fig. 2(d)). The T7 late protein that migrates just above 0.3 protein on 13% polyacrylamide gels (15 K protein) was also somewhat sensitive to ribosome concentration, but was only stimulated threefold by a tenfold increase in the concentration of ribosome.
(c) EJfects of in.hibitors
of translational
elongation
and initiation
If 0.3 mRNA is at a translational disadvantage relative to late T7 mRNAs when translational initiation is limiting, then partial inhibition of translational elongation should inhibit 0.3 protein synthesis less than synthesis of other T7 proteins (Lodish, 1974,1976). To test this, the effect of fusidic acid, an inhibitor of the ribosomeassociated GTPase activity of elongation factor G (Tanaka et al., 1968) was studied. T7 RNA was translated in a cell-free system in t’he presence of concentrations of fusidic acid that inhibited [35S]methionine incorporation into total protein by 20 to
440
S. STROMI”
;\NI)
E.
‘I’.
YOUNG
9()0,;,. Wllcn a lo~v concentration of T7 RNA (92 pg/ml) mns translated in vitro in the presence of fusidic acid, inhibition of 0.3 protein synthesis was very similar to inhibit,ion of total prot,rin synthesis and synthesis of other T7 prut,eins (Fig. 3(a)). This bvould be expected if. at R low conceIlt,r~~t,ion of RNA. all mRNAs ~vere translated as a function of their relatiw conc:ciitrations. which is t,hc basis for quantitation of mRNA by cell-free translation (Hopper et al.. 1975). However. at a concentration of RNA that saturated the translational machinery in the absence of inhibitor (650 pg/ml), fusidic acid inhibited 0.3 protein synthesis less than total protein synt,hesis or synthesis of other T7 protein (Fig. 3(b)). Conversely. inhibition of translational initiation should result in preferential inhibition of translation of those mRNAs with the lowest initiation r&e constants. When T7 RPU’A was translated in a cell-free system in t,he presence of concentrations of kasugamycin, an inhibit,or of initiation (Tai et al., 1973), that inhibited r3%]mcthionine incorporat,ion int)o protein by 35 to 75:/i,, 0.3 protein synthesis was preferentially reduced relat’ive to synthesis of totma1 protein and other T7 proteins (Fig. 3(c) and (d)). This was more pronounced when a low concentration of T7 RNA (92 pg/ml: Fig. 3(c)) was translated, perhaps because 0.3 mRNA translation was alrea,dy being discriminated against at t’he high RNA concent’ration (650 pg/ml; Fig. 3(d)). Since synt’hesis of t’he T7 late protein which migrat’es just above 0.3 protein on our gels (15K protein) was inhibited by fusidic acid and kasugamycin t’o about the same extent as t)otal protein synthesis, it was used as a reference. The ratio of the intensity of the 0.3 prot,ein band to the intensity of the 15K protein band was measured after translat’ion of T7 RNA in t,he absence and presence of inhibitor (Pig. 3(e) and (f)). This ratio, 0.3: 15K, decreased from 1.2 to 042 when increasing concentrations (92 and 650 pg/ml, rcspect~ivelp) of T7 RNA were translated in the absence of inhibitor (Figs 1 and 3(e) and (f)), presumably reflecting discrimination against 0.3 mRNA translation. At’ t)he low RN,A concentration (Fig. 3(e)), fusidic acid increased the ra,tio of 0.3 : 15K protein from 1.2 to 1.8, and kasugamycin decreased the ratio from 1.2 t’o 0.53. More int’crestingl,v. at the high concentration of RNA (Fig. 3(f)), fusidic acid increased the ratio from 0.42 to I .4. i.e. back t’o the ratio of 0.3 : 15K protein seen at the low RNA concentration where protein synthesis reflects mRNA concentration. A similar increase in the ratio of 0.3: 15K protein, from 0.3 to 1.4, was obtained in the previous section when the concent,ration of crude ribosomes was increased tenfold (Fig. 2). The effect’s of fusidic acid and kasugamycin on T7-infected E. coli were also examined in ‘cj~o. Kasugamycin or fusidic acid was added 6 minut,es aft,er infection with T7 at 42”C, a time when 0.3 mRNA translation \\-as being discriminated against. Two minutes later T7 prot,eins were pulse-labelled with [35S]metllionine. Kasugamycin resulted in preferential inhibition of 0.3 protein svnthesis (Pig. 4(b)), and fusidic acid inhibited 0.3 protein synthesis less than total protein synthesis (Fig. 4(a)). The ratio of 0.3 protein: 15K protein synthesis irr viva was decreased from 0.2 to 0.09 by kasugamycin and increased from 0.2 to 0.39 by fusidic acid (Fig. 4(c)). The similarity in the effects of the protein synthesis inhibitors on bn ~iwo and in vitro 0.3 mRPiA translation further validates our use of a cell-free t,ranslational systetn to mimic T7 infection in viva.
0.3
mRNA
DISCRIMINATION
441
100 80 60 40 20
Fusidic
acid
(mt.+)
100 80 60 40 20
Kasugomycln
i?
(pg/ml
)
--
0
I
I
0.01 3.3
0.02 IO
I
0.02 IO
I
0.04 100
I
0.06 1000
fus kos
and kasugamycin on cell-free synthesis of T7 proteins, especially (extraction described in legend to fig. 1) at a concentration of with 2.8 mg S30/ml at 37°C 92 &ml ((a), (0) and (0)) or 660 &ml ((b), (d) and (f)) was translated for 15 min in the presence of increasing concentrations of fusidic acid ((a) and (b)) or kasugamycin ((c) and (d)). The cell-free reactions were acetone-precipitated and processed for gel electrophoresis as described in Materials and Methods. Equal volumes of the 35S-labelled proteins synthesized in vitro were either trichloroacetic acid-precipitated to determine [35S]methionine incorporation into total protein or run on an SDS/13.576 polyacrylamide gel at 10 m-4 for 2 h and at 25 mA for 3 h. On an autoradiogram of the gel, the 0.3 protein band and 15K protein band were microdensitometrically scanned. (a) to (d) Both [%]methionine incorporation into total protein (-e-o---) and into 0.3 protein (--O--O--) are expressed as y. of the values obtained in the absence of inhibitor. (e) and (f) The ratio of 0.3 protein band intensity: 15K protein band intensity (from the autoradiogram of the gel described above) was plotted as a function of concentration of fusidic acid (-A-A-) or kasugamycin (--A--.h--) for both concentrations of RN.4: 92 pg/ml (e) and 650 &ml (f). 0.3
FIG. 3. Effects of fusidic acid protein. 14 min T7 RNA
I
44”
loo 80 60 40 20
Fusldlc
acid
(ml
20 t I
I 5
I IO Kasugamycln
I 15 (pg /mi
I 20 i
__-
0.6 r---
--
(c)
0.05 5
O-I IO
0.15 I5
0.2
20
fus kas
hG. 4. Effects of fusidic acid and kasugamycin on synthesis of T7 proteins, especially 0.3 protein, in oivo. I\ culture cd IC. coli BSt was u.v.-irradiated and infected with wild-type T7 with a multiplicity of infection of 10 at 42°C. (Similar results were obtained at a lower temperature.) At 6 min after infection, l-ml samples of infected-cell culture were atlrled to tubes containing either fusiclic acid (a) or knsugamycin (b), result,ing in the concrntr:tt.ions of inhibitor inclicittod in the Figure. Proteins wwe pulse-labellerl with [3”S]methionine from 8 to 10 min after infection, as described in Materials and Methodx. Cells WRI‘C collected and lysed, and the “5S-labellec~ proteins synthesized in viw wore prepared for gel electrophoresis. Equal volumes of the labelled proteinx mere either trichloroacetic tlcitl-l)recipit,atc,(i to determine [W]mcthionine incorpordion into total protein or run on an SDS/13’Jh polyacrylamide gel at 10 mA for 1 h 45 min and at 25 m;Z for 3 h. On an autordiogram of the gel, the 0.3 prutcin hand and 15K protein band were den&metrically scanned. (a) and (b) Both [358]mothionine incorporation into total protein (-+-a--) and into 0.3 protein (---C--C)--) are expressed as :$ of the value obtained in the absence of inhibitor. (c) Thr ratio of 0.3 band intensity: 15K band intensity was plotted as a function of concentration of fusitlic acid ( -A---A-) or kasugamycin (--,/1--,R--).
0.3
mRNA
DISCRIMINATION
443
(d) Distribution of T7 mRNA activities on polyribosomes Our hypothesis of translational discrimination against 0~3 mRNA in the presence of saturating levels of T7 late mRNA in T7-infected cells predicts that 0.3 mRNA should be shifted off of polysomes at late times in T7 infection and at least some T7 late mRNA should also be free of ribosomes. To test these predictions the association of T7 mRNA activity with polysomes was analyzed. Figure 5(a), (b) and (c) shows the absorbance profiles of polysomes extracted before T7 infection and after 6 and 20 minutes, respectively. It is apparent that T7 infection affects the polysome profile. resulting in an increase in ribosomal subunits and a shift of polysomes to higher molecular weight. As expected the polysomes were sensitive to RNase and were quantit,at’ively converted to monosomes (data not shown).
