Initiation of protein synthesis in a rabbit reticulocyte lysate system

526

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96720

INITIATION OF P R O T E I N SYNTHESIS IN A R A B B I T RETICULOCYTE LYSATE SYSTEM* W O L F R A M H O E R Z AND K E N N E T H

S. M c C A R T Y

Department o/ Biochemistry, Duke University Medical Center, Durham, N.C. 27706 (U.S.A.) (Received A u g u s t 24th, 197 o)

SUMMARY

Initiation of protein synthesis in a cell-free system from rabbit reticulocytes has been resolved into two discrete steps by the use of the inhibitors NaF and aurintricarboxylic acid (ATA) : (I) the attachment of a 4o-S ribosomal subunit to polysomes which takes place in the presence of NaF but is blocked by ATA. (2) the addition of the 6o-S ribosomal subunit in the formation of a complete ribosome that is prevented by NaF. Subunits are shown to be directly incorporated into polysomes during initiation of protein synthesis without evidence for the prior formation of a monosome.

INTRODUCTION

The concept that the mechanism of initiation of protein synthesis in eucaryotic cells proceeds in a fashion similar to bacterial protein synthesis is supported by a number of experiments. Initiation factors from reticulocyte ribosomes have been partially purified and appear to correspond fairly closely to those isolated from bacterial cells1. In addition, a particular species of methionyl-tRNA in mammalian cells has recently been implied to transfer methionine only into the amino terminal position 2, and as with Escherichia coli, AUG seems to have properties in this system characteristic of an initiator codon 3. Ribosomes from eucaryotic cells are more resistant to dissociation into subunits by low magnesium concentrations than ribosomes from procaryotic cells4. In spite of this difference, subunits can be derived from reticulocyte polyribosomes by exposure to high concentrations of KC1 and have been reported to be active in the in vitro synthesis of hemoglobin with endogenous mRNA 5-~ as well as the synthesis of polyphenylalanine directed by poly U (refs. 8, 9). With a cell-free system from skeletal muscle it was found that in the presence of GTP, tRNA, and initiation factors, the 4o-S subparticle binds to mRNA, prior to the addition of the 6o-S subunit l°. No rigorous proof is as yet available to demonstrate that subunits are the obligatory intermediates in protein synthesis in eucaryotic cells. Evidence for subunit participation in protein synthesis was suggested by a report from our laboratory on the isolation of polysome complexes consisting of 4o-S ribosomal subunits attached A b b r e v i a t i o n : ATA, a u r i n t r i c a r b o x y l i c acid. * S u b m i t t e d in partial fulfillment of t h e r e q u i r e m e n t s for t h e P h . D . degree in B i o c h e m i s t r y , D u k e U n i v e r s i t y (W.H.).

Biochim. Biophys. Acta, 228 (1971) 526-535

I N I T I A T I O N OF P R O T E I N S Y N T H E S I S

527

to mono-, di-, tri-, and tetrasomes which were formed upon incubation of rabbit reticulocyte lysates in the presence of IO mM NaF (ref. Ii). These structures were proposed to represent transient initiation complexes for protein synthesis in this system. We wish to report here additional evidence for the involvement of subunits in the initiation of protein synthesis. The use of two inhibitors, NaF and aurintricarboxylic acid (ATA), have permitted the resolution of initiation into two discrete steps: (I) the 4o-S ribosomal subunit recognition of mRNA on polyTibosomes which takes place in the presence of NaF but is blocked by ATA. (2) the addition of the 6o-S subunit in the formation of a complete ribosome that is prevented by NaF.

