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Defective initiation of f2 RNA translation by ribosomes from bacteriophage T4-infected cells
Biochimica et Biophysica Acta, 331 (1973) 102-116
~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97836
D E F E C T I V E I N I T I A T I O N OF f2 R N A T R A N S L A T I O N BY RIBOSOMES F R O M B A C T E R I O P H A G E T 4 - I N F E C T E D CELLS
ROBERT E. SINGER and THOMAS W. CONWAY Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Received June 4th, 1973)
SUMMARY Ribosomes from T4-infected Escherichia coli when isolated with their initiation factors still attached have negligible activity for the translation of f2 R N A but retain activity for the translation of late T4 RNA. When low concentrations of f2 R N A are used with T4 ribosomes which contain factors, formylmethionyl-tRNA does not bind in response to f2 RNA. Yet the latter ribosomes show substantial activity in binding formylmethionyl-tRNA in response to high concentrations of f2 RNA. Failure of the aberrant initiation complexes to function in translation does not appear to be related to the amount of initiator t R N A bound.
INTROD UCTION Infection ofEscherichia coli cells by bacteriophage T4 results in both the cessation of host protein synthesis 1'2 and the restricted development of R N A phages such as f2, R17, M12 or MS2 (ref. 3). Following the observation of Salser and Gesteland, reported by Hattman and Hofschneider 4 that extracts from T4-infected cells were inactive for the in vitro translation of R17 RNA, it was found that the ribosomes from T4-infected cells discriminated against the translation of MS2 R N A 5'6 or f2 R N A while apparently retaining activity with late T4 m R N A 5. Selectivity for utilization of these m R N A s likely resided in the ribosomal high-salt wash fraction, since this fraction from uninfected cells stimulated f2 R N A translation by T4 ribosomes 6 while that from T4-infected cells inhibited MS2 R N A translation by control ribosomes 5. Several studies using combinations of the high-salt wash fractions and high-salt washed ribosomes from T4 phage-infected and uninfected cells have shown that the specificity for initiation of protein synthesis on the R N A phage RNAs is determined by the source of the high-salt wash fraction 7'8. That is, the initiation factor preparation from the uninfected cells stimulated initiation with R N A phage RNAs while that from T4-infected cells did not. The subsequent resolution of multiple forms of the initiation factor IF3, each form specific for a different m R N A 9-1 ~ or cistron 12- ~4, suggested that translation was regulated by changes in the levels of the different IF3's 1°'15'16 after T4 infection. The conclusion from all of these studies is that the
DEFECTIVE INITIATION BY T4 RIBOSOMES
103
step in R N A phage R N A translation limited by T4 is involved in the formation of the initiation complex. In contrast to these findings, Goldman and Lodish x7 have reported that T4 ribosomes isolated with or without the initiation factors in place showed a similarly reduced activity f o r both translation and initiation whether either f2 RNA or T4 RNA was used as messenger. Furthermore, it has also been reported that infection of E. coli by phages T7, Qfl or ) resulted in a nonspecific reduction in the activity of the ribosomes for the translation of both Qfl and T7 RNAs (ref. 18). These studies have raised a question as to the selectivity of the T4-induced inhibition of in vitro RNA phage R N A translation. We have compared the relative activities of ribosomes from T4-infected cells for the translation of f2 and late T4 mRNAs and find that the activities vary according to the experimental conditions including the time and multiplicity of infection. These variations may be at least partly responsible for the apparently conflicting findings of others. To determine more precisely how the T4-induced defect affects the initiation process, experiments were performed to test the effectiveness of binding formyl[14C]methionyl-tRNA to low-salt washed ribosomes as a function of f2 RNA concentration. Although ribosomes from T4-infected cells fail to initiate efficiently at low concentrations of f2 RNA, at high concentrations of this R N A the label is bound very effectively. In spite of this further translation fails. MATERIALS AND METHODS
Phage infections and preparation of cell extracts E. coli B06 was obtained from E. Six and used for the preparation of uninfected (designated here as control) cells and T4 phage infected (designated here as T4) cells. All comparisons between control and T4 activities were done using extracts derived from a single culture which was grown in M9 medium 19 at 30 °C to a density of 5 • 108 cells/ml and then split into two parallel cultures just before infection of one with bacteriophage T4 (multiplicity of infection of 10 and L-tryptophan, 5/ig/ml). After 10 min &incubation with aeration, both the control and T4 cultures were poured onto 0.5 vol. of frozen and crushed 0.15 M NaC1. The cooled cells were collected by centrifugation at 10 0 0 3 x 9 for 10 rain. This step and all ensuing steps were carried out at 4 °C. The pelleted cells were resuspended in an equal volume of Buffer A (0.01 M Tris-HCl buffer (pH 7.4), 0.01 M MgCI2, and 10 mM 2-mercaptoethanol) which had been made 3/lg/ml in deoxyribonuclease (Sigma, electrophoretically purified free of ribonuclease). They were broken in an ice-cold French pressure cell at 15 000 to 20000 lb/inch 2 and the resultant suspension was centrifuged at 3 0 0 0 0 x 9 for 30 min. The supernatant fraction (S-30) was immediately centrifuged at 145 000 × 9 for 3 h. The upper half of the supernatant liquid (S-145) was dialyzed for 8 h against Buffer A; the ribosomal pellet was resuspended in Buffer A and sedimented again by centrifugation at 145 0 0 0 x g for 3 h. This pellet was resuspended in Buffer A and stored at 4 °C, if used within 2 weeks, or unfrozen in 50 ~ glycerol at --20 °C where it retained its activity for more than a month. The high-salt wash fractions were prepared by resuspending the ribosomal
104
R . E . SINGER, T. W. CONWAY
pellet in 2.0 M NH4CI, 0.01 M Tris-HCl buffer (pH 7.4), 0.01 M MgCI2 and 0.1 mM dithiothreitol and centrifuging for 3 h at 145 000 ×9. The supernatant fraction was dialyzed against Buffer A and fractionated with (NH4)zSO 4 as indicated in the text. The precipitated fraction was resuspended and dialyzed against 0.01 M Tris-HCl buffer (pH 7.4) and 10 mM 2-mercaptoethanol.
Preparations of mRNAs and aminoaeyl-tRNAs Phage f2 was grown and isolated as before s or purchased from Miles Laboratories. Prior to use phage particles were suspended in 20 mM EDTA (pH 7.2), and pelleted by centrifugation at 100 000 × 9 for 3 h; the pellet was resuspended and this step was repeated. These phage preparations sediment in sucrose density gradients as a single peak of A254 nm materials at approx. 79 S. Phenol extraction was done in the presence of 10 mM EDTA (pH 7.2), and 0.1 M sodium acetate (pH 5.2). The aqueous phase was extracted with phenol 3 times and then made 2.0 M in NaCI before adding 2 vol. of chilled ethanol. The suspension was stored at --20 °C for 3 h, centrifuged, and the RNA-containing pellet resuspended in water and dialyzed overnight against water. These preparations of f2 RNA consistently gave a single A254 nm peak on sucrose density gradients of 27 S. Late T4 m R N A was prepared after Wilhelm and Haselkorn 2° from cells infected by T4 phage for 18 min at 30 °C. It was found that much of the DNA in these preparations could be eliminated by the addition of deoxyribonuclease (5/~g/ml) before the EDTA used for cell lysis. All tRNA was charged according to Conway 21 but with the following changes: 75 mM Tris-HC1 buffer (pH 7.4), 5 mM ATP, 10 mM MgCl2 and 2.5 mM 2-mercaptoethanol. When charging methionine tRNAv, calcium leucovorin was present at 0.03 mg/ml; the reaction was for 20 rain at 37 °C and usually in a volume of 1 ml. Purified methionine tRNA v was generously provided by the Oak Ridge National Laboratory. The radioisotopes were purchased from Schwarz/Mann and their respective specific activities noted in the text.
Protein synthesis The reaction mixtures for amino acid incorporation contained: 20/~g of E.
eoli B tRNA, 3 mM ATP, 0.4 mM GTP, 5 mM phosphoenolpyruvate, 2/~g of pyruvate kinase, 75 mM NH4C1 , l0 mM 2-mercaptoethanol, 50 mM Tris-HC1 buffer pH 7.8, 0.04 mM of each 19 unlabeled amino acids, 0.02 mM of [14C]lysine (160 Ci/mole) in volumes from 0.1 to 0.15 ml as indicated for each experiment. In all experiments control S- 145 was employed and typically 180/~g was added to the amounts of ribosomes indicated in the figure legends. Ordinarily the reactions were limited by the concentration of ribosomes present. Immediately prior to their use the ribosomes and S-145 were preincubated 15 min at 37 °C in a buffer containing 225 mM NH4CI, 150 mM Tris-HCl buffer (pH 7.8), 30 mM 2-mercaptoethanol, and 30 mM MgC12. Addition of the remainder of reagents, mRNAs, labeled amino acids and water brought the reaction mixture to the concentrations indicated above. After 30 min incubation at 37 °C the reactions were terminated and counted as previously described 22 at 50 ~ efficiency.
The bindin9 of aminoacyl-tRNAs to ribosomes The binding of formyl-[a4C]methionyl-tRNA and other aminoacyl-tRNAs to
DEFECTIVE INITIATION BY T4 RIBOSOMES
105
ribosomes was measured in 50 mM Tris buffer (pH 7.8), 50 mM NH4C1, 10 mM 2mercaptoethanol, and 0.4 mM GTP (1 mM G T P was employed when measuring binding of other aminoacyl tRNAs) and assayed by binding to Millipore membranes similar to Nirenberg and Leder 23. Unless otherwise stated, binding was done with limiting amounts of ribosomes and relied upon the ribosomes' endogenous complement of initiation factors and elongation factors. Comparisons between the T4 and control ribosomes' relative abilities to bind the second aminoacyl-tRNAs were done with parallel preparations of ribosomes prepared from cells broken in at least 10 mM 2-mercaptoethanol in order to insure sufficient endogenous elongation factor activity. Immediately prior to their use, ribosomes were preincubated for 15 min at 37 °C in a buffer containing 150 mM Tris, 150 mM NH4C1, 30 mM 2-mercaptoethanol, and 15 mM MgCI 2. Again, additions of the remaining reactants brought all concentrations to those indicated above for the binding reaction. Reaction volumes were in a range of 0.1-0.15 ml as indicated for each experiment.
