Stage-specific initiation factors for protein synthesis during insect development

DEVELOPMENTAL

BIOLOGY

Stage-Specific

25, 280-292

(1971)

Initiation during

Factors for Protein Synthesis

Insect

Development’

JOSEPH ILAN~ AND JUDITH Department

of Biology,

Temple

University,

Accepted

January

ILAN~

Philadelphia,

Pennsylvania

19122

25,197l

INTRODUCTION

It is believed that in eukaryotic organisms protein synthesis is initiated in a manner similar to bacterial protein synthesis. First it was shown in protozoa (Ilan, 1969b; Ilan and IIan, 1970) and later in ascites tumor cells (Smith and Marcker, 1970; Brown and Smith, 1970) that in a cell free system containing all tRNAs only synthetic messenger RNA with the methionine AUG near their beginning are translated at low magnesium concentration. Eukaryotic cells contain two species of methionine tRNA (Brown and Smith, 1970; Caskey et al., 1967; Takeishi et al., 1968). One tRNA inserts methionine only internally in the growing polypeptide chain. The second acts as initiator and incorporates methionine into the NH,-terminal position. It differs from that of prokaryotes in that the methionine is not formylated. In eukaryotes natural messenger RNA also initiates with methionine as was shown recently for the alpha and beta globin chains and for protamine in trout testis cells (Jackson and Hunter, 1970; Wilson and Dintzis, 1970; Housman et al., 1970; Wigle and Dixon, 1970). In prokaryotes, for protein synthesis to start, mRNA binds to a 30 S ribosomal subunit and the initiator formylmethionyl-tRNA (fMettRNA) connects to its codon AUG, and concomitantly the 50 S subunit joins the 30 S to form a 70 S initiation complex. Three initiation factors function in the formation of the initiation complex. They can be isolated from ribosomes by extraction with 1 M NH&l. GTP is also needed to promote the binding of bacterial initiator fMettRNA to the initiation complex. It appears that during initiation of protein synthesis in prokaryotes the 70 S ribosomal unit formed by association of free 30 S and 50 S subunits arises from ribosomes ’ Supported by Grant GB-8464 from the National Science Foundation. *Present address: Department of Anatomy, Case Western Reserve School of Medicine, Cleveland, Ohio 44106. 280

University,

STAGE

SPECIFIC

INITIATION

FACTORS

281

which were dissociated after chain termination (Lipmann, 1969). In eukaryotes there is no such wealth of available information. However, studies involving the ribosomal-polysomal subunit cycle in mammalian systems and studies on the formation of the initiation complex suggest that this model for protein synthesis may also apply (Kaempfer, 1969; Hogan and Korner, 1968; Kabat and Rich, 1969; Joklik and Becker, 1965). Moreover, it was recently shown that three factors are required for initiating the synthesis of new and complete hemoglobin alpha and beta chains at low Mg*+ concentration (Prichard et al., 1970). An illustration summarizing initiation of mRNA translation is shown in Fig. 1. In a protein-synthesizing system from Tenebrio molitor, we observed that ribosomes capable of synthesizing cuticular protein in vitro (Ilan, 1968; Ilan et al., 1970) lost their ability to incorporate amino acids into the NH,-terminal position after extensive washing with 0.5 M KCl. Here we report evidence that the 40 S ribosomal subunit binds mRNA during the initiation of protein synthesis. Formation of the complete 80 S initiation complex in insects requires mRNA, GTP, aminoacyl-tRNA and initiation factors. The complex sediments as an 80 S band on sucrose gradient. Moreover, the initiation factors are stage specific and promote formation of the 80 S initiation complex only with mRNA extracted from the same stage of development. MATERIALS