1
(a)
(b)
40 20 0 40 20
IOh
0 40 20 0
FIG. 5. Comparison of polysome profiles and of the association of T7 mRNA activities with polysomes early and late in T7 infection of unirradiated E. coli BSt. Polysomes were extracted before infection (a) and at 6 min (b) and 20 rnin (c) after T7 infection of unirradiated E. cc& BSt at 30°C. Polysomes were sedimented in 15O4, to 459; sucrose gradients. Sedimentation is from left to right. Arrows indicate monosome peaks. The peak to the far right in (c) is presumably mature virions. The protein bands representing genes 0.3, 10 h, and 11 on the autoradiogram shown in Fig. 6 were microdensitometrically .xanned. For each protein the total mRNA activity per sucrose gradient, was set at loo%, and t,he mRNA activity per fraction is expressed as 3/o of the total mRNA activity.
S.
444
STRO3lE
ANI)
E.
1’.
YOUXG
RNA was extracted from the sucrose gradient fractions and translated in a cell-free translational system. The labelled proteins synthesized in vitro were analyzed by SDS/polyacrylamide gel electrophoresis and autoradiography (Fig. 6). The majority of host mRNA activity in uninfected cells was associated with polysomes (Fig. 6(a)). Early in T7 infection most of t,he T7 mRNA activity was also associated with polysomes, although there was some mRNA activity in the supernatant (La) and subunit (Ib) fractions of the sucrose gradient (Fig. 6(b)). By 20 minutes after infection, however, a substantial fraction of mRNA activity was dissociated from polysomes (Fig. 6(c)), which is consistent with T7 infection developing in mRNA excess. Several T7 protein bands on t)he autoradiogram shown in Figure 6 were analyzed quantitatively, and the histograms in the lower panel of Figure 5 display the distribution of T7
10057 -i
ii-
41 -
b x
i .F 5
A-
(b)
A Gene products -. II* -4m
c-1 PI9 -P8
PlOh P9 PO.7, PI0
PI.3
29 P29K PII
14.4
-
P 0.3
6. Autoradiogram of SDS/13”/0 polyaccrylamide gel of cell-free protein synthesis directed RNA extracted from SncloSe gradient fmctionation of polysomes. The Swrose gradients from Fig. 5 were collected as 5 fractions: Ia = supernatant, Ib = ribosomal subunits, II = monosomes, III = disomes, trisomes and tetrasomes, and IV = polysomes of 5 and larger. The RNA was extracted from each fraction and translated in a cell-free system, as described in Materials and Methods. Equal volumes of the 35S-labelled proteins synthesized in vitro were run on an SDS/13% polyacrylamide gel at 10 mA for 1.5 h and at 25mA for 3.5 h. (a) to (c) Correspond to (a] to (c) of Fig. 5, respectively. In (c) fractions Ia and Ib were collected as one fraction (I). FIQ.
by
0.3
mRNA
DISCRIMINATION
445
mRNA activities in sucrose gradient fractions. The association of O-3 mRNA activity with polysomes, monosomes and supernatant and subunits was 62%, 20% and IS:/,, respectively, at 6 minutes after infection (Fig. 5(b)) and 40%, 20% and 40%, respectively, at 20 minutes after infection (Fig. 5(c)). Thus early in infection (Fig. 5(b)) most of the 0.3 mRNA activity was associated with polysomes, a substantial fraction of that with polysomes of five or larger. Later in infection, 0.3 mRNA was shifted off of polysomes and 40 to 50% was found in the supernatant and ribosomal subunit fract’ions. The distribution of T7 late mRNA activities varied considerably. There were two basic patterns of T7 late mRNA distribution. Some T7 mRNAs, as exemplified by mRNA for genes 8, 11 and 19 were almost tota,lly associated with polysomes. This is shown for gene 11 mRNA in Figure 5(c). However, some T7 late mRNAs, such as genes 9 and 10 mRNAs, were found in t’he supernatant and subunit fractions (I) as well as t,he polysomal fractions of sucrose gradients (Fig. 5(c)). Late in T7 infection, the mRNA coding for the protein of about 29,000 M,, which appears to be a host protein, was found exclusively in the supernatant and subunit fractions of the sucrose gradient. If the shift of mRNA off of polysomes is caused by excess late mRNA, then a reduction of the amount of late mRNA should allow a larger proportion of both 0.3 mRNA activity and late T7 mRNA activity to remain associated with polysomes. Late mRNA levels can be reduced by infecting E. coli with the gene 1 temperaturesensitive mutant, ts 342, at a semi-restrictive temperature (Strome & Young, 1978). Polysomes were extracted from cells infected with wild-type T7 or ts 342 at 42°C and sedimented through sucrose gradients, and RNA was extracted from the sucrose gradient fractions and translated in a cell-free system as described above (Fig. 7). At 3 minutes after infection by wild-type T7 (Fig. 7(a))! 560,/, of the 0.3 mRNA activity was associated with polysomes, 26% was associated with monosomes, and 18% was found in t,he supernatant and subunit fractions of sucrose gradients. At 3 minutes after infection with ts 342 (Fig. 7(b)), h owever, 96% of the 0.3 mRNA activity was associated with polysomes and only 4% was free of rihosomes. Thus, as early as 3 minutes after infection by wild-type T7 at 42”C, 0.3 mRNA was being shifted off of polysomes, presumably due to the presence of late mRh’A. As late as 10 minutes aft’er infection at 42°C by ts 342, there was little 0.3 mRNA activity free of ribosomes (Fig. 7(d)), in contrast to the shift of 0.3 mRNA off of polysomes in cells infected by wild-type T7 (Fig. 7(c)). The association of 0.3 mRNA activity with polysomes, monosomes and supernatant plus subunits in ts 342-infected cells at 10 minutes was 520/,, 2776 and 21%, respectively, the same distribution as that observed at 3 minutes after infection by wild-type T7. The reduction in T7 late mRNA transcription in ts 342-infected cells also allowed a larger proportion of T7 late mRNA activity to remain associated with polysomes after infection by ts 342 than after infection by wild-type T7, as expected by our model of T7 infection developing in mRNA excess.
4. Discussion Results of the experiments described in this paper support our hypothesis that O-3 mRNA has a low initiation rate constant relative to late T7 mRNAs and is therefore
-
(a)
(b)
(c)
(d)
FIG. 7. Autorcldiogram of SDS/13.75% polyncrylamide gel showing cell-free translation of RX-1 extracted from polyson~~s from cells infected with wild-type T7 and ts 342 at 42°C. Polysomes were extracted at 3 min (a) and 10 min E. coli PF by wild-type T7 at 42°C and at 3 min (b) and 10 min (d) after infection of unirradiated ~011s by t’s 342 at fractions II, III and IV from fractionation of polysomen extracted from uninfected cells at 42°C. The extraction proteins svnt,hesized in and the numboring rrf fractions are as described in the legend to Fig. 6. The 35S-labelled polyacrylnm~rle gel at 8 m-4 for 1.75 h and at 25 md for 3 h.
PO-3
PI I
P9 PO, &7,P1.3
IOh
Pi9
Gene products
-----
.’
I
-
-
58
100
sucrose gradient fractionation of (c) after infection of u&radiated the same temperature. (e) Shows and cell-free translation of RNA vitro were run on an SDS/13.7576
(e)
0.3
mRN4
DISCRIMINSTION
447
at a translational disadvantage when it must compete with T7 late mRNAs for a limiting amount of translational machinery. Our results also provide strong support for Lodish’s model of translational control of protein synthesis. The discrimination against 0.3 mRNA translation seen in viva (Strome & Young, 1978) is also seen in vitro when the concentration of T7 RNA used to direct cell-free protein synthesis is increased beyond the level needed to saturate the translational apparatus. By fractionating the E. coli cell-free system into ribosome-free supernatant (SlOO) and crude ribosomes, it was possible to mimic the translational discrimination against, 0.3 mRNA seen in viva; the discrimination seen at low ribosome concentrations (low ribosome to mRNA ratio) could be relieved by increasing the concentration of ribosomes (increasing the ribosome to mRNA ratio). It appears that ribosomes (or some component contained in t,he crude ribosome fraction) limit kanslation of T7 mRNA by E. coli 530 extracts, since of the components tested, only ribosomes stimulated tot’al protein synthesis with these messages. The crude ribosome fraction has been further fractionated by NH,Cl into crude initiation factors and salt-washed ribosomes. Based on preliminary results, it appears that initiation factors do not limit translation of T7 mRNA by E. coli 530 extracts and do not have the same stimulatory effect on total protein synthesis or on 0.3 protein synthesis that ribosomes have (Strome, 1979). Using a DNA-dependent cell-free protein synthesis system, Benne 8r. Pouwels (1975) found that synthesis of T7 RNA polymerase and [14C]leucine incorporation into tot,al T7 protein showed no dependence on added IF-3. In contrast, synthesis of active enzymes encoded by the E. co& t,rp operon (using @O-trp DNA) and [ 14C]leucine incorporation into trp-protein depended strongly on added W-3. Our results are consistent with a low dependence of T7 protein synthesis on the concentration of initiation factors. Slowing of translational elongation by fusidic acid relieves translational discrimination against 0.3 mRNA in vivo and at high RNA concentrations in vitro (Figs 3 and 4). As seen with tl and ,!Iglobin (Lodish, 1971b) and ovalbumin and conalbumin (Palmiter, 1974), partial inhibition of translational elongation on T7 mRNA abolishes the inhibitJory effect caused by an apparently low initiation rate const,ant and allows 0.3 mRNA to be translated at a rate which is proportional to its relative concentration in the cell or in the cell-free extract. Consistent with a low initiat,ion rate constant for 0.3 mRNA, partial inhibition of pol.ypeptide chain initiation results in preferential inhibition of O-3 protein synthesis relative to tot,al protein synthesis both in viva and in vitro (Figs 3 and 4). In agreement with Mangiarot’ti & Schlessinger (1966) and Kennel1 (1970), our results indicate that in uninfected cells essentially all of the mRNA activity is associated with polysomes (Fig. 6(a)). I n uninfected E. coli there is a close correlation between mRNA concentrations and rates of protein synthesis in the gal operon (Miller et al., 1971), the lac operon (Varmus et al., 1970), and the trp operon (Imamoto et al., 1965). The presence of all E. coli mRNA on polysomes and the close correlation between mRNA concentration and protein synthesis in E. coli suggests that in uninfected cells there is an excess of translational machinery over mRNA and that mRNA may limit protein synthesis. In contrast to the situation in uninfected cells, a substantial fraction of T7 mRNA activity is found in the supernatant at the top of sucrose gradients and sedimenting in the region of t’he gradient~s where ribosomal
448
s.