METHODS

The procedures for preparing reticulocytes, in vivo labeling, and lysis have been previously described 11. Cell-free incubations: I vol. of lysate was diluted with 2 vol. modified Medium A (IO mM Tris-HC1 (pH 7.5)-5 ° mM KC1-3 mM magnesium acetate-I mM dithiothreitol). 5 mM phosphoenolpyruvate (adjusted to pH 7) and 50 #g/ml phosphoenolpyruvate kinase were added and E14Clvaline when required. For polysome profiles, aliquots were further diluted with I vol. Medium A and layered directly on isokinetic sucrose density gradients (15-32.4 %, w/w, sucrose) TMcontaining 3 mM magnesium acetate, 5 ° mM KC1, IO mM Tris-HC1 (pH 7.5). Centrifugation was performed in a SW 41 rotor at 41 ooo rev./min for 75 min unless otherwise indicated. Protein synthesis was assayed with the MANS AND NOVELLIls filter disk technique as described previously11. Ribosome-free lysate supernatant was obtained by centrifuging lysate for 90 rain at 45 ooo rev./min. Native ribosomal subunits were prepared by centrifuging lysate through 2 ml Medium A containing 0.5 M sucrose for 3 h at 45 ooo rev./min in a 50 Ti rotor. The ribosomal pellet was resuspended in Medium A, sedimented through a sucrose density gradient for 3 h at 41 ooo rev./min, monitored with a Gilford spectrophotometer, and the subunits collected. Sephadex chromatography was performed with I-ml samples on a io cm × 0.8 cm Sephadex G25 column equilibrated in Medium A. Radioactivity was determined as previously described 14.

RESULTS

Our previous conditions for protein synthesis were slightly modified to reproduce in vitro as nearly as possible the balance between initiation and elongation that prevails in the intact reticulocyte. Protein synthesis was linear over a period of 15 min, Fig. I, when the temperature was maintained at 3 o°, an energy regenerating system added and the lysate diluted with 2 vol. of Medium A, the ionic composition of which was critical. These conditions were established by experiments with lysates that had been passed through a Sepbadex G25 column equilibrated with different buffers. The medium that was selected yielded maximal protein synthesis with no initial polysome breakdown upon the addition of optimal concentrations of ATP, GTP, and amino acids. Under these conditions the rate of initiation exceeded that of Biochim. Biophys. Acta, 228 (1971) 526-535

528

W. HOERZ AND K. S. MCCARTY

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Fig. 2. I vol. lysate w a s i n c u b a t e d w i t h 2 vol. M e d i u m A c o n t a i n i n g r a d i o a c t i v e 4o-S r i b o s o m a l s u b u n i t s in t h e c o m p l e t e cell-free s y s t e m . A l i q u o t s were diluted, layered directly on sucrose dens i t y g r a d i e n t s a n d s e d i m e n t e d for 75 rain a t 41 ooo r e v . / m i n . (a) Before i n c u b a t i o n . (b) A f t e r 8 rain, (c) A f t e r 8 m i n in t h e presence of I m M cycloheximide. - - , a b s o r b a n c e ; Q - G , radioactivity.

As in our previous report 11 I . I O -2 M N a F decreased the synthesis of hemoglobin b y more than 5 ° °/o under these conditions, Fig. i, and the polysome profile was not only diminished but was characterized b y 4o-S ribosomal subunits attached to all the remaining polysomes, Fig. 3 b. To show that these complexes did not represent artificial associations, ribosomes which lack the capacity to reinitiate were examined. For these experiments Biochim. Biophys. Acta, 228 (i97 I) 526-535

INITIATION OF PROTEIN SYNTHESIS

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Fig. 3- L y s a t e was incubated u n d e r s t a n d a r d conditions for 2 mill and fractionated as in Fig. 2. Additions: (a) o.I mM ATA. (b) IO mlV~NaF, (c) io mM N a F and o.i mM ATA. The arrow denotes the disome in each figure.

use was made of the observation that during maturation in vivo, reticulocytes gradually lose the ability to reinitiate new polypeptide chains which leads to a conversion of polysomes into inactive monosomes 15-1s, Fig. 4a shows the polysome profile from reticulocytes in a later stage of maturation. When this lysate was incubated under the above conditions, polysomes were rapidly diminished which confirmed their inability to reinitiate (Fig. 4b). Incubation in the presence of NaF resulted in an identical o)