AND

METHODS

Insects were maintained as previously described (Ilan et al., 1966). When last-instar larvae were used, guts were removed prior to homogenization. Uridine-2-14C (30 mCi/mmole) was obtained from New England Nuclear Co. Animals were labeled in uiuo by injecting each with 1 PCi of uridine-14C and incubating for 30 minutes at 28O before homogenization. Polysomes were prepared as previously described for preparation of ribosomes (Ilan, 1968; Ilan et al., 1970) and were divided into two parts. The control was layered on a 10 to 40:; sucrose gradient (4.5 ml) in buffer A and centrifuged for 90 minutes at 39,000 rpm. Drops were collected from the bottom and analyzed for optical density and radioactivity as previously described (Ilan, 1968). The other polysomal portion was suspended in buffer (IIan et al., 1970) which contained 12 mM MgCl,, 1 M KCl, 10 mM Tris-HCl (pH 7.6), 10 mM dithiothreitol (DTT), and 5 pg/ml of polyvinyl sulfate. The suspension was analyzed on a 15 to 305;, sucrose gradient containing

282

ILAN

AND

ILAN DNA 4

5’

-3’

mt?NA

PROTEIN

r

60s POOL

TRAN ISLOCASE (G- FACTOR 1

AMINOACYLtRNA AND PEPTIDYL TRANSFERASE ( T- FACTOR I

40s

MET

INITATION

IN TERMEDlATE

I-PA-hAr-r

80s

COMPLETE

INITIATION

FIG. 1. Initiation

CCA-

COMPLEX

of mRNA

translation.

the same buffer. Centrifugation was carried out in a SW 65 rotor at 39,000 rpm for 3 hours at 28” (Martin et al., 1969) (Fig. 2b). Tubes Nos. 8-22 were concentrated by lyophilization and served as sources of ribosomal subunits. Tubes 23-28 were also concentrated and served as mRNA. Polysomal RNA was extracted with phenol and analyzed on sucrose gradient as previously described (Ran, 1969a). Sedimentation values of the labeled RNA extracted from ribosomes was determined according to Martin and Ames (1961), assuming the two ribosomal RNA components were 18 S and 28 S, respectively. Aminoacyl-

STAGE

0

4

8

SPECIFIC

INITIATION

12 162024280 FRACTION

4

FACTORS

8

283

12 I6202428

NUMBER

FIG. 2. Release of messenger

ribonucleoprotein from polysomes. Seventh day pupae were labeled for 30 minutes with uridine-“C. Polysomes were prepared as described in Materials and Methods. They were divided into two parts. One (A) was layered on a 10 to 40% sucrose gradient in buffer A and centrifuged for 90 minutes at 39,000 rpm in SW 65 rotor at 4°C. Drops were collected from the bottom and analyzed for optical density and radioactivity. Another portion (B) was suspended in buffer containing 12 mM MgCl,, 1 M KC1 10 mM DTT, and 5 fig/ml of polyvinyl sulfate. The suspension was layered onto 4.5 ml 15 to 307; sucrose containing the same buffer and centrifuged for 3 hours at 28” in the same rotor and speed as (A).

tRNA was extracted by phenol from first day pupae and used as such or after deacylation (IIan, 1968). Initiation factors were prepared from monosomes obtained from crude polysomes prepared by omitting the KC1 wash. These polysomes were incubated in a complete system for protein synthesis for 30 minutes at 30”. This treatment was shown to break the polysomes completely to monosomes (Ilan et al., 1970) KC1 was added to a final concentration of 1 M. The ribosomes were centrifuged for 1 hour at 135,000 g and the protein initiation factors were precipitated from the post ribosomal supernatant fluid by 80% saturation with (NHJ,SO,, dissolved in a small volume of medium A (IIan and Lipmann, 1966) containing 157; glycerol and stored at -70”. All activity was lost after 10 minutes incubation at 60” but was retained in cold storage at -70” at least 3 months. The reaction mixture for the formation of the complete initiation complex was carried out in 0.5 ml and contained 50 mM Tris-HCl (pH 7.6), 6 nnJ4 MgCl, 50 mM KCl, 10 mM DTT, 0.5 mg of 40 S and 60 S of ribosomal subunits in equal amounts, 0.5 mM GTP, 0.5 mg of