S’I’IiOAl
I’ 1 .\Sl)
I.:. ‘I’.
Y‘oIiSic:
subunits sediment (Fig. B(C)). Thus. after infection I)?; wild-type T7, T7 mRNA must be in excess of the functional protcitl synthetic machinery of the cell. This contention is supported by the tindin g that. \vtlrrl T7 Iatta mRSA 1~~~1s n,rc reduced using the ts 342 mutant, a larger fract’ion of T7 latch mRSX ;lctivity is associated with polysomcs (Fig. 7(d)). The shift of 0.3 mR,EA activity from polysomes to the supernatant and rihosomal subunit fractions of sucrose gradients as infe&ion proceeds (Figs 6 and 6) supports our model of translat’ional discrimination against 0.3 mRNA late in wild-type T7 infection. Dunn et al. (1978) observed that 0.3 mRNA is shifted off of polysomcs in cells infected with T7 mutants containing base changes in the initiation region of gene 0.3. These mutants synt,hesized wild-type levels of 0.3 mRNA, hut the mutant, 0.3 mR&As are translated much less efficiently than wild-type 0.3 mRNAs. even at early times during infect,ion. We observe that under conditions where 0.3 mRNA is translated more efficiently in viva. namely when T7 late mRNA levels are reduced (Strome & Young. 1978); a larger proportion of 0.3 mRKA activity is associat,ed with polvsomes (Fig. 7). Tn our polysome analyses, the distribution of other T7 mRNAs in sucrose gradients varies. A substantial amount of some mRKAs (gene 9 and 10 mRNB) is dissociated from polysomes, while ot,her T7 mRNBs (genes 8, 11 and 19 mRNAs) are associated exclusively with polysomes (Figs 5 and 6). The differences in mRNA distribution probably reflect differences in initiation rate constant’s and the polycistronic nature of t’he various T7 mRSAs. For instance. the gene 10 protein. the major capsid protein, is t.ranslat.ed f?om at least, t,hree mRNAs (Pa&l R: Young, 1976). In addiCon to the monocistronic gene 10 mRKA, there are two polycistronic mRNAs : one coding for genes 9 and 10 and the other coding for genes 8. 9 and 10. Thus. translation of gene 8 protein and gem 9 protein will affect the apparent association of gcnf 10 mRNA activity with polysomes. The change in polysomc absorbance profile brought about l)y T7 infect.ion is not that predicted for a transition from limiting mRNA t,o mRNX excess. If T7 infection led to the synthesis of much higher mRNA levels than are present’ in uninfected E. COG, one would expect (1) functional 30 S and 50 S subunits to be recruited for protein synthesis and (2) a general decrease in polysome size. Inst)ead, upon T7 infect,ion there is an increase in ribosomal subunits and an increase in average polysome size (Fig. 5). One possible explanation for the increase in ribosomal subunits is a, T7induced inactivation of some component of the translational machinery. Tn &her words, TS infection might develop in apparent mRNA excess by reducing the amount, of functional translational machinerg instead of by synthesizing concentrations of ‘I’7 mRNA large enough to saturate t’he host’ translational machinery. Several findings are consistent with this possibility. (1) When T7 infects unirrsdiated E. cd, the rate of protein synthesis, as measured by j 35S]methiotline incorporation, declines progressively throughout infection (Strome, 1979). Tf this decrease is not an effect caused by a change in the specific activity of the methionine pool, it suggests that, T7 mRXB directs protein synthesis at a lower rate than is observed in uninfected cells. (2) Yamada 8: Nakada (1976) found that’ cell-free translational systems prepared from cells early in T7 infection showod increased ac%vit,y over uninfected-ccl1 cxt,ract’s. However, as infection proceeded there was a general decline in the translational capacity of infected-cell extra&s. The T7 early gene 0.7 codes for a protein kinase,
0.3 mRNA
DISCRIMINATIOK
449
which has been found to phosphorylate a variety of host proteins including RNA polymerase and ribosomes (Zillig et al., 1975; Rothman-Denes et al., 1973). Phosphorylation of ribosomes after T7 infection could lower the activity of the ribosomes. In this respect, however, ribosomal subunits do accumulate after infection of E. coli with deletion mutants either in gene 0.7 or gene 0.3 (Strome, 1979), suggesting that, if the accumulation of ribosomal subunits is due to inactivation of some translational components, the product of gene 0.7 or gene 0.3 (or 0.4) is not responsible. It is not clear why, if T7 infection develops in mRNA excess, polysomes extracted from T7-infected cells are larger than polysomes from uninfected E. co&. One possibility is tha,t the average size of T7 mRNAs is larger than the average size of E. coli mRNAs, perhaps as a result of the increased stability of T7 mRNA compared to E. coli mRNA. Tf this were the case, more ribosomes could be associated with T7 mRNAs than with E. coli mRNAs even if the ribosome density (number of ribosomes per unit length of RNA) were reduced upon T7 infection. The initiation region of the 0.3 mRNA has been sequenced by Steitz & Bryan (1978). O-3 protein synbhesis is initiat’ed at an AUG initiation codon located 35 nucleotides from the 5’ end of t,he 0.3 mRNB. A purine-rich sequence (G-A-G-G-U), located 11 nucleotides upstream from the initiation codon. show;; five-base-pair complementarity with the 3’ end of 16 S rRNA. Such a purine-rich sequence 5’ t)o the initiation codon is thought to base-pair with the 3’ end of 16 S rRNA during initiation of protein synthesis (Shine & Dslgarno, 1974,1975). In fact, hhe importance of this purine-rich sequrncc. as well as the AUG initiation codon, in initiation of 0.3 mRNA translation has been demonstrated by the decreased rate of 0.3 protein synthesis in viva in cells infected with ‘I’7 mutants with single base changes in the initiator AUG or the G-A-G-G-U sequence 5’ to the initiator codon (Dunn et tzZ., 1978). All of our evidence is consistent wit’h 0.3 mRNA having a low initiation rate constant when compared t’o Iat:, mRNA, but it is not defective in the feat,ures of an mRNA known to contribute to translat~ional efficiency. namely the initiation codon and the purine-rich sequence 5’ to the initint,or codon. Based on studies of the RNA phages (Lodish. 1968,1970,1971a,D; Lodish et al., 1964; Capecchi, 1967 ; Piers et al.. 1976), secondary structure and tertiary conformation contribute to determining the efficiency with which an mRNA is translat’ed. The secondary structure or conformation of the 0.3 mRNA could result in a low initiatjion rate constant. However, 0.3 mRNA functions efficiently both 2:n viz:0 at early times and ii? vitro at low concentrations of RNS. A more likelg alternative t,o explain the low efficiency of translation of 0.3 mRNA late in infection is that T7 late mRNAs are simply more efficient messages. Since the 0.3 mRNA is the only T7 mRNA whose initiation region has been sequenced, it is not possible to compare the initiator regions of the various T7 mRKAs.
\t’e express our appreciation to Drs Richard Palmiter, Have Morris and Milt Gordon for constructive discussions during the course of this work and the preparation of this manuscript. Dr F. W. Studier generously supplied tlie bacteria and T7. We are also grateful to Lenore Bemmels for assiseance in preparing the manuscript. This research was supported by National Institutes of Health grant AI 09456, and one of us (S. S.) was partially supported by a National Tustitut,es of Health training grant GM 07270.
450
s.
S’L‘HC)ME
ANI)
E.