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Fig. 4. L y s a t e p r e p a r e d f r o m reticulocytes t h a t h a d been allowed to m a t u r e in vivo after the last injection of phenylhydrazine, was incubated for 2 rain u n d e r s t a n d a r d conditions, and fractionated as in Fig. 2. (a) Before incubation. (b) After 2 rain, (c) After 2 min in the presence of IO mM N a F . Biochim. Biophys. Acta, 228 (1971) 526-535

53 °

W. HOERZ AND K. S. MCCARTY

breakdown without a n y evidence of polysome-subunit association (Fig. 4c). These experiments demonstrate that initiation is a prerequisite for N a F to induce the formation of the intermediate peaks. In order to examine the action of N a F in greater detail, ATA, a triphenolic dye first described by GROLLMANAND STEWART19 was employed because of its reported selective inhibition of initiation of protein synthesis by blocking the attachment of ribosomes to m R N A in both, bacterial and mammalian protein synthesizing systems ~°. As shown in Fig. I, I . IO-a M ATA completely inhibited protein synthesis, whereas I . lO -5 M ATA was ineffective. Protein synthesis was inhibited by 75 % in the presence of I - l O -4 M ATA, and the polysome patterns demonstrated a rapid degradation at this concentration, Fig. 3a. These results are similar to those of N a F inhibition as shown in Fig. 3b, with the important exception, however, that N a F permits the formation of the intermediate peaks which represents the attachment of

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Fig. 5. (a) Lysate was p r e i n c u b a t e d for 4 min in the presence of IO mM NaF, passed t h r o u g h a Sephadex G25 column and fractionated as in Fig. 2. (b)-(d) This lysate was reincubated for 5 min with a d d e d ribosome-free s u p e r n a t a n t under s t a n d a r d conditions of protein synthesis. (b) I mM cycloheximide was added. (c) control incubation. (d) i mM cycloheximide and IO mIV[ N a F were present. The lysate in (d) had been passed t h r o u g h a Sephadex G25 column t h a t contained NaF. The positions of the di-, tri-, tetra-, and p e n t a s o m e s are indicated (- - -).

Biochim. Biophys. Acta, 228 (1971) 526-535

INITIATION OF PROTEIN SYNTHESIS

531

4o-S ribosomal subunits. When both ATA and NaF were added to the incubation mixture, as shown in Fig. 3c, ATA prevented the attachment of the 4o-S ribosomal subunit and thereby the formation of any intermediate peaks. If NaF specifically inhibits the attachment of 6o-S ribosomal subunits to the mRNA-4o-S ribosomal subunit complex, it ought to be possible to convert these intermediate polysome complexes to complete integral ribosomes after the removal of NaF. Thus for example, it should be possible to demonstrate the formation of a trisome from a 4o-S subunit~lisome complex. This hypothesis was tested by preincubating reticulocyte lysate for a short period in the presence of NaF to produce the intermediate polyribosome complexes as shown in Fig. 5a. At 4 ° the incubation mixture was then passed through a Sephadex G2 5 column equilibrated with Medium A to remove the sodium fluoride. A ribosome-free lysate supernatant was then added and the preparation reincubated in the presence, Fig. 5b, and absence of cycloheximide, Fig. 5c. Fig. 5 a shows a polysome distribution that represents almost exclusively the intermediate peaks with attached 4o-S ribosomal subunits. In both Figs. 5b and 5c, these intermediate complexes have completed the second stage of initiation, namely the attachment of the 6o-S ribosomal subunit to form the complete polysomes. In the presence of cycloheximide, when translation was inhibited, there was a slight shift towards heavier polyribosomes (note for example the increase in pentasomes), Fig. 5b. In the absence of cycloheximide as shown in Fig. 5c, a gradual polysome breakdown was observed which usually occurs after longer periods of protein synthesis. NaF prevented the conversion to integral ribosomes (Fig. 5d) when it was included in both the Sephadex column as well as the subsequent incubation. To firmly establish that the conversion of the 4o-S subunit-polysome complexes to integral polysomes proceeded by addition of a 6o-S subunit rather than dissociation of the 4o-S particle, a radioactive disome-4o-S subunit complex was o)