Analysis off2 RNA coded dipeptides The electrophoretic analysis of f2 RNA coded formylated dipeptides and determination of the mobility of marker dipeptides was performed after the procedure of Roufa and Leder 24. Following electrophoresis of the applied reaction mixtures, the paper was dried, cut into strips of 2 cm × 3 cm and counted. The electrophoresis of reaction mixtures done in the presence and absence of f2 RNA were done simultaneously. No peptide products were detected in the absence of f2 RNA. RESULTS
Inactivation o f f 2 RNA translation by T4 infection Our findings and those of others suggest that either dialysis of ribosomes after preincubation 25 or reconstitution of high-salt washed ribosomes with their high-salt wash fraction 14 results in an alteration of the ribosome's translational activity. For this reason, ribosomes were ordinarily isolated without washing at high concentrations of salt and were preincubated without dialysis immediately before use. Cells infected with T4 phage were always compared with parallel uninfected cultures. This eliminated the non-specific variability seen with ribosomes prepared from cells grown on different days. A typical comparison of translation activities is presented in Table I. The T4 ribosomes appear to have less activity than the control ribosomes. Previously, Goldman and Lodish 17 showed that both T4 and control ribosomes synthesize the same polypeptide products with late T4 mRNA. Consequently, we cannot assume that the reduced activity of T4 ribosomes with late T4 m R N A is due to the partial loss of a translation factor specific for certain T4 mRNAs. In order to determine whether there is a reduction in the T4 ribosome's ability to translate f2 RNA which exceeds that due to any non-specific decreases, f2 RNA-stimulated translation with T4 and control ribosomes must be normalized to their respective activities with late T4 RNA. The ratio of amino acid incorporated in response to f2 R N A to that incorporated in response to T4 R N A is designated the incorporation ratio in Table I. Comparison of the incorporation ratios of T4 and control ribosomes shows that the reduction in the T4 ribosome's ability to translate f2 RNA clearly exceeds any non-specific
106
R . E . S I N G E R , T. W. C O N W A Y
TABLE I R E D U C E D T R A N S L A T I O N O F f2 R N A O N T4 R I B O S O M E S Reaction mixtures (0.1 ml) for amino acid incorporation contained 35 #g of f2 R N A or 80/~g of T4 m R N A and either 400 t~g o f control ribosomes or 300 #g of T4 ribosomes. 100 pmoles equals 16 500 cpm.
Additions
[14C]Lysine incorporation (cpm/m# ribosomes)
mRNA- dependent incorporation (pmoles/mg ribosomes)
Incorporation ratio f2 RNA/T4 RNA*
Control ribosomes +f2 RNA +T4 RNA
9 338 6l 250 115 130
-315 642
0.49
T4 ribosomes +f2 RNA +T4 RNA
6 184 11 788 72 369
-34 401
0.08
* 0.08 0.49
= 0.16, or 84 ~ restriction o f translation of f2 R N A by T4 ribosomes.
decrease in translation activity. Therefore, the T4 infection results in a selective decrease that can be quantitated by comparing the activity of T4 ribosomes to control ribosomes in terms of their abilities to translate both f2 and T4 RNAs. This is done by dividing the incorporation ratio for T4 ribosomes by the incorporation ratio for control ribosomes (Table I). If this new ratio is subtracted from one and multiplied by 100, the percent restriction of translation is obtained. The percent restriction of initiation can be calculated using the analogous ratios for the mRNA-dependent binding of fMet-tRNA. The percent restriction of translation has been observed to vary over a range of values depending on the conditions of infection and preparation of the cells. The percent restriction was monitored as a function of the time of infection (Fig. 1) and the multiplicity of infection (Fig. 2). Fig. 1 shows that restriction of translation is maximal by 5 min or less. Some experiments showed a significant decrease in the ability of T4 ribosomes to translate f2 R N A after only 3.5 min of infection; however, the complete set of controls was not done in these cases. The fact that high restriction can be achieved at relatively low multiplicities of infection (Fig. 2) indicates that this phenomenon is not related to changes in nucleic acid metabolism produced by high multiplicities of infection z6. Nomura e t al. 26 suggested that at least two mechanisms exist by which T4 phage can shut off host nucleic acid synthesis. One requires synthesis of T4-coded proteins, and the other is dependent on high multiplicities of infection but is independent of phage protein synthesis. The occurrence of a selective inhibition of f2 RNA translation was highly sensitive to the degree to which the cells were infected. Typically, cultures in which 1 ~o or more of the cells survived 3 min of infection yielded ribosomes which did not show selective inhibition of f2 RNA translation even though a nonspecific decrease in the activity of the ribosomes sometimes as high as 50 ~ was detected. The T4 ribosomes whose activities are presented here were derived from cultures in which
DEFECTIVE INITIATION BY T4 RIBOSOMES
I I00
I ioof ~ o
~
5o
•.~ 75 ~
~. 25
0
107
5o
25
;0 Minutes after infection
° Mulliplicily of infection
Fig. 1. Reduction of translation efficiency of f2 R N A as a function of time after infection. Amino acid incorporation was measured in a volume of 0.1 ml and in a mixture described in Materials and Methods supplemented with the following: 75/~g of f2 R N A or 80/~g of T4 m R N A and where indicated 150 #g of control ribosomes, 160 #g of T4 ribosomes (prepared 5 min after infection), 150/tg of T4 ribosomes (10 min), and 160/~g of T4 ribosomes (15 min). All infections were at 30 °C and at a multiplicity of infection of 15. The cells were broken in the standard Buffer A modified to contain 2.5 mM 2-mercaptoethanol. Control ribosomes were stimulated to incorporate 331 pmoles/mg ribosomes with T4 m R N A and 437 pmoles/mg ribosomes with f2 RNA. In the absence of added m R N A 40 pmoles/mg ribosomes was incorporated. The specific activities of T4 ribosomes with T4 m R N A were 88, 85, and 85 % of the control ribosomes at 5, 10, and 15 min of infection, respectively. The specific activities of T4 ribosomes with f2 RNA were 28, 24, and 22 ~ of the control ribosomes at 5, 10, and 15 rain of infection, respectively. Fig. 2. Reduction in translation efficiency of f2 R N A as a function of the multiplicity of infection. Mixtures contained 90/~g of f2 R N A or 80/~g of T4 mRNA, and where indicated 165/~g of T4 ribosomes (multiplicity of infection = 5), 198/~g of T4 ribosomes (multiplicity of infection = 10), or 165#g of T4 ribosomes (multiplicity of infection = 15). All infections were for 10 min at 30 °C. Other components and conditions are given in Materials and Methods. Control ribosomes were stimulated to incorporate 280 pmoles]mg ribosomes with T4 mRNA, 340 pmoles]mg ribosomes with f2 RNA, and 39 pmoles/mg ribosomes in the absence of added f2 RNA. The specific activities of T4 ribosomes with T4 m R N A were 65, 70 and 92 ~ of the control ribosomes at multiplicities of infection of 5, 10, and 15, respectively. The specific activities of T4 ribosomes with f2 R N A were 7, 24, and 41 ~ of the control ribosomes at multiplicities of infection of 5, I0, and 15, respectively.
0.1 ~ o r less o f t h e cells s u r v i v e d 3 m i n o f i n f e c t i o n . I t h a s a l s o b e e n o u r e x p e r i e n c e t h a t c o n t a m i n a t i o n o f e x t r a c t s f r o m T 4 - i n f e c t e d cells w i t h e x t r e m e l y s m a l l p r o p o r t i o n s o f e x t r a c t s f r o m c o n t r o l cells g r e a t l y r e d u c e s t h e p e r c e n t r e s t r i c t i o n o f t r a n s lation of the T4 ribosomes, without eliminating a non-specific decrease in the activity of T4 ribosomes.
Reversal of T4-induced restriction o f f 2 RNA translation by the high-salt wash fraction of control ribosomes RNA
R i b o s o m e s f r o m T 4 - i n f e c t e d ceils c a n b e r e a c t i v a t e d f o r t r a n s l a t i o n o f f 2 b y t h e a d d i t i o n o f t h e h i g h - s a l t w a s h f r a c t i o n f r o m c o n t r o l r i b o s o m e s 6. T h e
108
R . E . S I N G E R , T. W. C O N W A Y
stimulation observed in these earlier experiments, however, was not normalized with respect to T4 RNA. Since the high-salt wash fraction from control ribosomes contains factors required for the translation of both f2 and T4 R N A 1°, the addition of such a fraction at saturating levels should maximize translation for both kinds of ribosomes with both kinds of m R N A . The control high-salt wash fraction was concentrated by (NH4)2SO 4 precipitation. The effect of adding this preparation to T4 and control ribosomes is seen in Table II to cause a restoration of translation efficiency. The factor-supplemented T4 ribosomes respond to f2 R N A stimulation but no stimulation is observed for T4 RNA-dependent translation. The reversal of restriction by the fractions from control ribosomes implicates the initiation factors in the restriction process and demonstrates that if a T4-induced ribonuclease specific for f2 R N A is present on the T4 ribosomes 27 it is not the sole limiting influence on the f2 R N A translation under these conditions. T A B L E II RESTORATION RIBOSOMES
OF TRANSLATION
EFFICIENCY
BY F R A C T I O N S
FROM
CONTROL
The material washed from control ribosomes with 2 M NH4CI was futher fractionated by precipitation with (NH4)2SO4 between 55 a n d 70 ~ o f saturation. W h e r e indicated 45 itg o f this fraction was added to the reaction mixtures. This was sufficient to produce the m a x i m u m level o f stimulation. Reaction mixtures (0.1 ml) contained 75/~g o f f 2 R N A or 80/~g o f T4 m R N A a n d where indicated either 105/~g o f control ribosomes or 120 ktg o f T4 ribosomes. The T4 a n d control ribosomes were prepared as in the previous experiments, without removing their initiation factors. 100 pmoles -- 16 500 cpm. B a c k g r o u n d incorporation was between 22 a n d 35 p m o l e s / m g ribosome.