284

ILAN

AND

ILAN

aminoacyl-tRNA, 200 pugof initiation protein and 1000 cpm of mRNA as RNP. Omissions from the complete reaction mixture are described in the figure legends. Incubations were carried out for 10 minutes at 30”. The tubes were rapidly cooled and layered on 4.5 ml 15 to 30% sucrose gradient containing medium A. The mixture was centrifuged for 1 hour at 60,000 rpm in a SW 65 rotor at 4’. Optical density was recorded automatically with an Isco analyzer. Drops were collected and the samples were filtered through Millipore filters (pore size 0.45 p in diameter) and washed with medium A. Radioactivity was determined by liquid scintillation counting. RESULTS

Isolation

of mRNP

Complex from Polyribosomes

Polysomes were isolated from larvae or animals 7 days after the larval-pupal molt (7&y pupae). They had been labeled in viuo for 30 minutes with uridine-I%. Previous experiments (Ilan, 1969a) had shown that no significant label was detectable on ribosomal RNA before 45 minutes of exposure to the radioisotope. After isolation the polysomes were divided into two parts. One portion served as a control, and the other was exposed to 1 M KCl-12 mM MgCl, in order to dissociate the ribosomes into subunits (Martin et al., 1969) and release the mRNA. Figure 2a shows sedimentation analysis of pupal polysomes labeled (for 30 minutes) in uivo with uridine-14C. Most of the label is associated with the polysomes, and is more abundant in the smaller polyribosomes. When the polyribosomes are exposed to 1 M KC1 (Fig. 2b) the ribosomes dissociate into subunits and the radioactive RNA is released as a polydisperse array of RNP complex sedimenting between 10 S and 60 S. The radioactive RNA extracted from pulse-labeled polyribosome by phenol is itself polydisperse (Fig. 31, giving a sedimentation profile similar to that described previously for HeLa cells (Penman et al., 19631, L cells (Perry and Kelley, 1963), and rat liver (Henshaw, 1968). Formation

of Initiation

Complex

For these studies labeled RNP released from the polysome with 1 M KC1 served as mRNA. Formation of the initiation complex was detected by determination of the position of the radioactivity on sucrose gradient. Figure 4A shows sedimentation analysis of the complete system which includes RNP-‘“C as mRNA, 40 S and 60 S ribosomal subunits, initiation factors, GTP, and aminoacyl-tRNA. Most

STAGE

RNA

EXTRACTED

SPECIFIC

INITIATION

FROM

POLYRIBDSDMES

285

FACTORS

0.6

a

0 0.4 8

0.2

iBS SEDIMENTATION

IAS

:s

VELOCITY

FIG. 3. Sedimentation analysis of polysomal mRNA. Polysomal RNA was prepared as in Fig. 2 and it was extracted with phenol and analyzed on a 5 to 20% sucrose gradient, using an SW 65 rotor for 4 hr at 50,000 rpm at 4O.

of the radioactivity is associated with the 80 S ribosomes. However, omission of GTP (Fig. 4B), initiation factors (Fig. 4C) or replacement of aminoacyl-tRNA with deacylated tRNA (Fig. 4D) resulted in association of the RNP with the 40 S ribosomal subunit, but no radioactivity is found to sediment with 80 S ribosomes. The association of mRNA with the 40 S subunit is not specific since labeled ribosomal RNA is also found to associate with the 40 S ribosomal subunit under similar conditions, but when added to the complete system did not sediment with the 80 S ribosomes. Similar observations were reported for embryonic chick muscle mRNA (Heywood, 1970). Stage-Specific

Initiation

Factors

Figure 5A shows the formation of the complete initiation complex when all added components belonged to the same stage of development. However, when mRNA-“C is taken from 7-day pupae and initiation factors from larvae (Fig. 5B) no initiation complex is formed. Similar results are obtained when mRNA-W is from larvae and initiation factors are from 7-day pupae. Again no initiation complex is formed (Fig. 5C). On the other hand, when both mRNA and initia-

286

ILAN

A. COMPLETE 80s +

1.0

AND

ILAN

B LESS 60s I

GTP

40s *

-250

-200

-150

-100

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2

1

C. LESS

INITIATION

FACTORS

D. WITH

DEACYLATED

tRNA 5 F :: 250i2

4 a

,200

Q6

I50

100

50

5 FRACTION

IO

I5

20

NO.