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Fig. 6. I voh lysate was incubated w i t h 2 vol. Medium A containing radioactive disome~4o-S s u b u n i t complexes and sedimented for 2 h a t 41 ooo rev./min. (a) Before incubation. {b) After i n c u b a t i o n for 5 m i n in t h e presence of I mM cycloheximide. (c) After i n c u b a t i o n for 5 rain w i t h IO miY[ N a F a n d i mM cycloheximide. The lysate in (c) had been p r e i n c u b a t e d w i t h o u t dilution for 2 rain in the presence of N a F before the addition of the radioactive material which in this case h a d been isolated in the presence of N a F . - - , absorbance; O - O , radioactivity. The positions of the di-, tri-, a n d t e t r a s o m e s are indicated (- - -).

Biochim. Biophys. Acta, 228 (197 I) 526-535

532

W. HOERZ AND K. S. MCCARTY

isolated and its fate followed during an incubation in fluoride-free medium. Reticulocytes were labeled in vivo with **Pt as previously described n. An aliquot of the lysate was incubated in the presence of N a F and sedimented through a sucrose density gradient. The ribosome peak between the disome and trisome was collected, passed through a Sephadex G2 5 column and added to an unlabeled lysate under standard conditions of protein synthesis. The ~2p count distribution and the optical density tracing at zero time is shown in Fig. 6a and after 5 rain of incubation in the presence of cycloheximide, in Fig. 6b. This experiment illustrates significant conversion of the subunit-disome-complex to a trisome and rules out the loss of a 4o-S subunit which would result in a disome. To demonstrate that N a F prevented this conversion, the intermediate peak was isolated from a gradient and passed through a Sephadex G2 5 column, both containing NaF. In addition the unlabeled lysate was also preincubated with NaF. Under these conditions there was no conversion of the intermediate complex to a trisome, Fig. 6c. This demonstrates that N a F must be continuously present to prevent the attachment of the 6o-S ribosomal subunit. The proposed mechanism of initiation in eucaryotic cells as described here requires that subunits be incorporated directly into polysomes without prior monosome formation. In order to test this hypothesis native subunits were prepared from a 82p labeled lysate as described in METHODS and passed through a Sephadex G2 5 column. The capacity of these subunits to participate in protein synthesis was examined b y incubating them with unlabeled reticulocyte lysate under conditions optimal for reinitiation. The whole reaction mixture was then layered on a sucrose density gradient and centrifuged. Fig. 2a shows the polyribosome profile before incubation and Fig. 2b after 8 rain of incubation, where a substantial amount of the subunit label appears in the polysome region. To rule out non-specific association of subunits and polysomes, cycloheximide was added to the incubation (Fig. 2c) and prevented most of the incorporation of ribosomal subunits into polysomes. The remaining low level of incorporation which was also observed after very short periods of incubation in the absence of cycloheximide m a y be explained by the fact that at most one subunit could attach per polysome peak. The longer protein synthesis was allowed to proceed, the more asp label appeared in the heavy polyribosomes, as would be expected if these polysomes incorporated several radioactive ribosomal subunits during several rounds of reinitiation. If I- Io -4 M ATA was added as a control, no ribosomal subunit attachment to polysomes was observed, and the count distribution was identical to Fig. 2a. In no case was there a n y subunit label in the monosome area. Additional studies not shown here with the 6o-S subunit demonstrated that these behave in an identical manner. Under optimal conditions for reinitiation, the monosome peak effectively decreases during protein synthesis. We therefore proceeded to examine monosomes labeled with 32p. Radioactive lysate was centrifuged through a sucrose density gradient, the monosome collected and tested in the same way as the ribosomal subunits in Fig. 2. Fig. 7 shows that radioactivity appeared in the polysome region during protein synthesis which indicates that at least some of the monosomes participated in initiation. We would suggest, however, that this occurred by a prior dissociation into subunits which were then capable of reinitiation. A rigorous proof for this mechanism still remains to be established, even though the evidence that subunits directly participate in reinitiation strongly favors this theory. Biochim. Biophys. Acta, 228 (1971) 526-535