Ribosomes
Control T4 Control T4
Supplement
None None (NH4)zSO2 fraction (NI-I4)zSO4 fraction
mRNA-dependent incorporation (pmoles lysine/mg ribosomes) f2 RNA
T4 RNA
392 95 562 233
526 405 1031 36l
Incorporation ratio
0.75 0.24 0.55 0.65
% restriction
68
--18
Specificity of the initiation of translation Should formation of the initiation complex be the rate-limiting step in determining the percent restriction, then the restriction of initiation should be approximately equal to the restriction of translation. This appears to be true under some conditions but not others since the selectivity of initiation was found to vary with the concentration of f2 RNA. The initial velocity of f M e t - t R N A binding to T4 ribosomes at low concentrations of f2 R N A (Fig. 3) resembles the observations of others 7's and ourselves, in experiments not shown here, that high-salt washed ribosomes reconstituted with initiation factors from T4 ribosomes are unable to initiate the translation of R N A phage RNA. In contrast to these results the activity of T4 ribosomes for initiation always approached that of the control ribosomes with increasing amounts of f2 RNA; although they did not always exhibit equivalent activity at the maximal levels. The more sluggish response of the T4 ribosomes in binding f M e t - t R N A may
D E F E C T I V E I N I T I A T I O N BY T4 R I B O S O M E S
109
12,000
8,000
i
4,00C
z~S
t ,,.Z~ I I I I 50
0
I I I
I I
I
I
I
I
I00 jag of f2 RNA
I
150
Fig. 3. Initial velocities o f f M e t - t R N A binding by T4 a n d control r i b o s o m e s in response to f2 R N A . Reaction mixtures (0.15 ml) contained 3 5 # g o f [ 1 4 C ] f M e t - t R N A a n d either 350/~g o f control r i b o s o m e s or 410ftg o f T4 ribosomes. I n c u b a t i o n was for 90 s at 37 °C after addition o f isotope. Q - O , control ribosomes; / x - A , T4 ribosomes. B a c k g r o u n d for control r i b o s o m e s was 1200 c p m a n d for T4 r i b o s o m e s was 800 cpm.
result from a T4-induced modification of a ribosomal component so that it now has a lowered affinity for f2 RNA. It should be noted that the saturating concentration of f2 RNA varied between preparations of T4 ribosomes and was somewhat lower if the binding reaction was allowed to go to completion; furthermore, some preparations of T4 ribosomes showed a more linear than sigmoidal response to f2 RNA. Fig. 4 demonstrates that the initial binding velocities of T4 and control ribosomes respond similarly to increasing concentrations of late T4 RNA in contrast to their behavior with f2 RNA (Fig. 3).
I0,000
2,000
,/
,s~'~' I
50
I
I
100 .ug of T 4
150
I
200
RNA
Fig. 4. Initial velocities o f f M e t - t R N A binding by T4 a n d control r i b o s o m e s in response to late T4 m R N A . Binding reactions (0.1 ml) contained 25 # g o f [ 1 4 C ] f M e t - t R N A a n d either 280 # g o f T4 r i b o s o m e s or 250 # g o f control ribosomes. I n c u b a t i o n was as in Fig. 7. O - O , control ribosomes; A - / x , T4 ribosomes. B a c k g r o u n d for control r i b o s o m e s was 650 c p m a n d for T4 r i b o s o m e s was 420 cpm.
110
R . E . SINGER, T. W. CONWAY
Characterization of the activiO, of the T4-ribosome-f2 RNA initiation complex Since the initiation assay used above also serves as a measure of mRNA-dependent attachment of fMet-tRNA to the 30-S ribosomes, it is significant to note that when sucrose density gradient centrifugation is used to measure the binding of formyl-[~4C]Met-tRNA to T4 ribosomes, the radioactivity was found on the 70-S initiation complex (Figs 5a and 5b). That the initiation complex is active for forming peptidyl puromycin is indicated by Fig. 5c. Puromycin apparently is capable of reacting with virtually all of the bound formyl-[14C]Met-tRNA. 500 T4 Rilx~n~ " 0'4 fMei tRNA
400
A
TOS
I l!lq
300
~
08
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04~
200
100
02 ,~
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10 15 20 Fraction Number
1
25
30
500 T4 Ribosomes + f 2 400
-
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D E F E C T I V E I N I T I A T I O N BY T4 R I B O S O M E S 500
111
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i
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fMe,-, RNA
70S 3B
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3.6
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"~~l°'e- e ' ~ e l ° - °
25
30
Fig. 5. Sucrose gradient analysis of puromycin-induced release of fMet-tRNA bound to T4 ribosomes in response to f2 RNA. Reaction mixtures (0.1 ml) contained 30#g of fMet-tRNA, 350/~g of T4 ribosomes and, where indicated 50/tg of f2 RNA and 54/~g of puromycin. The binding reactions were started in the absence of puromycin. After a 10-min incubation, puromycin was added and the incubation continued for 5 min. After incubation with the labeled substrate, samples were applied to 5 ml sucrose gradient (15-30 ~o (w/w)) and were centrifuged in a Spinco SW 50L rotor at 48 000 rev./min for 2.5 h. The gradients were monitored with an ISCO ultraviolet analyzer at A25~ ,m and fractions collected for scintillation counting. Solid line indicates absorbance at 254 nm. Sedimentation was from left to right and Fractions 20-25 correspond to the 70-S monosome peak. If f o r m a t i o n e f the 70-S T4 r i b o s o m e - f 2 R N A initiation complex limits translation, it w o u l d be expected t h a t the percent restriction o f t r a n s l a t i o n w o u l d decrease as the c o n c e n t r a t i o n o f f2 R N A is increased. However, the d a t a shown in Table I I I indicate t h a t the percent restriction o f t r a n s l a t i o n is i n d e p e n d e n t o f the c o n c e n t r a t i o n o f f2 R N A ; yet the restriction o f initiation is m a r k e d l y decreased. The results suggest t h a t the 70-S r i b o s o m e - f M e t - t R N A - f 2 R N A c o m p l e x is unable to continue translation. O r d i n a r i l y the next step in t r a n s l a t i o n after the p r o p e r p o s i t i o n i n g o f the form y l m e t h i o n y l - t R N A is the b i n d i n g o f the next o r second a m i n o a c y l - t R N A . U s i n g the s a m e p r e p a r a t i o n o f r i b o s o m e s as in T a b l e I, it was f o u n d that t h e T4 r i b o s o m e s have a r e d u c e d affinity for each o f the second c o d e d a m i n o a c y l - t R N A s ( T a b l e IV) o f the three f2 R N A cistrons. The A cistron codes for a structural m i n o r protein, the B cistron for the m a j o r c o a t protein, a n d the C cistron for the R N A synthetase T h e s e c o n d N - t e r m i n a l a m i n o acids are arginine, alanine, a n d serine, respectively. To d e t e r m i n e the relative b i n d i n g efficiencies, the a m o u n t o f the s e c o n d - c o d e d a m i n o a c y l - t R N A s b o u n d m u s t be n o r m a l i z e d to the a m o u n t o f f o r m y l m e t h i o n y l - t R N A . b o u n d (Table V). The relative b i n d i n g efficiency o f the T4 r i b o s o m e s divided by t h a t o f the c o n t r o l r i b o s o m e s is s u b t r a c t e d f r o m one a n d m u l t i p l i e d b y 100 to o b t a i n a m e a s u r e o f the extent o f restriction o f b i n d i n g o f the second a m i n o a c y l - t R N A . These results were confirmed b y d e t e r m i n i n g the respective a m o u n t s o f dipeptides f o r m e d (Fig. 6). T h e relative a m o u n t s o f f M e t - A l a a n d f M e t - A r g detected by the electro-
112
R . E . SINGER, T. W. C O N W A Y
TABLE III R E S T R I C T I O N OF f M e t - t R N A B I N D I N G AS A F U N C T I O N OF f2 R N A C O N C E N T R A T I O N Reaction mixtures for amino acid incorporation (0.15 ml) contained either 200 Ftg T4 ribosomes or 200/~g of control ribosomes. The binding of f M e t - t R N A was measured in reaction mixtures (0.1 ml) which contained 200 pg of either control or T4 ribosomes.
mRNA added (Itg/O.1 ml)
Control ribosomes
T4 ribosomes
% restriction
18 588 763 2 604 4 721 4 685
66 60 62 60
A. mRNA-dependent incorporation (cpm) T4 R N A (160) f2 R N A (11.3) f2 R N A (22.6) f2 R N A (45.3) f2 R N A (136)
26 908 3 161 9 300 17 713 16 732
B. mRNA-dependent fMet-tRNA binding (cpm) T4 R N A (240) f2 R N A (17) f2 R N A (34) f2 R N A (68) f2 R N A (136) f2 R N A (204)
3 478 2 317 4 191 5 417 7 862 7 501
3 452 1 275 2 615 4 170 6 338 6 035
46 36 22 19 19
TABLE IV D I M I N I S H E D B I N D I N G OF T H E SECOND A M I N O A C Y L - t R N A TO f2 R N A C O N T A I N I N G T4 RIBOSOMES The assay mixtures (0.1 ml) contained 30 pg of unlabeled fMet-tRNA, 60/~g of f2 RNA, and either 320/~g of control ribosomes or 350/~g of T4 ribosomes. Where indicated 50/~g of [14C] A r g - t R N A (316 Ci/mole), [aH]Ala-tRNA (2000 Ci/mole), or [14C]Ser-tRNA (156 Ci/mole) were used. Incubation was for 20 min at 37 °C. In a similar reaction mixture with [t4C]fMet-tRNA present the control ribosomes bound a net 23.15 pmoles of f M e t - t R N A and the T4 ribosomes a net 22.71 pmoles of f M e t - t R N A in response to f2 RNA.