FIG. 4. Requirements for the formation of the complete initiation complex. The complete mixture was contained in 0.5 ml and incubated for 10 minutes at 30”. It consisted of 50 mM Tris-HCl (pH 7.6), 6 mM MgCI,, 50 mM KCl, 10 mM DTT, 0.5 mg ribosomal subunits (an equal mixture of 60 S and 40 S), 0.5 mM GTP 0.5 mg aminoacyl-tRNA, 200 pg initiation protein, and 1000 cpm of RNP as mRNA. After incubation the tubes were rapidly cooled and layered on 15-309; sucrose gradient in buffer A. The mixture was centrifuged for 1 hour at 66,000 rpm in an SW 65 rotor at 4”. Optical density was recorded automatically, and drops were collected for determination of radioactivity. (A) The complete system; (B) less GTP; (Cl less protein initiation factor; (D) deacylated-tRNA replaced aminoacyl-tRNA.

STAGE

A PUPAL PUPAL

mRNA AND INITIATION FACTORS 00s

IO

SPECIFIC

605

1 1 PI9

00 d 1

INITIATION

287

FACTORS

B PUPAL LARVAL

mRNA AND INITIATION

FACXRS

40s

1 200

’ 1

a6

150

04

loo I

I 0.2

x 2

50

C. LARVAL PUPAL

mRNA AND INITIATION FACTORS,

z 5 I= 2 P

C. LARVAL mRNA LARVAL INITIATION FACTORS

IO

2502

a : ‘, d 0 I ’

0.0 I’

I I 200

:

0.6

I50

0.4

100

0.2

50

I 5

s $

1 IO

15

I 20

FIG. 5. Stage-specific initiation factor during development. Experimental conditions used as described in the legend for Fig. 4. In all cases seventh day pupae ribosomal subunits were used. RNP was used as mRNA, and initiation protein factors were prepared from last instar larvae of ‘i-day pupae and used in combinations as described above. (A) Pupae mRNA and pupae initiation factor; (B) pupae mRNA and larval initiation factor; (C) larval mRNA and pupal initiation factor and; (D) larval mRNA and larval initiation factors.

tion factors from larvae are used (Fig. 5D), a complete initiation complex is formed. These results suggest the existence of stage-specific initiation factors. The source of ribosomes is not important since identical results are obtained with ribosomal subunits from larvae or from pupae.

288

ILAN

AND

ILAN

DISCUSSION

Our results indicate that in insects, as in bacteria, there are protein factors extractable from ribosomes with 1 M KCl, which appear to promote initiation of protein synthesis. This is judged by their ability to facilitate binding of mRNA to ribosomes. Our observations suggest that the mRNA-40 S subunit complex is the first intermediate in initiation and that GTP and aminoacyl-tRNA are essential to promote the association of the 40 S and 60 S ribosomal subunits into a complete initiation complex. However, in contrast to embryonic muscle the binding of mRNA to 40 S ribosomal subunits is neither dependent on initiation factors nor on GTP and appears to be nonspecific. Alternatively, it may be as a result of impurity of the system which may contain limiting amounts of initiation factors and GTP that can promote this binding. The difficulty of cleaning ribosomes from GTP is well known. Further experiments, however, are clearly necessary before we can delineate with any clarity the role of initiation factors in binding the mRNA to the 40 S subunit. Under our experimental conditions ribosomal RNA also binds to the 40 S subunit but does not form an 80 S initiation complex. Such nonspecific binding of ribosomal RNA to 40 S particles has been reported for embryonic muscle ribosomes (Heywood, 1970). The reason for this is not clear. Our results indicate that there is a stage-specific initiation factor which promotes the formation of the complete initiation complex only with mRNA extracted from the same stage of development. Since initiation factors are part of the ribosomal protein, it follows that ribosomes do not recognize identically all cistrons of mRNA. Indeed, Lodish (1969), by comparing bacterial species, demonstrated that ribosomes differentiate the three cistrons of f2 RNA. Also, IIsu and Weiss (1969) showed that after infection with phage T4, E. coli ribosomes translate T4 mRNA much more efficiently than f2 RNA. Therefore, these experiments suggest that mRNA contains, beside AUG, a specific signal which is recognized by some element of the initiation machinery. It was shown recently (Revel et al., 1970) that in E. coli initiation factor B(F3) plays a role in recognition of this signal and there is a cistron-specific fraction of this factor. Moreover, modification in template specificity after T4 infection which leads to preferential initiation of late T4 mRNA translation, can be accounted for by change in initiation factor B(F3) activity (Pollack et al., 1970). Since initiation factors are part of the ribosomal protein it can be