INITIATION OF PROTEIN SYNTHESIS

533 ¢)

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Fig. 7. I vol. lysate was incubated with 2 vol. 1V~ediumA containing radioactive monosomes under standard conditions and fractionated as in Fig. 2. (a) Before incubation. (b) After 8 mill, (C) After 8 min in the presence of i mlV[ cyclohexilnide. - - . , absorbance; O - O , radioactivity. At high concentrations of ATA (I.IO -a M) not only was protein synthesis completely inhibited, as shown in Fig. I, but in addition the majority of the monosomes were dissociated into 4o-S and 6o-S subunits while polysomes remained unchanged. At I . lO -2 M ATA all ribosomal material was converted to 3o-S and 5o-S particles. This is probably due to the depletion of free Mgz+, in analogy with other Mg ~+ complexing agents. Increasing the monovalent ion concentration or lowering the Mg ~+ concentration b y known chelating agents such as ATP and phosphoenolpyruvate enhances this dissociation in the presence of ATA. ATA appears to bind to the ribosomal subunits and is not removed b y gradient centrifugation or b y Sephadex G25 chromatography at 4 °. When these ribosomal subunits were tested for activity b y the technique outlined in Fig. 2, the bound ATA proved to inhibit initiation when present at concentrations higher than 1.1o -5 M. When the effective ATA concentration was lowered, however, either by adding fewer ribosomal subunits or diluting the protein synthesizing system, protein synthesis was not inhibited and the derived ribosomal subunits were as active as the native species. Thus monosomes can be dissociated into active subunits under conditions t h a t leave polysomes intact. This is an additional argument in favor of the concept t h a t subunits are the sole ribosomal species active in initiation. DISCUSSION The sequence of events during initiation of new polypeptide chains has not been firmly established in eucaryotic cells. N a F has been a useful tool for investigating this process in an i n vitro protein synthesizing system. One of the common difficulties observed with eucaryotic cells has been the fact that only low levels of initiation compared to translation have been obtained in most cell-free protein synthesizing systems. This problem has been overcome b y slight modifications of the protein synthesizing system. Thus in the absence of any inhibitor, polysomes are Biochim. Biophys. Aaa, 228 (1971) 526-535