Additions
f2 RNA-dependent binding (pmoles aminoacyl-tRNA)
Control ribosomes + f M e t - t R N A T4 ribosomes + f M e t - t R N A
[14C]Arg-tRNA (A)
[aH]AIa-tRNA (B)
[14C]Ser-tRNA (C)
0.90 0.25
9.62 3.09
0.80 0.39
TABLE V R E S T R I C T I O N OF B I N D I N G T H E SECOND A M I N O A C Y L - t R N A S TO T H E f2 R N A CIST R O N S ON T4 RIBOSOMES The ratios were obtained from the data presented in Table IV.
f2 RNA cistrons
A B C
Second aminoacyl-tRNA
[14C]Arg-tRNA [aH]AIa-tRNA. [14C]Ser-tRNA
pmoles aminoacyl-tRNA/ pmoles fMet-tRNA r a t i o Control ribosomes
T4 ribosomes
0.039 0.416 0.035
0.011 0.136 0.017
Ratio T4 % restriction ribosomes/ of binding control ribosomes 0.28 0.33 0.49
72 67 51
DEFECTIVE INITIATION BY T4 RIBOSOMES
113
phoresis were close to the a m o u n t s shown in Table IV; however, the correspondence between the f M e t - S e r detected and seryl-tRNA b o u n d was poor. The a m o u n t o f seryl-tRNA b o u n d might be the result of binding at the third c o d o n of the coat cistron. In any case the relative p r o p o r t i o n o f seryl-tRNA b o u n d to total second aminoacylt R N A b o u n d is very small, which is expected in the absence of translation of the coat cistron.
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E)
Fig. 6. (a) Electrophoretic analysis of f2 RNA coded initiation dipeptides. The reaction mixtures were performed in parallel to those in Table IV and electrophoresis performed as indicated in Materials and Methods at 1.33 kV for 3 h. The relative migration of fMet-tRNA, A; fMet-Arg, B; and fMet-Ser, C, are indicated by the arrows. (b) The reaction was performed in the same manner as the [aH]Ala-tRNA binding in Table IV with the exception that [14C] fMet-tRNA replaced the unlabeled fMet-tRNA. The distribution of [t4C]methionine products has been adjusted for any spill over of tritium counts into the 14C counting channel. The large peaks of formyl-methionine result from the free fMet-tRNA and Met-tRNA present in the reaction mixtures. The decrease in the binding of second a m i n o a c y l - t R N A was f o u n d to be similar to the percent restriction of translation for the parallel preparations of T4 and control (Fig. 1). However, the decrease in binding o f the second a m i n o a c y l - t R N A was not solely dependent on the percent restriction o f translation. Preparations o f T4 ribosomes with a low percent restriction o f translation ( < 60 ~ ) were not restricted for binding the second aminoacyl-tRNAs, possibly indicating that multiple T4-induced effects operate when translation is more severely inhibited. DISCUSSION Ribosomes f r o m T4-infected cells have a reduced ability to translate f2 R N A . The reduction o f the translation activity has been quantitated and shown to clearly exceed a m u c h smaller reduction in activity with late T4 m R N A . The fact that restriction of the translation o f f2 R N A can be observed early in infection, at low mul-
114
R.E. SINGER, T. W. CONWAY
tiplicities, and is of a large magnitude, indicates that it could potentially provide the T4 phage with a significant mechanism to restrict RNA phage development in vivo. It is probable that a number of T4-induced functions 27 result in the restriction of RNA phage development. The coupling of several such restrictive mechanisms might account for the complete shut-down of RNA phage development 28, while any single T4-induced effect probably would fall short of producing the complete restriction. The observations of Goldman and Lodish 17 and those described here, that T4 ribosomes typically have a reduced translational activity for late T4 mRNA suggests that the restriction of f2 RNA translation is not accomplished by a concomitant increase in activity with late T4 mRNA 7'29. General reductions in initiation factor activity noted with chloramphenicol-treated 3° and stationary phase cells 31-33 coupled with the existence of multiple forms of mRNA-specific IF3 factors 9'1°,12,34 indicate a possible role of these factors in the control of translation in bacteria. The recent demonstration of an inhibitory " i " factor-IF3 complex from ribosomes of stationary phase cells 3~'35 suggests that the initiation factors may be modulated to a cells advantage. This is supported by the report that the initiation factor IF3-~, which is specific for early T4 m R N A and MS2 RNA, and IF3-~, which is selective for late T4 mRNA, have separate interference factors, i-~ and i-E, respectively, which can be isolated from the high-salt wash of ribosomes from uninfected cells 35. The fact that the IF3-~ and IF3-fl have similar antigenic determinants 1° and that the total 1F3 antigen is not decreased during T4 phage infection 36 indicate that bacteriophage T4 may alter the ribosome's messenger specificity without synthesizing a new IF3-/~ factor. Two previous reports investigated the T4 ribosome's specificity for the initiation of protein synthesis on the three cistrons of f2 RNA. By measuring the relative amounts of each intiation sequence protected from ribonuclease digestion by T4 and control ribosomes, Steitz et al. ~6 concluded that the coat and synthetase regions (the B and C cistrons) were most reduced while the A cistron was better initiated. In contrast, the analysis of Goldman and Lodish iv of the relative amounts of the three f2 dipeptides synthesized by T4 and control ribosomes was taken to mean that translation of all three cistrons was equally reduced with T4 ribosomes. Our determinations of the amounts of the three dipeptides synthesized resembles the results of Goldman and Lodish a7 as do the data on the relative amounts of the three secondcodded aminoacyl-tRNAs bound to the ribosomes. Perhaps these apparently conflicting results may be due in part to the quite different assays used. The affinity of T4 and control initiation complexes for a particular second-coded aminoacyl-tRNA may not be equivalent to their ability to protect that cistron from ribonuclease. A similar interpretation has also been suggested by Yoshida and Rudland 14 for explaining differences in the two assays when cistron-specific IF3 factors were employed. We have shown here that the T4 ribosomes require rather high concentrations of f2 RNA to approach the initiation activity of control ribosomes. Yet at relatively low concentrations of f2 RNA, the percent restriction of initiation clearly indicates a defect in the initiation process. Comparison of T4 and control ribosomes under these conditions would lead to the conclusion that the inability of T4 ribosomes to translate f2 RNA is due solely to their inability to bind f2 RNA. However, when high concentrations of f2 RNA are employed, the T4 ribosomes, though still restricted
DEFECTIVE INITIATION BY T4 RIBOSOMES
115
for translation, can bind f M e t - t R N A to a much greater degree than would be predicted. It should also be noted in this connection that an excess o f MS2 R N A can overcome the inhibiting effect o f i factor on IF3 function 32. Several interpretations of our data are possible. First, T4 might cause the formation of an abortive initiation complex which fails to translate f2 R N A . Formation of such an abortive complex with high concentrations o f f2 R N A would be consistent with the observation that a T4 superinfection of M 12-infected cells prevents coat antigen synthesis f r o m the M12 R N A (ref. 28). It also fits the observations of Hattman and Hofschneider 4 and G o l d m a n and Lodish 27 that in R N A phage infected bacteria the R N A phage R N A can be f o u n d bound to E. coli ribosomes even t h o u g h a T4 superinfection has shut off synthesis of the R N A phage-coded proteins. A second interpretation considers the proposal that phage T4 induces an endonuclease 27 which in vitro would produce f2 R N A fragments which might compete for the three natural initiation sites o f f2 R N A . However, the ability o f the high-salt wash fraction from control ribosomes to reverse the T4-induced restriction of f2 R N A translation argues against this interpretation. A third possibility would be that infection by phage T4 causes a reduction in the a m o u n t or activity of elongation factors which remain tightly b o u n d to the T4 ribosomes thereby causing an apparent inhibition o f the binding o f the second aminoacyl-tRNAs. This would not explain, however, the inability o f such T4 ribosomes to translate efficiently with high concentrations of f2 R N A in the presence of a large excess o f elongation factors added with the supernatant fraction as is shown in Table III. Furthermore, there is no evidence that T4 infection affects the activity o f cellular elongation factors. Present investigations are directed to resolving these possibilities and the mechanism by which T4 mediates a selective decrease in f2 R N A translation. ACKNOWLEDGMENTS This work was made possible by grants from the American Cancer Society, I o w a Division, and the National Institutes o f Health, A I 11301.