‘1’. Young:
REFERFNCESA Belill, D. & Epstein, R. H. (1977). c’iroZog:y, 78, 537~-553. l?enne, R. & Youwels, P. H. (1975). :voZ. /:ew. Genenet. 139. 311 -319. Caprcchi, M. R. (1967). J. Mol. Biol. 30, 213-217. Dunn, J. J., Buzasll-Pollort, K. & Studier, F. W. (1978). I’TOC. Nat. Acad. 8ci., U.S.A. 75, 2741-2745. Fiers, W., Contreras, R., Duerinck, I?., Haegeman, G., Iserentant, D., Merregaert, J., Min Jou, W., Molrmans, F., Rseymarkers, A., Va11 den Berghe, A., Volckaert, G. & Ysebaert, M. (1976). N&ure (London), 260, 500~507. Hagon, F. S. & Young, E. T. (1973). I’iroloyy, 55, 231-241. Hagen, F. S. & Young, E. ‘I’. (1978). J. Viral. 26, 793-804. Hopper, J. E., Ko, C. & Young, E. T. (1975). J. Mol. Biol. 94, 539-554. Imamoto, F., Morikawa, N. & Sat.0, K. (1965). J. Mol. Hiol. 13, 169-182. Kennel], D. (1970). J. C’irol. 6, 208 -217. Lindahl, L. & For&hammer, J. (1969). J. Mol. Riol. 43, 593-606. Lodish, H. F. (1968). J. ~lrlol. Biol. 32, 681-685. Lodish, H. F. (1970). J. 11foZ. Riol. 50, 689%702. Lodish, H. F. (197la) J. ,%foZ. Biol. 56, 627-632. Lodish, H. F. (197lb). J. Biol. Chem. 246, 7131-7138. Lodish, H. F. (1974). Nature (London), 251, 385-388. Lodish, H. F. (1976). Annu. Rev. Riochem. 45, 39 -72. Lodish, H. F. & Jacobsen, M. (1972). J. Biol. Chem. 247, 3622-3629. Lodish, H. F., Cooper, S. & Zinder, N. D. (1964). T’irology, 24, 60-70. Mangiarotti, G. & Schlessinger, D. (1966). ,J. Mol. Biol. 20, 123-143. McKeehan, W. L. (1974). J. Biol. Chem. 249, 6517-6526. Miller, Z., Varmus, H. E., Parks, J S., Pcrlman, R. L. & Pastan, I. ( 1971). .I. Rio/. Chem. 246, 2898.-2903. Pactll, C. A. Sr. Young, E. T. (1976). I’roc. ivat. Acad. Sci.. II.X.A. 73, 312-316. Palmiter, R. D. (1974). J. Biol. Chem. 249, 6779-6787. Rothman-Denes, L. B.,. Muthukrishnan, S., Haselkorn, It. & Studier, F. W. (1973). In T’i~u8 Research. (Fox, C. P. & Robinson, W. S., eds), pp. 227-239, Academic Press, New York. Shine, J. & Dalgarno 1,. (1974). I’roc. Sat. dcud.Sci., U.S.A. 71, 1342m1346. Shine, J. & Dalgarno L. (1975). rlla&rre (London), 254, 34.-38. Steit,z, J. A. & Bryan, R. A. (1978). J. Mol. Biol. 114, 527-543. Strome, S. (1979). Doctoral thesis, University of Washington. Strome, S. & Yourlg, E. ‘I’. (1978). b. Mol. Biol. 125, 75-93. Strome, S. KS Yorulg. E. ‘1’. (1980). J. ,)10(. Rio{. 36, 417 432. Studier, F. W. (1973). .1. Mol. BioZ. 79, 2:)7-248. Tai, P. C., Wallace, B. J. & Davis, B. D. (1973). Biochemistry, 12, 616-620. Tanaka, N., Kineshita. 1’. 85 Masnkawa, H. (1968). Riochem. Hiophys. Res. Commun. 30, 278-283. Varmus, H. E., Perlman, R. L. & Pastarl, I. (1970). ,J. Hiol. Chem. 245, 2259-2267. Yamada, Y. & Nakada, D. (1976). .J. Mol. BioZ. 100, 35-45. Zillig, W., Fujiki, H., Blum, W., ,Janekevic, D., Schweiger, M., Rahmsdorf, H. .J., Ponta, H. & Hirsh-Kauffmann, M. (1975). 1’roc. Nat. ilcad. &‘ci., I7.S.A. 72, 2506-2510.
Biol.
(1980)
Translational
136, 433450
Discrimination Against Bacteriophage T7 Gene 0.3 Messenger RNA SUSAN STROME-~AND
ELTON T.YouNG’~
Department of Biochem,istry University of Washington Seattle, Wash. 98195, U.S.A. (Received
8 August
1979)
Based on genet’ic manipulation of T7 l&e messenger RNA levels in viva, we previously hypothesized that wild-type T7 infection of Escherichia coli develops in mRNA excess and that there is translational discrimination against T7 gene 0.3 mRNA (Strome & Young, 1978). The results presented here support our hypothesis. The discrimination against 0.3 mRN,4 translation observed in. viva can be mimicked in a cell-free system by increasing the concentration of T7 RNA beyond the level needed to saturate the translational machinery or by translating T7 RN,4 with a low concentration of ribosomes. This discrimination can be overcome by adding ribosomes to the cell-free system (increasing the ribosome to mRNA ratio) or by slowing the rate of polypeptide chain elongation. In addition 0.3 mRNA activity as well as a substantial fraction of T7 late mRNA activity is found to be shifted off of polysomes late in T7 infection, Our results are indicative of a low initiation rate constant for 0.3 mRNA compared to T7 late mRNAs.
1. Introduction As described in the accompanying paper (Strome & Young, 1980), T7 gene 0.3 protein synthesis is shut-off at lat’e times during T7 infection when chemical and functional 0.3 messenger RNA persists. The hypothesis that O-3 gene expression is at least partially controlled at the level of translation was initially tested by genetically manipulating T7 late mRNA levels ill vivo (Strome & Young, 1978). Reduction of T7 late mRNA levels in T7-infected Escherichia coli did not result in reduced T7 late protein synthesis, suggesting that wild-type T7-infected cells are normally in mRNA excess. In the presence of reduced levels of T7 late mRNA, O-3 mRNA was translated much more efficiently than in the presence of wild-type levels of late mRNA, which is consistent with discrimination against 0.3 mRh’A translation in the presence of T7 late mRNA levels that saturate the translational machinery of the cell. It is not necessary to invoke a specific mRNA-discriminatory factor to explain the translational discrimination against 0.3 mRNA. Rat’her, mRNAs differ in the inherent rates at which they initiate protein synthesis, as best illustrated by studies of ‘/’ Present of Colorado, $ Author
address: Boulder, to whom
Department of Molecular, CO 80309, U.S.A. reprint requests should
Cellular
and
Development.al
Biology,
University
be addressed. 433
0022-2836/80/040433-18~$02.00/0
Q 1980 Academic
Press Inc.
(London)
Ltd.
S.
434
RTROillll:
h&I)
E.
T.
YOUNG
lysates /I globin mRNA than CCglobin mRNA (Lodish & ,Jacobsen, 1972; McKeehan, 1974: Lodish, 197lh). A kinet’ic analysis of protein synthesis (Lodish, 1974,1976) predicts that if a component, required for initiation of translation were limit’ing, those mRNAs wibh the lowest rate constants for initiation would be discriminat’ed against. Non-specific reduction of polypeptide chain alongabion should prevent discrimination at the initiaCon stage of protein synthesis and result in protein synthesis more as a funct,ion of relative mRNA concentrations and less as a function of initiation rate constant,s. These predictions have been borne out nit,h GCand /3 globin synthesis (McKeehan, 1974; Lodish, 1971b,1974), as well as with the translation of ovalbumin and conalbumin mRNA, t,wo oviduct mRNAs that differ in their rates of translational initiation (Palmiter, 1974). In this paper, experiments are described that test the applicability of Lodish’s hypothesis of differential mRNA t,ranslation to control of 0.3 mRNA translation. The results demonstrate t,hat the t’ranslational discrimination against 0.3 mRNA observed in z&o can be mimicked in a cell-free translational system and that this discrimination can be overcome by increasing the ribosome to mRNA ratio or by slowing the rate of polypeptide chain elongation. The results are also consistent with T7 infection developing in mRNA excess. C( and
fl globin
initiates
synthesis.
protein
In
synthesis
ret,iculocytes
400/:,
rnort
2. Materials (a) E’. coli
BSt and
wild-type
Bacterial T71,
and
chemicals
are described
ret~iculocytc
and Methods
cmd bacteriophage
(wild-t,ype (b) Media
Media
and
rfficicntly
T7) and
wcrc
paper
(d) of S30 extracts for fractionation (1977). (e)
(Strome
F. W.
Studier.
& Young,
1980).