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w . HOERZ AND K. S. MCCARTY

maintained over a period of 15 min of active protein synthesis. Under these conditions it is possible to establish that N a F inhibits initiation, and that its characteristic event is the association of a small subunit with polysomes. We propose that this is a part of the physiological initiation process in view of the fact that when maturing reticulocytes become deficient in initiation 17, they lose the capacity to carry out this step. Therefore, the formation of the intermediate peaks can be utilized as an assay for initiation as shown in these studies. ATA has also been shown to inhibit initiation but at a different level 10. With these two inhibitors then it is possible to resolve initiation into two discrete steps. ATA prevents the formation of the first initiation complex of m R N A with the 4o-S ribosomal subunit, whereas N a F permits the addition of the small subunit to a polysome, inhibits, however, the subsequent addition of the 6o-S ribosomal subunit. As soon as N a F is removed, the large subunit does attach and the next larger polysome is formed apparently without prior dissociation of the polysome-4o-S complex. The inhibitory action of N a F seems to reside in the first of these two steps, because once N a F is removed and then added back, this initiation complex is no longer sensitive to fluoride inhibition and readily accepts the 6o-S subunit even in the presence of newly added NaF. The mechauism of initiation proposed here requires subunits to be the active species and 8o-S ribosomes to represent only temporarily associated subunits. This hypothesis was tested here, and it was found that the subunits were readily incorporated into polysomes during protein synthesis, apparently without prior formation of monosomes. This is in agreement with our earlier findings 21 and the results of HENSHAW et al. 22. The fact that 8o-S ribosomes were also capable of participating in initiation was anticipated and observed. Puromycin and ethionine experiments have shown that polysomes can reform from monosomes after the inhibitor has been washed out 2s,~. It appears that the rapid interconversion between 8o-S ribosomes and their subunits is a normal event. This is supported by the artificial dissociation of monosomes by high concentration of ATA described here and b y high ionic strength buffers as shown in other laboratories 25 under conditions where polysomes remain intact. These derived subunits were found active in initiation of protein synthesis. N a F has been postulated to act in whole cell systems from reticulocytes and H e L a cells by inhibiting the dissociation of monosomes with a subsequent depletion of subunits 2e, an observation that has been confirmed b y VESCO AND COLOMBO 27 but is in disagreement with BISHOPm. In these experiments 27 a complex was reported in-reticulocytes which was formed under the influence of N a F that could correspond to a complex of a monosome with an attached 40-S subunit. It is difficult, however, to compare our results to these studies, as these investigators have not observed a n y effect of N a F on subunits in cell-free systems whereas all our work has been done with reticulocyte lysates. We have consistently observed a decrease in the quantity of small subunits and an increase in large subunits after incubation of a cell-free system in the presence of NaF. In the absence of a n y information on the mechanism of dissociation and reaggregation of monosomes, however, it is difficult at this time to assess the value of these observations considering that the breakdown of polysomes is easily induced b y various inhibitors of protein synthesis and always results in the accumulation of monosomes and not subunits 23,24,29. In addition, the ionic composition of the medium in which the ribosomes are suspended m a y influence polysome proBiochim. Biophys. Aaa, 228 (i97 I) 526-535

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files, as it h a s b e e n r e p o r t e d for b a c t e r i a w h e r e s o d i u m s u b s t i t u t e d for p o t a s s i u m p r o d u c e s d i f f e r e n t m o n o s o m e - s u b u n i t r a t i o s a°. W e w o u l d p r o p o s e t h e n t h a t p r o t e i n s y n t h e s i s in e u c a r y o t i c a n d p r o c a r y o t i c cells p r o c e e d s in a s i m i l a r f a s h i o n . I n b o t h cases, t h e a t t a c h m e n t of a 40-S s u b u n i t t o m R N A - p o l y s o m e c o m p l e x e s c o n s t i t u t e s t h e first s t e p in i n i t i a t i o n , w h i c h c a n be p r e v e n t e d b y A T A . D u r i n g t h e n e x t step, a 60-S r i b o s o m a l s u b u n i t a t t a c h e s t o t h e 40-S p a r t i c l e - m R N A - p o l y s o m e c o m p l e x , a n e v e n t t h a t is s p e c i f i c a l l y i n h i b i t e d b y N a F . U n d e r n o r m a l c o n d i t i o n s t h e s e t w o steps are r a p i d a n d a p p e a r as a single e v e n t . T h u s , o n l y b y e m p l o y i n g a p p r o p r i a t e i n h i b i t o r s h a s it b e e n possible t o v i s u a l i z e t h e i n t e r m e d i a t e stages. T h e a t t a c h m e n t of 40-S s u b u n i t s t o p o l y s o m e s c o n s t i t u t e s a s i m p l e a s s a y i n i t i a t i o n for a n d t h e i n v o l v e m e n t of i n i t i a t i o n f a c t o r s in t h i s process is c u r r e n t l y u n d e r i n v e s t i g a t i o n . Since t h e s u b m i s s i o n of t h i s m a n u s c r i p t , LEBLEU et al. 31 r e p o r t e d o b s e r v a t i o n s in a g r e e m e n t w i t h o u r s on t h e effect of A T A t o i n h i b i t b i n d i n g of m R N A t o t h e ribosomal subunit whereas NaF did not prevent this binding.

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