REFERENCES 1 Levinthal, C., Hosoda, J. and Shub, D. (1967) in The Molecular Biology o f Viruses, (Colter, S. J. and Paranchych, W., eds), pp. 71-87, Academic Press, New York 2 McCorquodale, D. J., Oleson, A. E. and Buchanan, J. M. (1967) in The Molecular Biology of Viruses (Colter, S. J. and Paranchych, W., eds), pp. 31-54, Academic Press, New York 3 Zinder, N. (1963) Perspectives in Virology (Pollard, M., ed.), pp. 58-67, Harper and Row, New York 4 Hattman, S. and Hofschneider, P. H (1968) J. Mol. Biol. 35, 513-522 5 Hsu, W. T. and Weiss, S. B. (1969) Proc. NatL Acad. Sci. U.S. 64, 345-351 6 Schedl, P. D., Singer, R. E. and Conway, T. W. (1970) Biochem. Biophys. Res. Commun. 38, 631-637 7 Dube, S. K. and Rudland, P. S. (1970) Nature 226, 820-823 8 Klem, E. B., Hsu, W. T. and Weiss, S. B. (1970) Proc. Natl. Acad. Sci. U.S. 67, 696-701 9 Grunberg-Manago, M., Rabinowitz, J. C., Dondon, J., Lelong, J. C. and Gros, F. (1971) FEBS Lett. 19, 193-200 10 Lee-Haung, S. and Ochoa, S. (1971) Nature 234, 236-239
116 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
R . E . SINGER, T. W. CONWAY
Vermeer, C., Talens, J., Bloemsma-Jonkman, F. and Bosch, L. (1971) FEBS Lett. 19, 201-206 Berissi, H., Groner, Y. and Revel, M. (1971) Nat. New Biol. 234, 44-47 Revel, M., Aviv, H., Groner, Y. and Pollack, Y. (1970) F E B S L e t t . 9, 213-217 Yoshida, M. and Rudland, P. S. (1972) J. MoL Biol. 68,465-481 Pollack, Y., Groner, Y., Aviv, t-I. and Revel, M. (1970) F E B S L e t t . 9, 218-221 Steitz, J. A., Dube, S. K. and Rudland, P. S. (1970) Nature 226, 824-827 Goldman, E. and Lodish, H. F. (1972) J. Mol. Biol. 67, 37--47 Leder, P., Skogerson, L. S., Callahan, R. (1973) Arch. Biochem. Biophys. 153, 814--822 Adams, M. I-[. (1969) Bacteriophages, p. 446, Interscience Publishers, New York Wilhelm, J. H. and Haselkorn, R. (1970) Virology 43, 198-208 Conway, T. W. (1964) Proc. Natl. Acad. Sci. U.S. 51, 1216-1220 Celis, J. E. and Conway, T. W. (1968) Proc. Natl. Acad. Sci. U.S. 59, 923-929 Nirenberg, M. W. and Leder, P. (1964) Science 145, 1399-1407 Roufa, D. J. and Leder, P. (1971) J. Biol. Chem. 246, 3160-3167 Coolsma, J. and Haselkorn, R. (1969) Biochem. Biophys. Res. Commun. 34, 253-259 Nomura, M., Witten, C., Mantei, N. and Echols, C. (1966) J. Mol. Biol. 17, 273-278 Goldman, E. and Lodish, H. F. (1971) J. Virol. 8,417-429 Hattman, S. and Hofschneider, P. H. (1967) J. Mol. Biol. 29, 173-190 Ihler, G. and Nakada, D. (1970) Nature 228,239-242 Young, R. M. and Nakada, D. (1971) J. Mol. Biol. 57, 457-473 Groner, Y., Pollack, Y., Berissi, Y. and Revel, M. (1972) FEBSLett. 21,223-228 Scheps, R. and Revel, M. (1971) Biochim. Biophys. Acta 232, 140-150 Scheps, R., Wax, R. and Revel, M. (1971) Biochim Biophys. Acta 232, 140--150 Groner, Y., Pollack, Y., Berissi, H. and Revel, M. (1972) Nat. New Biol. 239, 16-19 Lee-l-[uang, S. and Ochoa, S. (1972) Biochem. Biophys. Res. Commun. 49, 371-376 Scheps, R., Zeller, H. and Revel, M. (1972) FEBS Lett. 27, 1-4
~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97836
D E F E C T I V E I N I T I A T I O N OF f2 R N A T R A N S L A T I O N BY RIBOSOMES F R O M B A C T E R I O P H A G E T 4 - I N F E C T E D CELLS
ROBERT E. SINGER and THOMAS W. CONWAY Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Received June 4th, 1973)
SUMMARY Ribosomes from T4-infected Escherichia coli when isolated with their initiation factors still attached have negligible activity for the translation of f2 R N A but retain activity for the translation of late T4 RNA. When low concentrations of f2 R N A are used with T4 ribosomes which contain factors, formylmethionyl-tRNA does not bind in response to f2 RNA. Yet the latter ribosomes show substantial activity in binding formylmethionyl-tRNA in response to high concentrations of f2 RNA. Failure of the aberrant initiation complexes to function in translation does not appear to be related to the amount of initiator t R N A bound.
INTROD UCTION Infection ofEscherichia coli cells by bacteriophage T4 results in both the cessation of host protein synthesis 1'2 and the restricted development of R N A phages such as f2, R17, M12 or MS2 (ref. 3). Following the observation of Salser and Gesteland, reported by Hattman and Hofschneider 4 that extracts from T4-infected cells were inactive for the in vitro translation of R17 RNA, it was found that the ribosomes from T4-infected cells discriminated against the translation of MS2 R N A 5'6 or f2 R N A while apparently retaining activity with late T4 m R N A 5. Selectivity for utilization of these m R N A s likely resided in the ribosomal high-salt wash fraction, since this fraction from uninfected cells stimulated f2 R N A translation by T4 ribosomes 6 while that from T4-infected cells inhibited MS2 R N A translation by control ribosomes 5. Several studies using combinations of the high-salt wash fractions and high-salt washed ribosomes from T4 phage-infected and uninfected cells have shown that the specificity for initiation of protein synthesis on the R N A phage RNAs is determined by the source of the high-salt wash fraction 7'8. That is, the initiation factor preparation from the uninfected cells stimulated initiation with R N A phage RNAs while that from T4-infected cells did not. The subsequent resolution of multiple forms of the initiation factor IF3, each form specific for a different m R N A 9-1 ~ or cistron 12- ~4, suggested that translation was regulated by changes in the levels of the different IF3's 1°'15'16 after T4 infection. The conclusion from all of these studies is that the
DEFECTIVE INITIATION BY T4 RIBOSOMES
103
step in R N A phage R N A translation limited by T4 is involved in the formation of the initiation complex. In contrast to these findings, Goldman and Lodish x7 have reported that T4 ribosomes isolated with or without the initiation factors in place showed a similarly reduced activity f o r both translation and initiation whether either f2 RNA or T4 RNA was used as messenger. Furthermore, it has also been reported that infection of E. coli by phages T7, Qfl or ) resulted in a nonspecific reduction in the activity of the ribosomes for the translation of both Qfl and T7 RNAs (ref. 18). These studies have raised a question as to the selectivity of the T4-induced inhibition of in vitro RNA phage R N A translation. We have compared the relative activities of ribosomes from T4-infected cells for the translation of f2 and late T4 mRNAs and find that the activities vary according to the experimental conditions including the time and multiplicity of infection. These variations may be at least partly responsible for the apparently conflicting findings of others. To determine more precisely how the T4-induced defect affects the initiation process, experiments were performed to test the effectiveness of binding formyl[14C]methionyl-tRNA to low-salt washed ribosomes as a function of f2 RNA concentration. Although ribosomes from T4-infected cells fail to initiate efficiently at low concentrations of f2 RNA, at high concentrations of this R N A the label is bound very effectively. In spite of this further translation fails. MATERIALS AND METHODS
Phage infections and preparation of cell extracts E. coli B06 was obtained from E. Six and used for the preparation of uninfected (designated here as control) cells and T4 phage infected (designated here as T4) cells. All comparisons between control and T4 activities were done using extracts derived from a single culture which was grown in M9 medium 19 at 30 °C to a density of 5 • 108 cells/ml and then split into two parallel cultures just before infection of one with bacteriophage T4 (multiplicity of infection of 10 and L-tryptophan, 5/ig/ml). After 10 min &incubation with aeration, both the control and T4 cultures were poured onto 0.5 vol. of frozen and crushed 0.15 M NaC1. The cooled cells were collected by centrifugation at 10 0 0 3 x 9 for 10 rain. This step and all ensuing steps were carried out at 4 °C. The pelleted cells were resuspended in an equal volume of Buffer A (0.01 M Tris-HCl buffer (pH 7.4), 0.01 M MgCI2, and 10 mM 2-mercaptoethanol) which had been made 3/lg/ml in deoxyribonuclease (Sigma, electrophoretically purified free of ribonuclease). They were broken in an ice-cold French pressure cell at 15 000 to 20000 lb/inch 2 and the resultant suspension was centrifuged at 3 0 0 0 0 x 9 for 30 min. The supernatant fraction (S-30) was immediately centrifuged at 145 000 × 9 for 3 h. The upper half of the supernatant liquid (S-145) was dialyzed for 8 h against Buffer A; the ribosomal pellet was resuspended in Buffer A and sedimented again by centrifugation at 145 0 0 0 x g for 3 h. This pellet was resuspended in Buffer A and stored at 4 °C, if used within 2 weeks, or unfrozen in 50 ~ glycerol at --20 °C where it retained its activity for more than a month. The high-salt wash fractions were prepared by resuspending the ribosomal
104
R . E . SINGER, T. W. CONWAY
pellet in 2.0 M NH4CI, 0.01 M Tris-HCl buffer (pH 7.4), 0.01 M MgCI2 and 0.1 mM dithiothreitol and centrifuging for 3 h at 145 000 ×9. The supernatant fraction was dialyzed against Buffer A and fractionated with (NH4)zSO 4 as indicated in the text. The precipitated fraction was resuspended and dialyzed against 0.01 M Tris-HCl buffer (pH 7.4) and 10 mM 2-mercaptoethanol.
Preparations of mRNAs and aminoaeyl-tRNAs Phage f2 was grown and isolated as before s or purchased from Miles Laboratories. Prior to use phage particles were suspended in 20 mM EDTA (pH 7.2), and pelleted by centrifugation at 100 000 × 9 for 3 h; the pellet was resuspended and this step was repeated. These phage preparations sediment in sucrose density gradients as a single peak of A254 nm materials at approx. 79 S. Phenol extraction was done in the presence of 10 mM EDTA (pH 7.2), and 0.1 M sodium acetate (pH 5.2). The aqueous phase was extracted with phenol 3 times and then made 2.0 M in NaCI before adding 2 vol. of chilled ethanol. The suspension was stored at --20 °C for 3 h, centrifuged, and the RNA-containing pellet resuspended in water and dialyzed overnight against water. These preparations of f2 RNA consistently gave a single A254 nm peak on sucrose density gradients of 27 S. Late T4 m R N A was prepared after Wilhelm and Haselkorn 2° from cells infected by T4 phage for 18 min at 30 °C. It was found that much of the DNA in these preparations could be eliminated by the addition of deoxyribonuclease (5/~g/ml) before the EDTA used for cell lysis. All tRNA was charged according to Conway 21 but with the following changes: 75 mM Tris-HC1 buffer (pH 7.4), 5 mM ATP, 10 mM MgCl2 and 2.5 mM 2-mercaptoethanol. When charging methionine tRNAv, calcium leucovorin was present at 0.03 mg/ml; the reaction was for 20 rain at 37 °C and usually in a volume of 1 ml. Purified methionine tRNA v was generously provided by the Oak Ridge National Laboratory. The radioisotopes were purchased from Schwarz/Mann and their respective specific activities noted in the text.
Protein synthesis The reaction mixtures for amino acid incorporation contained: 20/~g of E.
eoli B tRNA, 3 mM ATP, 0.4 mM GTP, 5 mM phosphoenolpyruvate, 2/~g of pyruvate kinase, 75 mM NH4C1 , l0 mM 2-mercaptoethanol, 50 mM Tris-HC1 buffer pH 7.8, 0.04 mM of each 19 unlabeled amino acids, 0.02 mM of [14C]lysine (160 Ci/mole) in volumes from 0.1 to 0.15 ml as indicated for each experiment. In all experiments control S- 145 was employed and typically 180/~g was added to the amounts of ribosomes indicated in the figure legends. Ordinarily the reactions were limited by the concentration of ribosomes present. Immediately prior to their use the ribosomes and S-145 were preincubated 15 min at 37 °C in a buffer containing 225 mM NH4CI, 150 mM Tris-HCl buffer (pH 7.8), 30 mM 2-mercaptoethanol, and 30 mM MgC12. Addition of the remainder of reagents, mRNAs, labeled amino acids and water brought the reaction mixture to the concentrations indicated above. After 30 min incubation at 37 °C the reactions were terminated and counted as previously described 22 at 50 ~ efficiency.