Fractionation
Strome & Young (1978), light irradiation of cells, cells, determination of of 35S-labelled proteins,
of 830
leas been described elsewhere (Hagen of S30 extract into SlOO and crude
Cell-free
Dr
procedures
The following methods are described by Hopper et al. (1975), Studier (1973), and Hagen & Young (1973,1978): ultraviolet labelling T7 proteins in viva, RNA extraction from T7-infected [35S]methionine incorporation, preparation and electrophoresis densitometric scanning of autoradiograms.
The prepasation and the procedure Belin & Epstein
from
chemicals
in the accompanying (c) General
strains
obtained
protein
& Young, ribosomes
1973) is from
synthesis
The details of cell-free protein synthesis are essentially those described by Hagen & Young (1973) with the following changes. Instead of 48 mm-NH,Cl in t,he cell-free synthesis reaction, 110 mM-NH,Ac was used. DNase at a final concentration of 2.5 pg/ml was added to inhibit synthesis of endogenous E. coli RNA in t,he S30 extract. The concentration of 530 (or SlOO plus ribosomes) used in the protein synthesis reactions varied as indicated in the Figure legends. Synthesis of labelled polypeptides was achieved by adding 2 to 10 &i [35S]metliionine to 100 ~1 total reaction mixture. No unlabelled
0.3
mRNA
435
DIRCKIMIKATION
methionine was added to the reactions. At the end of tlio synthesis, the reaction mixtures were immediately chilled and processed for olectrophoresis. Tlre duration of linear [35S]mrthionine incorporation varied somewhat between S30 preparations. Wlren necessary the length of incubation was reduced below the usual 20 min to maintain the linear relationship between time and incorporation. (f) The
procedure
for
Extraction
of polysomes
extracting
polysomes is essentially t)hat of Linda111 & Forchhammer wit11 aeration in MQ medium, at 37”(.‘, to a cell density of 6 x 10E/ml. The cells were infect,ed with T7 with a multiplicity of infection of 10. Before and at irltervals during infection l&ml samples were removed to tubes wit,h IO g ice and 1.5 ml I M-NaN,. The cells were harvested by centrifugat)ion at, 10,000 revs/min for 5 min at 5°C. x.\-aslled wit11 1 ml lysis buffer (5 mM-Tris (pH 7.5), 5 m>l-MpSO,, GO m&l-KCl, ISIS, (w/w) sucrose, 0.01 M-NaN,, and 300 pg lysozyme/ml), and collected again by centrifuRation. The pellet was frozen in solid COz/acetone and could be st’ored at -70°C. The pellet, was thawed at O”C, resuspended in 50 ~1 lysis buffer, incubated 10 min at O”C, frozen ill solid CO,/acetone, and thawed at 0°C. The cells were lysed by adding 0.6 ml TMK hufk (5 mM-Tris (pH 7.5), 5 m&f-MgSO,. 60 mnr-K(J). 60 ~1 5:/, Rrij 58, 60 ~1 59, deoxycholate, and 15 ~1 DNase (1 mg/ml). The lysatc was incubated for 10 min at 0°C and cerltrlfilged at 10.000 revs/mill for 10 min at 5’C. The supernatant was layered ont)o an II-ml 15”‘, t,o 4i0t ,;, (w/w) sucrose gradient in TMK buffer. Tile gradient was cent.rifllped for 90 min at 4O:OOO revs/min at 5°C and collected using an Isco gradient fractionator. The absorbance at 254 nm was monit,ored continuously as tile gradient, was collected. Tllr fractiorls were made 0.5% in sodium dodecyl sulplmte and 0.2 ~1 in NH,Ac and tllcrr exkacted wit’h an equal volume of phenol saturated in 50 mM-NH,Ac and an equal volume of chloroform. Phases were separated by cenkifugation at 2000 revs/min for 5 min. The aqrleous phase was re-extracted with 2 l-01. chloroform. and the RNA was precipitated with 2.5 1.01. cold 95% ethanol. Tile precipitate was collected by centrifugal&r either at 3000 Ir>vs/min for 90 min or at 10.000 revs/min for 20 to 30 min, washed with 2 vol. cold 95:/o ettlanol, lyophilized, and resuspended in an appropriato volume of water for cell-free translat~i~)t1.
(1969). E. coli RSt was grown
3. Results (a) RNA
saturation
of the translational
machinery
in vitro
In an att,empt to mimic in vitro the situation late in T7 infection, when mRNA is apparently in excess of the translational machinery, increasing concentrations of T7 were translated in a cell-free translational system with a constant amount of E. coli 530 extract. The RNA has been extractled from cells 14 minutes after T7 infection, a time at which the majority of T7 late gene transcription is complete (Hopper et al., 1975). Since O-3 mRNA is relatively stable, both chemically and functionally (see accompanying paper, Strome & Young, 1980), 14 minute RNA contained a substantial amount of functional 0.3 mRNA. The [35S]methionine-labelled proteins that were sYynthesized in vitro were analyzed by SDSt/polyacrylamide gel electrophoresis followed by autoradiography (Fig. 1 (a)) ; q uantitation of individual mRNA activities is shown in Figure I(b). As the concentration of T7 mRNA was increased beyond the level needed to saturate the translational apparatus, [35S]methionine incorporation into total protein and individual late proteins reached a maximum and then remained t Abbreviations prokin. 21
used:
SDS,
sodium
rtodecyl
sulphate,
15K
protein,
15,000
molecular
weight
4x
S. STROME
ANI)
E.
T.
YOCNG
too _
Gene products
41
”
-
-P9 -
PlOh
PO.7, PI0
‘-
P (DUP),
-
PII
-
P3.5
-
Pl5K PO-3
PI.3,
P6
0
‘, ;
14.4
-
-
FIG. 1. Cell-free translation of increasing concentrations of T7 RNA with a constant concentration of S30. The RNA used in this experiment was extracted from u.v.-irradiated E. coli Bat 14 min after T7 infection at 30°C. The cell-free synthesis reactions (100 ~1) wore carried out at
0.3
6
mRNA
16 24
DISCRTMINRTION
32
40
56
48
RNA translated
FIG.
ii, vitro
437
64
72
80
80
(pg)
l(b).
constant. However, synthesis of 0.3 protein reached a maximum and then declined as the translational machinery became saturated. In several experiments the rate of 0.3 protein synthesis using high RNA concentrations ranged from 35 to 50% of the maximal O-3 protein synthetic rate. Based on the experiments described in the accompanying paper (Strome & Young, 1980), 0.3 mRPu’A is translated at 25 t,o 300/b efficiency in vivo late in T7 infection. Thus the discrimination against 0.3 mRNA translation in vitro is similar t’o that seen in vivo at late times during T7 infection. (h)
Sensitivity
of 0.3
mRNA
translation
to rihosome
concentration
The sensitivity of protein synthesis, especially 0.3 protein synthesis, to the concentration of translational component’s was tested in two ways. In the first experiment, 14 minute T7 RNA was translated using half the normal concentration of X30 extract, with either additional ribosome-free supernatant (SlOO) or additional crude ribosomes. In the second experiment, T7 RNA was translated using a constant amount of ribosome-free SlOO and various concentrations of crude ribosomes. The labelled proteins synthesized in the reconstituted systems were electrophoresed on SDS/ polyacrylamide gels, an autoradiogram of which is shown in Figure 2. The results are quantitated in Table 1.
37°C’ for 20 min using the RNA concentrations indicated and an 630 concentration of 2.8 mg/ml. A portion (10 ~1) of each reaction was trichloroncetic acid-precipitated to determine [?!J]methionine incorporation into total protein, and the remainder was acetone-precipitated and processed for electrophoresis as described in Materials and Methods. (a) Equal volumes of the 9. labelled proteins synthesized in vitro were run on an SDS/13.61& polyacrylamidc gel at 10 mA for 1 h and at 25 rnA for 3 h. (b) The intensities of individual protein bands from an autoradiogram equivalent to the one shown in (a) were quantitated by microdensitometric scanning as described in Materials and Methods. (0) [35S]methionine incorporation into total protein; (O), PO.3; (A) , 1’3.5. 3 (C), I’(Dvl’).
-.
(a I (b)
.*,r
Cd)
-Pl5K -PO.3
0.3
mRNA
DISCRIMINATTON
TABLE In vitro
Crude ribosomes
synthesis of T7 proteins [%]methionine incorporation total proteint LOWS
s30
1
at different
into
0.3
Highs
439
concentrations
of crude ribosomes
proteinj!
LOWS
15K High
5
Low
proteinf $
Hi&S
35,000
94,000
2.3
2.3
6.7
12.2
2 mg/ml
76,000
117,000
21.4
10.6
18.0
26.2
0.5 mg/ml
41,000
94,000
11.0
16.3
2 mg/ml
72,000
116,000
22.4
15.0
23.3
28.2
5 mg/ml
76,000
130,000
29.6
24.6
21.6
47.2
SlOO
3000
6000 3.3
t Trichloroacetic acid-precipitable [Wlmethionine $ Relative densitometric units. 5 Low RNA concentration, 92 pg/ml; high RNA The data are from the experiment) shown in Fig.
3.1
incorporation concentration, 2.
(cts/min). 650
pg/ml.