The bindin9 of aminoacyl-tRNAs to ribosomes The binding of formyl-[a4C]methionyl-tRNA and other aminoacyl-tRNAs to
DEFECTIVE INITIATION BY T4 RIBOSOMES
105
ribosomes was measured in 50 mM Tris buffer (pH 7.8), 50 mM NH4C1, 10 mM 2mercaptoethanol, and 0.4 mM GTP (1 mM G T P was employed when measuring binding of other aminoacyl tRNAs) and assayed by binding to Millipore membranes similar to Nirenberg and Leder 23. Unless otherwise stated, binding was done with limiting amounts of ribosomes and relied upon the ribosomes' endogenous complement of initiation factors and elongation factors. Comparisons between the T4 and control ribosomes' relative abilities to bind the second aminoacyl-tRNAs were done with parallel preparations of ribosomes prepared from cells broken in at least 10 mM 2-mercaptoethanol in order to insure sufficient endogenous elongation factor activity. Immediately prior to their use, ribosomes were preincubated for 15 min at 37 °C in a buffer containing 150 mM Tris, 150 mM NH4C1, 30 mM 2-mercaptoethanol, and 15 mM MgCI 2. Again, additions of the remaining reactants brought all concentrations to those indicated above for the binding reaction. Reaction volumes were in a range of 0.1-0.15 ml as indicated for each experiment.
Analysis off2 RNA coded dipeptides The electrophoretic analysis of f2 RNA coded formylated dipeptides and determination of the mobility of marker dipeptides was performed after the procedure of Roufa and Leder 24. Following electrophoresis of the applied reaction mixtures, the paper was dried, cut into strips of 2 cm × 3 cm and counted. The electrophoresis of reaction mixtures done in the presence and absence of f2 RNA were done simultaneously. No peptide products were detected in the absence of f2 RNA. RESULTS
Inactivation o f f 2 RNA translation by T4 infection Our findings and those of others suggest that either dialysis of ribosomes after preincubation 25 or reconstitution of high-salt washed ribosomes with their high-salt wash fraction 14 results in an alteration of the ribosome's translational activity. For this reason, ribosomes were ordinarily isolated without washing at high concentrations of salt and were preincubated without dialysis immediately before use. Cells infected with T4 phage were always compared with parallel uninfected cultures. This eliminated the non-specific variability seen with ribosomes prepared from cells grown on different days. A typical comparison of translation activities is presented in Table I. The T4 ribosomes appear to have less activity than the control ribosomes. Previously, Goldman and Lodish 17 showed that both T4 and control ribosomes synthesize the same polypeptide products with late T4 mRNA. Consequently, we cannot assume that the reduced activity of T4 ribosomes with late T4 m R N A is due to the partial loss of a translation factor specific for certain T4 mRNAs. In order to determine whether there is a reduction in the T4 ribosome's ability to translate f2 RNA which exceeds that due to any non-specific decreases, f2 RNA-stimulated translation with T4 and control ribosomes must be normalized to their respective activities with late T4 RNA. The ratio of amino acid incorporated in response to f2 R N A to that incorporated in response to T4 R N A is designated the incorporation ratio in Table I. Comparison of the incorporation ratios of T4 and control ribosomes shows that the reduction in the T4 ribosome's ability to translate f2 RNA clearly exceeds any non-specific
106
R . E . S I N G E R , T. W. C O N W A Y
TABLE I R E D U C E D T R A N S L A T I O N O F f2 R N A O N T4 R I B O S O M E S Reaction mixtures (0.1 ml) for amino acid incorporation contained 35 #g of f2 R N A or 80/~g of T4 m R N A and either 400 t~g o f control ribosomes or 300 #g of T4 ribosomes. 100 pmoles equals 16 500 cpm.
Additions
[14C]Lysine incorporation (cpm/m# ribosomes)
mRNA- dependent incorporation (pmoles/mg ribosomes)
Incorporation ratio f2 RNA/T4 RNA*
Control ribosomes +f2 RNA +T4 RNA
9 338 6l 250 115 130
-315 642
0.49
T4 ribosomes +f2 RNA +T4 RNA
6 184 11 788 72 369
-34 401
0.08
* 0.08 0.49
= 0.16, or 84 ~ restriction o f translation of f2 R N A by T4 ribosomes.
decrease in translation activity. Therefore, the T4 infection results in a selective decrease that can be quantitated by comparing the activity of T4 ribosomes to control ribosomes in terms of their abilities to translate both f2 and T4 RNAs. This is done by dividing the incorporation ratio for T4 ribosomes by the incorporation ratio for control ribosomes (Table I). If this new ratio is subtracted from one and multiplied by 100, the percent restriction of translation is obtained. The percent restriction of initiation can be calculated using the analogous ratios for the mRNA-dependent binding of fMet-tRNA. The percent restriction of translation has been observed to vary over a range of values depending on the conditions of infection and preparation of the cells. The percent restriction was monitored as a function of the time of infection (Fig. 1) and the multiplicity of infection (Fig. 2). Fig. 1 shows that restriction of translation is maximal by 5 min or less. Some experiments showed a significant decrease in the ability of T4 ribosomes to translate f2 R N A after only 3.5 min of infection; however, the complete set of controls was not done in these cases. The fact that high restriction can be achieved at relatively low multiplicities of infection (Fig. 2) indicates that this phenomenon is not related to changes in nucleic acid metabolism produced by high multiplicities of infection z6. Nomura e t al. 26 suggested that at least two mechanisms exist by which T4 phage can shut off host nucleic acid synthesis. One requires synthesis of T4-coded proteins, and the other is dependent on high multiplicities of infection but is independent of phage protein synthesis. The occurrence of a selective inhibition of f2 RNA translation was highly sensitive to the degree to which the cells were infected. Typically, cultures in which 1 ~o or more of the cells survived 3 min of infection yielded ribosomes which did not show selective inhibition of f2 RNA translation even though a nonspecific decrease in the activity of the ribosomes sometimes as high as 50 ~ was detected. The T4 ribosomes whose activities are presented here were derived from cultures in which
DEFECTIVE INITIATION BY T4 RIBOSOMES
I I00
I ioof ~ o
~
5o
•.~ 75 ~
~. 25
0
107
5o
25
;0 Minutes after infection
° Mulliplicily of infection
Fig. 1. Reduction of translation efficiency of f2 R N A as a function of time after infection. Amino acid incorporation was measured in a volume of 0.1 ml and in a mixture described in Materials and Methods supplemented with the following: 75/~g of f2 R N A or 80/~g of T4 m R N A and where indicated 150 #g of control ribosomes, 160 #g of T4 ribosomes (prepared 5 min after infection), 150/tg of T4 ribosomes (10 min), and 160/~g of T4 ribosomes (15 min). All infections were at 30 °C and at a multiplicity of infection of 15. The cells were broken in the standard Buffer A modified to contain 2.5 mM 2-mercaptoethanol. Control ribosomes were stimulated to incorporate 331 pmoles/mg ribosomes with T4 m R N A and 437 pmoles/mg ribosomes with f2 RNA. In the absence of added m R N A 40 pmoles/mg ribosomes was incorporated. The specific activities of T4 ribosomes with T4 m R N A were 88, 85, and 85 % of the control ribosomes at 5, 10, and 15 min of infection, respectively. The specific activities of T4 ribosomes with f2 RNA were 28, 24, and 22 ~ of the control ribosomes at 5, 10, and 15 rain of infection, respectively. Fig. 2. Reduction in translation efficiency of f2 R N A as a function of the multiplicity of infection. Mixtures contained 90/~g of f2 R N A or 80/~g of T4 mRNA, and where indicated 165/~g of T4 ribosomes (multiplicity of infection = 5), 198/~g of T4 ribosomes (multiplicity of infection = 10), or 165#g of T4 ribosomes (multiplicity of infection = 15). All infections were for 10 min at 30 °C. Other components and conditions are given in Materials and Methods. Control ribosomes were stimulated to incorporate 280 pmoles]mg ribosomes with T4 mRNA, 340 pmoles]mg ribosomes with f2 RNA, and 39 pmoles/mg ribosomes in the absence of added f2 RNA. The specific activities of T4 ribosomes with T4 m R N A were 65, 70 and 92 ~ of the control ribosomes at multiplicities of infection of 5, 10, and 15, respectively. The specific activities of T4 ribosomes with f2 R N A were 7, 24, and 41 ~ of the control ribosomes at multiplicities of infection of 5, I0, and 15, respectively.
0.1 ~ o r less o f t h e cells s u r v i v e d 3 m i n o f i n f e c t i o n . I t h a s a l s o b e e n o u r e x p e r i e n c e t h a t c o n t a m i n a t i o n o f e x t r a c t s f r o m T 4 - i n f e c t e d cells w i t h e x t r e m e l y s m a l l p r o p o r t i o n s o f e x t r a c t s f r o m c o n t r o l cells g r e a t l y r e d u c e s t h e p e r c e n t r e s t r i c t i o n o f t r a n s lation of the T4 ribosomes, without eliminating a non-specific decrease in the activity of T4 ribosomes.
Reversal of T4-induced restriction o f f 2 RNA translation by the high-salt wash fraction of control ribosomes RNA
R i b o s o m e s f r o m T 4 - i n f e c t e d ceils c a n b e r e a c t i v a t e d f o r t r a n s l a t i o n o f f 2 b y t h e a d d i t i o n o f t h e h i g h - s a l t w a s h f r a c t i o n f r o m c o n t r o l r i b o s o m e s 6. T h e
108
R . E . S I N G E R , T. W. C O N W A Y
stimulation observed in these earlier experiments, however, was not normalized with respect to T4 RNA. Since the high-salt wash fraction from control ribosomes contains factors required for the translation of both f2 and T4 R N A 1°, the addition of such a fraction at saturating levels should maximize translation for both kinds of ribosomes with both kinds of m R N A . The control high-salt wash fraction was concentrated by (NH4)2SO 4 precipitation. The effect of adding this preparation to T4 and control ribosomes is seen in Table II to cause a restoration of translation efficiency. The factor-supplemented T4 ribosomes respond to f2 R N A stimulation but no stimulation is observed for T4 RNA-dependent translation. The reversal of restriction by the fractions from control ribosomes implicates the initiation factors in the restriction process and demonstrates that if a T4-induced ribonuclease specific for f2 R N A is present on the T4 ribosomes 27 it is not the sole limiting influence on the f2 R N A translation under these conditions. T A B L E II RESTORATION RIBOSOMES
OF TRANSLATION
EFFICIENCY
BY F R A C T I O N S
FROM
CONTROL
The material washed from control ribosomes with 2 M NH4CI was futher fractionated by precipitation with (NH4)2SO4 between 55 a n d 70 ~ o f saturation. W h e r e indicated 45 itg o f this fraction was added to the reaction mixtures. This was sufficient to produce the m a x i m u m level o f stimulation. Reaction mixtures (0.1 ml) contained 75/~g o f f 2 R N A or 80/~g o f T4 m R N A a n d where indicated either 105/~g o f control ribosomes or 120 ktg o f T4 ribosomes. The T4 a n d control ribosomes were prepared as in the previous experiments, without removing their initiation factors. 100 pmoles -- 16 500 cpm. B a c k g r o u n d incorporation was between 22 a n d 35 p m o l e s / m g ribosome.
Ribosomes
Control T4 Control T4
Supplement
None None (NH4)zSO2 fraction (NI-I4)zSO4 fraction
mRNA-dependent incorporation (pmoles lysine/mg ribosomes) f2 RNA
T4 RNA
392 95 562 233
526 405 1031 36l
Incorporation ratio
0.75 0.24 0.55 0.65
% restriction
68
--18
Specificity of the initiation of translation Should formation of the initiation complex be the rate-limiting step in determining the percent restriction, then the restriction of initiation should be approximately equal to the restriction of translation. This appears to be true under some conditions but not others since the selectivity of initiation was found to vary with the concentration of f2 RNA. The initial velocity of f M e t - t R N A binding to T4 ribosomes at low concentrations of f2 R N A (Fig. 3) resembles the observations of others 7's and ourselves, in experiments not shown here, that high-salt washed ribosomes reconstituted with initiation factors from T4 ribosomes are unable to initiate the translation of R N A phage RNA. In contrast to these results the activity of T4 ribosomes for initiation always approached that of the control ribosomes with increasing amounts of f2 RNA; although they did not always exhibit equivalent activity at the maximal levels. The more sluggish response of the T4 ribosomes in binding f M e t - t R N A may
D E F E C T I V E I N I T I A T I O N BY T4 R I B O S O M E S
109
12,000
8,000
i
4,00C
z~S
t ,,.Z~ I I I I 50
0
I I I
I I
I
I
I
I
I00 jag of f2 RNA
I
150
Fig. 3. Initial velocities o f f M e t - t R N A binding by T4 a n d control r i b o s o m e s in response to f2 R N A . Reaction mixtures (0.15 ml) contained 3 5 # g o f [ 1 4 C ] f M e t - t R N A a n d either 350/~g o f control r i b o s o m e s or 410ftg o f T4 ribosomes. I n c u b a t i o n was for 90 s at 37 °C after addition o f isotope. Q - O , control ribosomes; / x - A , T4 ribosomes. B a c k g r o u n d for control r i b o s o m e s was 1200 c p m a n d for T4 r i b o s o m e s was 800 cpm.
result from a T4-induced modification of a ribosomal component so that it now has a lowered affinity for f2 RNA. It should be noted that the saturating concentration of f2 RNA varied between preparations of T4 ribosomes and was somewhat lower if the binding reaction was allowed to go to completion; furthermore, some preparations of T4 ribosomes showed a more linear than sigmoidal response to f2 RNA. Fig. 4 demonstrates that the initial binding velocities of T4 and control ribosomes respond similarly to increasing concentrations of late T4 RNA in contrast to their behavior with f2 RNA (Fig. 3).
I0,000
2,000
,/
,s~'~' I
50
I
I
100 .ug of T 4
150
I
200
RNA
Fig. 4. Initial velocities o f f M e t - t R N A binding by T4 a n d control r i b o s o m e s in response to late T4 m R N A . Binding reactions (0.1 ml) contained 25 # g o f [ 1 4 C ] f M e t - t R N A a n d either 280 # g o f T4 r i b o s o m e s or 250 # g o f control ribosomes. I n c u b a t i o n was as in Fig. 7. O - O , control ribosomes; A - / x , T4 ribosomes. B a c k g r o u n d for control r i b o s o m e s was 650 c p m a n d for T4 r i b o s o m e s was 420 cpm.
110
R . E . SINGER, T. W. CONWAY
Characterization of the activiO, of the T4-ribosome-f2 RNA initiation complex Since the initiation assay used above also serves as a measure of mRNA-dependent attachment of fMet-tRNA to the 30-S ribosomes, it is significant to note that when sucrose density gradient centrifugation is used to measure the binding of formyl-[~4C]Met-tRNA to T4 ribosomes, the radioactivity was found on the 70-S initiation complex (Figs 5a and 5b). That the initiation complex is active for forming peptidyl puromycin is indicated by Fig. 5c. Puromycin apparently is capable of reacting with virtually all of the bound formyl-[14C]Met-tRNA. 500 T4 Rilx~n~ " 0'4 fMei tRNA
400
A
TOS
I l!lq
300
~
08
D6
04~
200
100
02 ,~
o-,m..oe~.o~.o I
0
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1
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10 15 20 Fraction Number
1
25
30
500 T4 Ribosomes + f 2 400
-
RNA
"--["4 fN" - ~
300 -
A
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t
200 -
v
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-
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Fraction
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20
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I
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~'~1 -J 30
D E F E C T I V E I N I T I A T I O N BY T4 R I B O S O M E S 500
111
!
i
T4 Ribosomes + f2 RNA + Puromycin ~-['4C1
fMe,-, RNA
70S 3B
400 -
f 30C -
3.6
200 -
0.4 "~
t00 -
0.2 . ~
"e'°" w "e~ °-"~°--o. o ..g, o •
0- I
0 Top
1
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10 15 20 Froction Number
I
I
"~~l°'e- e ' ~ e l ° - °
25
30
Fig. 5. Sucrose gradient analysis of puromycin-induced release of fMet-tRNA bound to T4 ribosomes in response to f2 RNA. Reaction mixtures (0.1 ml) contained 30#g of fMet-tRNA, 350/~g of T4 ribosomes and, where indicated 50/tg of f2 RNA and 54/~g of puromycin. The binding reactions were started in the absence of puromycin. After a 10-min incubation, puromycin was added and the incubation continued for 5 min. After incubation with the labeled substrate, samples were applied to 5 ml sucrose gradient (15-30 ~o (w/w)) and were centrifuged in a Spinco SW 50L rotor at 48 000 rev./min for 2.5 h. The gradients were monitored with an ISCO ultraviolet analyzer at A25~ ,m and fractions collected for scintillation counting. Solid line indicates absorbance at 254 nm. Sedimentation was from left to right and Fractions 20-25 correspond to the 70-S monosome peak. If f o r m a t i o n e f the 70-S T4 r i b o s o m e - f 2 R N A initiation complex limits translation, it w o u l d be expected t h a t the percent restriction o f t r a n s l a t i o n w o u l d decrease as the c o n c e n t r a t i o n o f f2 R N A is increased. However, the d a t a shown in Table I I I indicate t h a t the percent restriction o f t r a n s l a t i o n is i n d e p e n d e n t o f the c o n c e n t r a t i o n o f f2 R N A ; yet the restriction o f initiation is m a r k e d l y decreased. The results suggest t h a t the 70-S r i b o s o m e - f M e t - t R N A - f 2 R N A c o m p l e x is unable to continue translation. O r d i n a r i l y the next step in t r a n s l a t i o n after the p r o p e r p o s i t i o n i n g o f the form y l m e t h i o n y l - t R N A is the b i n d i n g o f the next o r second a m i n o a c y l - t R N A . U s i n g the s a m e p r e p a r a t i o n o f r i b o s o m e s as in T a b l e I, it was f o u n d that t h e T4 r i b o s o m e s have a r e d u c e d affinity for each o f the second c o d e d a m i n o a c y l - t R N A s ( T a b l e IV) o f the three f2 R N A cistrons. The A cistron codes for a structural m i n o r protein, the B cistron for the m a j o r c o a t protein, a n d the C cistron for the R N A synthetase T h e s e c o n d N - t e r m i n a l a m i n o acids are arginine, alanine, a n d serine, respectively. To d e t e r m i n e the relative b i n d i n g efficiencies, the a m o u n t o f the s e c o n d - c o d e d a m i n o a c y l - t R N A s b o u n d m u s t be n o r m a l i z e d to the a m o u n t o f f o r m y l m e t h i o n y l - t R N A . b o u n d (Table V). The relative b i n d i n g efficiency o f the T4 r i b o s o m e s divided by t h a t o f the c o n t r o l r i b o s o m e s is s u b t r a c t e d f r o m one a n d m u l t i p l i e d b y 100 to o b t a i n a m e a s u r e o f the extent o f restriction o f b i n d i n g o f the second a m i n o a c y l - t R N A . These results were confirmed b y d e t e r m i n i n g the respective a m o u n t s o f dipeptides f o r m e d (Fig. 6). T h e relative a m o u n t s o f f M e t - A l a a n d f M e t - A r g detected by the electro-
112
R . E . SINGER, T. W. C O N W A Y
TABLE III R E S T R I C T I O N OF f M e t - t R N A B I N D I N G AS A F U N C T I O N OF f2 R N A C O N C E N T R A T I O N Reaction mixtures for amino acid incorporation (0.15 ml) contained either 200 Ftg T4 ribosomes or 200/~g of control ribosomes. The binding of f M e t - t R N A was measured in reaction mixtures (0.1 ml) which contained 200 pg of either control or T4 ribosomes.