The addition of SlOO to the S30 extract did not significantly stimulate total incorporation into protein or specific incorporation into 0.3 protein (Fig. Z(a) and (b)). In contrast, synthesis of 0.3 protein was very sensitive to the concentration of ribosomes (Fig. Z(a) to (d)). As previously observed by Belin & Epstein (1977), total [35S]methionine incorporation into protein did not vary proport!ionally to ribosome concentration, and most of the change in protein synthesis was seen at low ribosome concentrations. Total protein synthesis only increased by a factor of about two between 0.5 and 2 mg/ml ribosome concentration, and most of the T7 proteins analyzed by gel electrophoresis were stimulated only slightly by higher concentrations of crude ribosomes (Fig. 2(a) to (d)). A benfold increase in the concentration of ribosomes caused a ninefold increase in 0.3 protein synthesis (Fig. 2(c)). A slightly lower (eightfold) stimulation was observed at the higher of the two RNA concentrations tested (Fig. 2(d)). The T7 late protein that migrates just above 0.3 protein on 13% polyacrylamide gels (15 K protein) was also somewhat sensitive to ribosome concentration, but was only stimulated threefold by a tenfold increase in the concentration of ribosome.
(c) EJfects of in.hibitors
of translational
elongation
and initiation
If 0.3 mRNA is at a translational disadvantage relative to late T7 mRNAs when translational initiation is limiting, then partial inhibition of translational elongation should inhibit 0.3 protein synthesis less than synthesis of other T7 proteins (Lodish, 1974,1976). To test this, the effect of fusidic acid, an inhibitor of the ribosomeassociated GTPase activity of elongation factor G (Tanaka et al., 1968) was studied. T7 RNA was translated in a cell-free system in t’he presence of concentrations of fusidic acid that inhibited [35S]methionine incorporation into total protein by 20 to
440
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9()0,;,. Wllcn a lo~v concentration of T7 RNA (92 pg/ml) mns translated in vitro in the presence of fusidic acid, inhibition of 0.3 protein synthesis was very similar to inhibit,ion of total prot,rin synthesis and synthesis of other T7 prut,eins (Fig. 3(a)). This bvould be expected if. at R low conceIlt,r~~t,ion of RNA. all mRNAs ~vere translated as a function of their relatiw conc:ciitrations. which is t,hc basis for quantitation of mRNA by cell-free translation (Hopper et al.. 1975). However. at a concentration of RNA that saturated the translational machinery in the absence of inhibitor (650 pg/ml), fusidic acid inhibited 0.3 protein synthesis less than total protein synt,hesis or synthesis of other T7 protein (Fig. 3(b)). Conversely. inhibition of translational initiation should result in preferential inhibition of translation of those mRNAs with the lowest initiation r&e constants. When T7 RPU’A was translated in a cell-free system in t,he presence of concentrations of kasugamycin, an inhibit,or of initiation (Tai et al., 1973), that inhibited r3%]mcthionine incorporat,ion int)o protein by 35 to 75:/i,, 0.3 protein synthesis was preferentially reduced relat’ive to synthesis of totma1 protein and other T7 proteins (Fig. 3(c) and (d)). This was more pronounced when a low concentration of T7 RNA (92 pg/ml: Fig. 3(c)) was translated, perhaps because 0.3 mRNA translation was alrea,dy being discriminated against at t’he high RNA concent’ration (650 pg/ml; Fig. 3(d)). Since synt’hesis of t’he T7 late protein which migrat’es just above 0.3 protein on our gels (15K protein) was inhibited by fusidic acid and kasugamycin t’o about the same extent as t)otal protein synthesis, it was used as a reference. The ratio of the intensity of the 0.3 prot,ein band to the intensity of the 15K protein band was measured after translat’ion of T7 RNA in t,he absence and presence of inhibitor (Pig. 3(e) and (f)). This ratio, 0.3: 15K, decreased from 1.2 to 042 when increasing concentrations (92 and 650 pg/ml, rcspect~ivelp) of T7 RNA were translated in the absence of inhibitor (Figs 1 and 3(e) and (f)), presumably reflecting discrimination against 0.3 mRNA translation. At’ t)he low RN,A concentration (Fig. 3(e)), fusidic acid increased the ra,tio of 0.3 : 15K protein from 1.2 to 1.8, and kasugamycin decreased the ratio from 1.2 t’o 0.53. More int’crestingl,v. at the high concentration of RNA (Fig. 3(f)), fusidic acid increased the ratio from 0.42 to I .4. i.e. back t’o the ratio of 0.3 : 15K protein seen at the low RNA concentration where protein synthesis reflects mRNA concentration. A similar increase in the ratio of 0.3: 15K protein, from 0.3 to 1.4, was obtained in the previous section when the concent,ration of crude ribosomes was increased tenfold (Fig. 2). The effect’s of fusidic acid and kasugamycin on T7-infected E. coli were also examined in ‘cj~o. Kasugamycin or fusidic acid was added 6 minut,es aft,er infection with T7 at 42”C, a time when 0.3 mRNA translation \\-as being discriminated against. Two minutes later T7 prot,eins were pulse-labelled with [35S]metllionine. Kasugamycin resulted in preferential inhibition of 0.3 protein svnthesis (Pig. 4(b)), and fusidic acid inhibited 0.3 protein synthesis less than total protein synthesis (Fig. 4(a)). The ratio of 0.3 protein: 15K protein synthesis irr viva was decreased from 0.2 to 0.09 by kasugamycin and increased from 0.2 to 0.39 by fusidic acid (Fig. 4(c)). The similarity in the effects of the protein synthesis inhibitors on bn ~iwo and in vitro 0.3 mRPiA translation further validates our use of a cell-free t,ranslational systetn to mimic T7 infection in viva.
0.3
mRNA
DISCRIMINATION
441
100 80 60 40 20
Fusidic
acid
(mt.+)
100 80 60 40 20
Kasugomycln
i?
(pg/ml
)
--
0
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I
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0.02 IO
I
0.02 IO
I
0.04 100
I
0.06 1000
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and kasugamycin on cell-free synthesis of T7 proteins, especially (extraction described in legend to fig. 1) at a concentration of with 2.8 mg S30/ml at 37°C 92 &ml ((a), (0) and (0)) or 660 &ml ((b), (d) and (f)) was translated for 15 min in the presence of increasing concentrations of fusidic acid ((a) and (b)) or kasugamycin ((c) and (d)). The cell-free reactions were acetone-precipitated and processed for gel electrophoresis as described in Materials and Methods. Equal volumes of the 35S-labelled proteins synthesized in vitro were either trichloroacetic acid-precipitated to determine [35S]methionine incorporation into total protein or run on an SDS/13.576 polyacrylamide gel at 10 m-4 for 2 h and at 25 mA for 3 h. On an autoradiogram of the gel, the 0.3 protein band and 15K protein band were microdensitometrically scanned. (a) to (d) Both [%]methionine incorporation into total protein (-e-o---) and into 0.3 protein (--O--O--) are expressed as y. of the values obtained in the absence of inhibitor. (e) and (f) The ratio of 0.3 protein band intensity: 15K protein band intensity (from the autoradiogram of the gel described above) was plotted as a function of concentration of fusidic acid (-A-A-) or kasugamycin (--A--.h--) for both concentrations of RN.4: 92 pg/ml (e) and 650 &ml (f). 0.3
FIG. 3. Effects of fusidic acid protein. 14 min T7 RNA
I
44”
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Fusldlc
acid
(ml
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__-
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hG. 4. Effects of fusidic acid and kasugamycin on synthesis of T7 proteins, especially 0.3 protein, in oivo. I\ culture cd IC. coli BSt was u.v.-irradiated and infected with wild-type T7 with a multiplicity of infection of 10 at 42°C. (Similar results were obtained at a lower temperature.) At 6 min after infection, l-ml samples of infected-cell culture were atlrled to tubes containing either fusiclic acid (a) or knsugamycin (b), result,ing in the concrntr:tt.ions of inhibitor inclicittod in the Figure. Proteins wwe pulse-labellerl with [3”S]methionine from 8 to 10 min after infection, as described in Materials and Methodx. Cells WRI‘C collected and lysed, and the “5S-labellec~ proteins synthesized in viw wore prepared for gel electrophoresis. Equal volumes of the labelled proteinx mere either trichloroacetic tlcitl-l)recipit,atc,(i to determine [W]mcthionine incorpordion into total protein or run on an SDS/13’Jh polyacrylamide gel at 10 mA for 1 h 45 min and at 25 m;Z for 3 h. On an autordiogram of the gel, the 0.3 prutcin hand and 15K protein band were den&metrically scanned. (a) and (b) Both [358]mothionine incorporation into total protein (-+-a--) and into 0.3 protein (---C--C)--) are expressed as :$ of the value obtained in the absence of inhibitor. (c) Thr ratio of 0.3 band intensity: 15K band intensity was plotted as a function of concentration of fusitlic acid ( -A---A-) or kasugamycin (--,/1--,R--).
0.3
mRNA
DISCRIMINATION
443
(d) Distribution of T7 mRNA activities on polyribosomes Our hypothesis of translational discrimination against 0~3 mRNA in the presence of saturating levels of T7 late mRNA in T7-infected cells predicts that 0.3 mRNA should be shifted off of polysomes at late times in T7 infection and at least some T7 late mRNA should also be free of ribosomes. To test these predictions the association of T7 mRNA activity with polysomes was analyzed. Figure 5(a), (b) and (c) shows the absorbance profiles of polysomes extracted before T7 infection and after 6 and 20 minutes, respectively. It is apparent that T7 infection affects the polysome profile. resulting in an increase in ribosomal subunits and a shift of polysomes to higher molecular weight. As expected the polysomes were sensitive to RNase and were quantit,at’ively converted to monosomes (data not shown).
1
(a)
(b)
40 20 0 40 20
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0 40 20 0
FIG. 5. Comparison of polysome profiles and of the association of T7 mRNA activities with polysomes early and late in T7 infection of unirradiated E. coli BSt. Polysomes were extracted before infection (a) and at 6 min (b) and 20 rnin (c) after T7 infection of unirradiated E. cc& BSt at 30°C. Polysomes were sedimented in 15O4, to 459; sucrose gradients. Sedimentation is from left to right. Arrows indicate monosome peaks. The peak to the far right in (c) is presumably mature virions. The protein bands representing genes 0.3, 10 h, and 11 on the autoradiogram shown in Fig. 6 were microdensitometrically .xanned. For each protein the total mRNA activity per sucrose gradient, was set at loo%, and t,he mRNA activity per fraction is expressed as 3/o of the total mRNA activity.
S.
444
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RNA was extracted from the sucrose gradient fractions and translated in a cell-free translational system. The labelled proteins synthesized in vitro were analyzed by SDS/polyacrylamide gel electrophoresis and autoradiography (Fig. 6). The majority of host mRNA activity in uninfected cells was associated with polysomes (Fig. 6(a)). Early in T7 infection most of t,he T7 mRNA activity was also associated with polysomes, although there was some mRNA activity in the supernatant (La) and subunit (Ib) fractions of the sucrose gradient (Fig. 6(b)). By 20 minutes after infection, however, a substantial fraction of mRNA activity was dissociated from polysomes (Fig. 6(c)), which is consistent with T7 infection developing in mRNA excess. Several T7 protein bands on t)he autoradiogram shown in Figure 6 were analyzed quantitatively, and the histograms in the lower panel of Figure 5 display the distribution of T7
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6. Autoradiogram of SDS/13”/0 polyaccrylamide gel of cell-free protein synthesis directed RNA extracted from SncloSe gradient fmctionation of polysomes. The Swrose gradients from Fig. 5 were collected as 5 fractions: Ia = supernatant, Ib = ribosomal subunits, II = monosomes, III = disomes, trisomes and tetrasomes, and IV = polysomes of 5 and larger. The RNA was extracted from each fraction and translated in a cell-free system, as described in Materials and Methods. Equal volumes of the 35S-labelled proteins synthesized in vitro were run on an SDS/13% polyacrylamide gel at 10 mA for 1.5 h and at 25mA for 3.5 h. (a) to (c) Correspond to (a] to (c) of Fig. 5, respectively. In (c) fractions Ia and Ib were collected as one fraction (I). FIQ.
by
0.3
mRNA
DISCRIMINATION
445
mRNA activities in sucrose gradient fractions. The association of O-3 mRNA activity with polysomes, monosomes and supernatant and subunits was 62%, 20% and IS:/,, respectively, at 6 minutes after infection (Fig. 5(b)) and 40%, 20% and 40%, respectively, at 20 minutes after infection (Fig. 5(c)). Thus early in infection (Fig. 5(b)) most of the 0.3 mRNA activity was associated with polysomes, a substantial fraction of that with polysomes of five or larger. Later in infection, 0.3 mRNA was shifted off of polysomes and 40 to 50% was found in the supernatant and ribosomal subunit fract’ions. The distribution of T7 late mRNA activities varied considerably. There were two basic patterns of T7 late mRNA distribution. Some T7 mRNAs, as exemplified by mRNA for genes 8, 11 and 19 were almost tota,lly associated with polysomes. This is shown for gene 11 mRNA in Figure 5(c). However, some T7 late mRNAs, such as genes 9 and 10 mRNAs, were found in t’he supernatant and subunit fractions (I) as well as t,he polysomal fractions of sucrose gradients (Fig. 5(c)). Late in T7 infection, the mRNA coding for the protein of about 29,000 M,, which appears to be a host protein, was found exclusively in the supernatant and subunit fractions of the sucrose gradient. If the shift of mRNA off of polysomes is caused by excess late mRNA, then a reduction of the amount of late mRNA should allow a larger proportion of both 0.3 mRNA activity and late T7 mRNA activity to remain associated with polysomes. Late mRNA levels can be reduced by infecting E. coli with the gene 1 temperaturesensitive mutant, ts 342, at a semi-restrictive temperature (Strome & Young, 1978). Polysomes were extracted from cells infected with wild-type T7 or ts 342 at 42°C and sedimented through sucrose gradients, and RNA was extracted from the sucrose gradient fractions and translated in a cell-free system as described above (Fig. 7). At 3 minutes after infection by wild-type T7 (Fig. 7(a))! 560,/, of the 0.3 mRNA activity was associated with polysomes, 26% was associated with monosomes, and 18% was found in t,he supernatant and subunit fractions of sucrose gradients. At 3 minutes after infection with ts 342 (Fig. 7(b)), h owever, 96% of the 0.3 mRNA activity was associated with polysomes and only 4% was free of rihosomes. Thus, as early as 3 minutes after infection by wild-type T7 at 42”C, 0.3 mRNA was being shifted off of polysomes, presumably due to the presence of late mRh’A. As late as 10 minutes aft’er infection at 42°C by ts 342, there was little 0.3 mRNA activity free of ribosomes (Fig. 7(d)), in contrast to the shift of 0.3 mRNA off of polysomes in cells infected by wild-type T7 (Fig. 7(c)). The association of 0.3 mRNA activity with polysomes, monosomes and supernatant plus subunits in ts 342-infected cells at 10 minutes was 520/,, 2776 and 21%, respectively, the same distribution as that observed at 3 minutes after infection by wild-type T7. The reduction in T7 late mRNA transcription in ts 342-infected cells also allowed a larger proportion of T7 late mRNA activity to remain associated with polysomes after infection by ts 342 than after infection by wild-type T7, as expected by our model of T7 infection developing in mRNA excess.
4. Discussion Results of the experiments described in this paper support our hypothesis that O-3 mRNA has a low initiation rate constant relative to late T7 mRNAs and is therefore
-
(a)
(b)
(c)
(d)
FIG. 7. Autorcldiogram of SDS/13.75% polyncrylamide gel showing cell-free translation of RX-1 extracted from polyson~~s from cells infected with wild-type T7 and ts 342 at 42°C. Polysomes were extracted at 3 min (a) and 10 min E. coli PF by wild-type T7 at 42°C and at 3 min (b) and 10 min (d) after infection of unirradiated ~011s by t’s 342 at fractions II, III and IV from fractionation of polysomen extracted from uninfected cells at 42°C. The extraction proteins svnt,hesized in and the numboring rrf fractions are as described in the legend to Fig. 6. The 35S-labelled polyacrylnm~rle gel at 8 m-4 for 1.75 h and at 25 md for 3 h.
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sucrose gradient fractionation of (c) after infection of u&radiated the same temperature. (e) Shows and cell-free translation of RNA vitro were run on an SDS/13.7576
(e)
0.3
mRN4
DISCRIMINSTION
447
at a translational disadvantage when it must compete with T7 late mRNAs for a limiting amount of translational machinery. Our results also provide strong support for Lodish’s model of translational control of protein synthesis. The discrimination against 0.3 mRNA translation seen in viva (Strome & Young, 1978) is also seen in vitro when the concentration of T7 RNA used to direct cell-free protein synthesis is increased beyond the level needed to saturate the translational apparatus. By fractionating the E. coli cell-free system into ribosome-free supernatant (SlOO) and crude ribosomes, it was possible to mimic the translational discrimination against, 0.3 mRNA seen in viva; the discrimination seen at low ribosome concentrations (low ribosome to mRNA ratio) could be relieved by increasing the concentration of ribosomes (increasing the ribosome to mRNA ratio). It appears that ribosomes (or some component contained in t,he crude ribosome fraction) limit kanslation of T7 mRNA by E. coli 530 extracts, since of the components tested, only ribosomes stimulated tot’al protein synthesis with these messages. The crude ribosome fraction has been further fractionated by NH,Cl into crude initiation factors and salt-washed ribosomes. Based on preliminary results, it appears that initiation factors do not limit translation of T7 mRNA by E. coli 530 extracts and do not have the same stimulatory effect on total protein synthesis or on 0.3 protein synthesis that ribosomes have (Strome, 1979). Using a DNA-dependent cell-free protein synthesis system, Benne 8r. Pouwels (1975) found that synthesis of T7 RNA polymerase and [14C]leucine incorporation into tot,al T7 protein showed no dependence on added IF-3. In contrast, synthesis of active enzymes encoded by the E. co& t,rp operon (using @O-trp DNA) and [ 14C]leucine incorporation into trp-protein depended strongly on added W-3. Our results are consistent with a low dependence of T7 protein synthesis on the concentration of initiation factors. Slowing of translational elongation by fusidic acid relieves translational discrimination against 0.3 mRNA in vivo and at high RNA concentrations in vitro (Figs 3 and 4). As seen with tl and ,!Iglobin (Lodish, 1971b) and ovalbumin and conalbumin (Palmiter, 1974), partial inhibition of translational elongation on T7 mRNA abolishes the inhibitJory effect caused by an apparently low initiation rate const,ant and allows 0.3 mRNA to be translated at a rate which is proportional to its relative concentration in the cell or in the cell-free extract. Consistent with a low initiat,ion rate constant for 0.3 mRNA, partial inhibition of pol.ypeptide chain initiation results in preferential inhibition of O-3 protein synthesis relative to tot,al protein synthesis both in viva and in vitro (Figs 3 and 4). In agreement with Mangiarot’ti & Schlessinger (1966) and Kennel1 (1970), our results indicate that in uninfected cells essentially all of the mRNA activity is associated with polysomes (Fig. 6(a)). I n uninfected E. coli there is a close correlation between mRNA concentrations and rates of protein synthesis in the gal operon (Miller et al., 1971), the lac operon (Varmus et al., 1970), and the trp operon (Imamoto et al., 1965). The presence of all E. coli mRNA on polysomes and the close correlation between mRNA concentration and protein synthesis in E. coli suggests that in uninfected cells there is an excess of translational machinery over mRNA and that mRNA may limit protein synthesis. In contrast to the situation in uninfected cells, a substantial fraction of T7 mRNA activity is found in the supernatant at the top of sucrose gradients and sedimenting in the region of t’he gradient~s where ribosomal
448
s.