mRNA added (Itg/O.1 ml)
Control ribosomes
T4 ribosomes
% restriction
18 588 763 2 604 4 721 4 685
66 60 62 60
A. mRNA-dependent incorporation (cpm) T4 R N A (160) f2 R N A (11.3) f2 R N A (22.6) f2 R N A (45.3) f2 R N A (136)
26 908 3 161 9 300 17 713 16 732
B. mRNA-dependent fMet-tRNA binding (cpm) T4 R N A (240) f2 R N A (17) f2 R N A (34) f2 R N A (68) f2 R N A (136) f2 R N A (204)
3 478 2 317 4 191 5 417 7 862 7 501
3 452 1 275 2 615 4 170 6 338 6 035
46 36 22 19 19
TABLE IV D I M I N I S H E D B I N D I N G OF T H E SECOND A M I N O A C Y L - t R N A TO f2 R N A C O N T A I N I N G T4 RIBOSOMES The assay mixtures (0.1 ml) contained 30 pg of unlabeled fMet-tRNA, 60/~g of f2 RNA, and either 320/~g of control ribosomes or 350/~g of T4 ribosomes. Where indicated 50/~g of [14C] A r g - t R N A (316 Ci/mole), [aH]Ala-tRNA (2000 Ci/mole), or [14C]Ser-tRNA (156 Ci/mole) were used. Incubation was for 20 min at 37 °C. In a similar reaction mixture with [t4C]fMet-tRNA present the control ribosomes bound a net 23.15 pmoles of f M e t - t R N A and the T4 ribosomes a net 22.71 pmoles of f M e t - t R N A in response to f2 RNA.
Additions
f2 RNA-dependent binding (pmoles aminoacyl-tRNA)
Control ribosomes + f M e t - t R N A T4 ribosomes + f M e t - t R N A
[14C]Arg-tRNA (A)
[aH]AIa-tRNA (B)
[14C]Ser-tRNA (C)
0.90 0.25
9.62 3.09
0.80 0.39
TABLE V R E S T R I C T I O N OF B I N D I N G T H E SECOND A M I N O A C Y L - t R N A S TO T H E f2 R N A CIST R O N S ON T4 RIBOSOMES The ratios were obtained from the data presented in Table IV.
f2 RNA cistrons
A B C
Second aminoacyl-tRNA
[14C]Arg-tRNA [aH]AIa-tRNA. [14C]Ser-tRNA
pmoles aminoacyl-tRNA/ pmoles fMet-tRNA r a t i o Control ribosomes
T4 ribosomes
0.039 0.416 0.035
0.011 0.136 0.017
Ratio T4 % restriction ribosomes/ of binding control ribosomes 0.28 0.33 0.49
72 67 51
DEFECTIVE INITIATION BY T4 RIBOSOMES
113
phoresis were close to the a m o u n t s shown in Table IV; however, the correspondence between the f M e t - S e r detected and seryl-tRNA b o u n d was poor. The a m o u n t o f seryl-tRNA b o u n d might be the result of binding at the third c o d o n of the coat cistron. In any case the relative p r o p o r t i o n o f seryl-tRNA b o u n d to total second aminoacylt R N A b o u n d is very small, which is expected in the absence of translation of the coat cistron.
• • [3HI AIo%ne
IOO
i
i i Control R~bosomes
f Met fMet-Ala
slo
Control Ribosomes
IO
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Control Ribosomes + f2 RNA
®
I ÷ f2 L
RNA
2O I0 0 I0 20 Oislonce From Origin (cm)
E)
Fig. 6. (a) Electrophoretic analysis of f2 RNA coded initiation dipeptides. The reaction mixtures were performed in parallel to those in Table IV and electrophoresis performed as indicated in Materials and Methods at 1.33 kV for 3 h. The relative migration of fMet-tRNA, A; fMet-Arg, B; and fMet-Ser, C, are indicated by the arrows. (b) The reaction was performed in the same manner as the [aH]Ala-tRNA binding in Table IV with the exception that [14C] fMet-tRNA replaced the unlabeled fMet-tRNA. The distribution of [t4C]methionine products has been adjusted for any spill over of tritium counts into the 14C counting channel. The large peaks of formyl-methionine result from the free fMet-tRNA and Met-tRNA present in the reaction mixtures. The decrease in the binding of second a m i n o a c y l - t R N A was f o u n d to be similar to the percent restriction of translation for the parallel preparations of T4 and control (Fig. 1). However, the decrease in binding o f the second a m i n o a c y l - t R N A was not solely dependent on the percent restriction o f translation. Preparations o f T4 ribosomes with a low percent restriction o f translation ( < 60 ~ ) were not restricted for binding the second aminoacyl-tRNAs, possibly indicating that multiple T4-induced effects operate when translation is more severely inhibited. DISCUSSION Ribosomes f r o m T4-infected cells have a reduced ability to translate f2 R N A . The reduction o f the translation activity has been quantitated and shown to clearly exceed a m u c h smaller reduction in activity with late T4 m R N A . The fact that restriction of the translation o f f2 R N A can be observed early in infection, at low mul-
114
R.E. SINGER, T. W. CONWAY
tiplicities, and is of a large magnitude, indicates that it could potentially provide the T4 phage with a significant mechanism to restrict RNA phage development in vivo. It is probable that a number of T4-induced functions 27 result in the restriction of RNA phage development. The coupling of several such restrictive mechanisms might account for the complete shut-down of RNA phage development 28, while any single T4-induced effect probably would fall short of producing the complete restriction. The observations of Goldman and Lodish 17 and those described here, that T4 ribosomes typically have a reduced translational activity for late T4 mRNA suggests that the restriction of f2 RNA translation is not accomplished by a concomitant increase in activity with late T4 mRNA 7'29. General reductions in initiation factor activity noted with chloramphenicol-treated 3° and stationary phase cells 31-33 coupled with the existence of multiple forms of mRNA-specific IF3 factors 9'1°,12,34 indicate a possible role of these factors in the control of translation in bacteria. The recent demonstration of an inhibitory " i " factor-IF3 complex from ribosomes of stationary phase cells 3~'35 suggests that the initiation factors may be modulated to a cells advantage. This is supported by the report that the initiation factor IF3-~, which is specific for early T4 m R N A and MS2 RNA, and IF3-~, which is selective for late T4 mRNA, have separate interference factors, i-~ and i-E, respectively, which can be isolated from the high-salt wash of ribosomes from uninfected cells 35. The fact that the IF3-~ and IF3-fl have similar antigenic determinants 1° and that the total 1F3 antigen is not decreased during T4 phage infection 36 indicate that bacteriophage T4 may alter the ribosome's messenger specificity without synthesizing a new IF3-/~ factor. Two previous reports investigated the T4 ribosome's specificity for the initiation of protein synthesis on the three cistrons of f2 RNA. By measuring the relative amounts of each intiation sequence protected from ribonuclease digestion by T4 and control ribosomes, Steitz et al. ~6 concluded that the coat and synthetase regions (the B and C cistrons) were most reduced while the A cistron was better initiated. In contrast, the analysis of Goldman and Lodish iv of the relative amounts of the three f2 dipeptides synthesized by T4 and control ribosomes was taken to mean that translation of all three cistrons was equally reduced with T4 ribosomes. Our determinations of the amounts of the three dipeptides synthesized resembles the results of Goldman and Lodish a7 as do the data on the relative amounts of the three secondcodded aminoacyl-tRNAs bound to the ribosomes. Perhaps these apparently conflicting results may be due in part to the quite different assays used. The affinity of T4 and control initiation complexes for a particular second-coded aminoacyl-tRNA may not be equivalent to their ability to protect that cistron from ribonuclease. A similar interpretation has also been suggested by Yoshida and Rudland 14 for explaining differences in the two assays when cistron-specific IF3 factors were employed. We have shown here that the T4 ribosomes require rather high concentrations of f2 RNA to approach the initiation activity of control ribosomes. Yet at relatively low concentrations of f2 RNA, the percent restriction of initiation clearly indicates a defect in the initiation process. Comparison of T4 and control ribosomes under these conditions would lead to the conclusion that the inability of T4 ribosomes to translate f2 RNA is due solely to their inability to bind f2 RNA. However, when high concentrations of f2 RNA are employed, the T4 ribosomes, though still restricted
DEFECTIVE INITIATION BY T4 RIBOSOMES
115
for translation, can bind f M e t - t R N A to a much greater degree than would be predicted. It should also be noted in this connection that an excess o f MS2 R N A can overcome the inhibiting effect o f i factor on IF3 function 32. Several interpretations of our data are possible. First, T4 might cause the formation of an abortive initiation complex which fails to translate f2 R N A . Formation of such an abortive complex with high concentrations o f f2 R N A would be consistent with the observation that a T4 superinfection of M 12-infected cells prevents coat antigen synthesis f r o m the M12 R N A (ref. 28). It also fits the observations of Hattman and Hofschneider 4 and G o l d m a n and Lodish 27 that in R N A phage infected bacteria the R N A phage R N A can be f o u n d bound to E. coli ribosomes even t h o u g h a T4 superinfection has shut off synthesis of the R N A phage-coded proteins. A second interpretation considers the proposal that phage T4 induces an endonuclease 27 which in vitro would produce f2 R N A fragments which might compete for the three natural initiation sites o f f2 R N A . However, the ability o f the high-salt wash fraction from control ribosomes to reverse the T4-induced restriction of f2 R N A translation argues against this interpretation. A third possibility would be that infection by phage T4 causes a reduction in the a m o u n t or activity of elongation factors which remain tightly b o u n d to the T4 ribosomes thereby causing an apparent inhibition o f the binding o f the second aminoacyl-tRNAs. This would not explain, however, the inability o f such T4 ribosomes to translate efficiently with high concentrations of f2 R N A in the presence of a large excess o f elongation factors added with the supernatant fraction as is shown in Table III. Furthermore, there is no evidence that T4 infection affects the activity o f cellular elongation factors. Present investigations are directed to resolving these possibilities and the mechanism by which T4 mediates a selective decrease in f2 R N A translation. ACKNOWLEDGMENTS This work was made possible by grants from the American Cancer Society, I o w a Division, and the National Institutes o f Health, A I 11301.
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