S’I’IiOAl
I’ 1 .\Sl)
I.:. ‘I’.
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subunits sediment (Fig. B(C)). Thus. after infection I)?; wild-type T7, T7 mRNA must be in excess of the functional protcitl synthetic machinery of the cell. This contention is supported by the tindin g that. \vtlrrl T7 Iatta mRSA 1~~~1s n,rc reduced using the ts 342 mutant, a larger fract’ion of T7 latch mRSX ;lctivity is associated with polysomcs (Fig. 7(d)). The shift of 0.3 mR,EA activity from polysomes to the supernatant and rihosomal subunit fractions of sucrose gradients as infe&ion proceeds (Figs 6 and 6) supports our model of translat’ional discrimination against 0.3 mRNA late in wild-type T7 infection. Dunn et al. (1978) observed that 0.3 mRNA is shifted off of polysomcs in cells infected with T7 mutants containing base changes in the initiation region of gene 0.3. These mutants synt,hesized wild-type levels of 0.3 mRNA, hut the mutant, 0.3 mR&As are translated much less efficiently than wild-type 0.3 mRNAs. even at early times during infect,ion. We observe that under conditions where 0.3 mRNA is translated more efficiently in viva. namely when T7 late mRNA levels are reduced (Strome & Young. 1978); a larger proportion of 0.3 mRKA activity is associat,ed with polvsomes (Fig. 7). Tn our polysome analyses, the distribution of other T7 mRNAs in sucrose gradients varies. A substantial amount of some mRKAs (gene 9 and 10 mRNB) is dissociated from polysomes, while ot,her T7 mRNBs (genes 8, 11 and 19 mRNAs) are associated exclusively with polysomes (Figs 5 and 6). The differences in mRNA distribution probably reflect differences in initiation rate constant’s and the polycistronic nature of t’he various T7 mRSAs. For instance. the gene 10 protein. the major capsid protein, is t.ranslat.ed f?om at least, t,hree mRNAs (Pa&l R: Young, 1976). In addiCon to the monocistronic gene 10 mRKA, there are two polycistronic mRNAs : one coding for genes 9 and 10 and the other coding for genes 8. 9 and 10. Thus. translation of gene 8 protein and gem 9 protein will affect the apparent association of gcnf 10 mRNA activity with polysomes. The change in polysomc absorbance profile brought about l)y T7 infect.ion is not that predicted for a transition from limiting mRNA t,o mRNX excess. If T7 infection led to the synthesis of much higher mRNA levels than are present’ in uninfected E. COG, one would expect (1) functional 30 S and 50 S subunits to be recruited for protein synthesis and (2) a general decrease in polysome size. Inst)ead, upon T7 infect,ion there is an increase in ribosomal subunits and an increase in average polysome size (Fig. 5). One possible explanation for the increase in ribosomal subunits is a, T7induced inactivation of some component of the translational machinery. Tn &her words, TS infection might develop in apparent mRNA excess by reducing the amount, of functional translational machinerg instead of by synthesizing concentrations of ‘I’7 mRNA large enough to saturate t’he host’ translational machinery. Several findings are consistent with this possibility. (1) When T7 infects unirrsdiated E. cd, the rate of protein synthesis, as measured by j 35S]methiotline incorporation, declines progressively throughout infection (Strome, 1979). Tf this decrease is not an effect caused by a change in the specific activity of the methionine pool, it suggests that, T7 mRXB directs protein synthesis at a lower rate than is observed in uninfected cells. (2) Yamada 8: Nakada (1976) found that’ cell-free translational systems prepared from cells early in T7 infection showod increased ac%vit,y over uninfected-ccl1 cxt,ract’s. However, as infection proceeded there was a general decline in the translational capacity of infected-cell extra&s. The T7 early gene 0.7 codes for a protein kinase,
0.3 mRNA
DISCRIMINATIOK
449
which has been found to phosphorylate a variety of host proteins including RNA polymerase and ribosomes (Zillig et al., 1975; Rothman-Denes et al., 1973). Phosphorylation of ribosomes after T7 infection could lower the activity of the ribosomes. In this respect, however, ribosomal subunits do accumulate after infection of E. coli with deletion mutants either in gene 0.7 or gene 0.3 (Strome, 1979), suggesting that, if the accumulation of ribosomal subunits is due to inactivation of some translational components, the product of gene 0.7 or gene 0.3 (or 0.4) is not responsible. It is not clear why, if T7 infection develops in mRNA excess, polysomes extracted from T7-infected cells are larger than polysomes from uninfected E. co&. One possibility is tha,t the average size of T7 mRNAs is larger than the average size of E. coli mRNAs, perhaps as a result of the increased stability of T7 mRNA compared to E. coli mRNA. Tf this were the case, more ribosomes could be associated with T7 mRNAs than with E. coli mRNAs even if the ribosome density (number of ribosomes per unit length of RNA) were reduced upon T7 infection. The initiation region of the 0.3 mRNA has been sequenced by Steitz & Bryan (1978). O-3 protein synbhesis is initiat’ed at an AUG initiation codon located 35 nucleotides from the 5’ end of t,he 0.3 mRNB. A purine-rich sequence (G-A-G-G-U), located 11 nucleotides upstream from the initiation codon. show;; five-base-pair complementarity with the 3’ end of 16 S rRNA. Such a purine-rich sequence 5’ t)o the initiation codon is thought to base-pair with the 3’ end of 16 S rRNA during initiation of protein synthesis (Shine & Dslgarno, 1974,1975). In fact, hhe importance of this purine-rich sequrncc. as well as the AUG initiation codon, in initiation of 0.3 mRNA translation has been demonstrated by the decreased rate of 0.3 protein synthesis in viva in cells infected with ‘I’7 mutants with single base changes in the initiator AUG or the G-A-G-G-U sequence 5’ to the initiator codon (Dunn et tzZ., 1978). All of our evidence is consistent wit’h 0.3 mRNA having a low initiation rate constant when compared t’o Iat:, mRNA, but it is not defective in the feat,ures of an mRNA known to contribute to translat~ional efficiency. namely the initiation codon and the purine-rich sequence 5’ to the initint,or codon. Based on studies of the RNA phages (Lodish. 1968,1970,1971a,D; Lodish et al., 1964; Capecchi, 1967 ; Piers et al.. 1976), secondary structure and tertiary conformation contribute to determining the efficiency with which an mRNA is translat’ed. The secondary structure or conformation of the 0.3 mRNA could result in a low initiatjion rate constant. However, 0.3 mRNA functions efficiently both 2:n viz:0 at early times and ii? vitro at low concentrations of RNS. A more likelg alternative t,o explain the low efficiency of translation of 0.3 mRNA late in infection is that T7 late mRNAs are simply more efficient messages. Since the 0.3 mRNA is the only T7 mRNA whose initiation region has been sequenced, it is not possible to compare the initiator regions of the various T7 mRKAs.
\t’e express our appreciation to Drs Richard Palmiter, Have Morris and Milt Gordon for constructive discussions during the course of this work and the preparation of this manuscript. Dr F. W. Studier generously supplied tlie bacteria and T7. We are also grateful to Lenore Bemmels for assiseance in preparing the manuscript. This research was supported by National Institutes of Health grant AI 09456, and one of us (S. S.) was partially supported by a National Tustitut,es of Health training grant GM 07270.
450
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