STAGE

SPECIFIC

INITIATION

FACTORS

289

argued that in our system we are dealing with ribosomal specificity for mRNA binding. That is, a template-specific initiation reaction is taking place, by which ribosomes could recognize selectively the proper mRNA to be translated. This may provide a mechanism of gene expression controlled at the level of translation. It could be efficiently operated with short-lived mRNA. We have already demonstrated that in insects translation of long-lived mRNA can be controlled at the chain elongation level by affecting the rate of translation through a rate-limiting species of aminoacyl-tRNA (Ilan et al., 1970). There is experimental support for the idea of ribosome specificity in translation during development and embryogenesis. It appears that in sea urchin embryos after fertilization maternal mRNA is translated on maternal ribosomes and translation of newly synthesized mRNA coincides with a burst of new ribosome synthesis (Gross et al., 1964). This may be true also with Xenopus embryos (Brown and Littna, 1964) and mouse embryos (Tasca and Hillman, 1970). Therefore, our results are in agreement with the “masked messenger” or “informosomes” theory (Spirin and Nemer, 1965). Two possible interpretations may be found for the results presented above: (1) A group of mRNAs from a given stage of development may have a specific sequence of oligonucleotides preceding the AUG codon recognized by a unique initiation factor. Such a sequence is known for viral mRNA. Protein synthesis directed by virus RNA does not begin immediately at the 5’-end of phage RNA because the sequence of the first few nucleotides in viral chains do not contain one of the fMet codons AUG or GUG necessary for initiating phage protein synthesis (De Wachter et al., 1968; De Wachter and Fiers, 1968; Adams and Cory, 1970, Steitz, 1969). Moreover, recently the nucleotide sequence of the three ribosomal binding sites in R17 RNA was described (Steitz, 1969). These untranslated sequences extend many nucleotides on the 5’ end of each of the initiation codons for three R17 proteins. Namely, the phage coat protein, the A protein, and R17 RNA replicase. (2) The secondary structure of mRNA may be stage specific and thus recognized by a unique initiation factor on the ribosomes. Here again, such a possibility was shown in the regulation of initiation of translation of three f2 phage proteins by phage RNA. When the secondary structure of the RNA was partially disrupted by mild reaction with formaldehyde, the ability of the RNA to direct synthesis of both RNA polymerase and maturation protein increases up to 20-fold (Lodish, 1970). We have no experimental evidence to

290

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support either of the possibilities, and this is a subject for further experiments in our laboratory. Finally, our observation may explain the stimulation in ribosome synthesis by almost all hormones studied. It may be that new ribosomes containing new initiation factor are needed to facilitate translation of hormone-specific mRNA. If this is true, a qualitative change in at least one of the ribosomal proteins should be noted. Such a change was seen recently on insect ribosomes after treatment with ecdysone (Pate1 et al., in preparation). SUMMARY

In insects, as in bacteria, the smaller (40 S) ribosomal subunit binds messenger RNA during initiation of protein synthesis. An 80 S ribosomal unit is formed by association of free 40 S and 60 S subunits. Formation of the complete initiation complex requires GTP, aminoacyl-tRNA, protein initiation factors and messenger RNA. The complex sediments as an 80 S band on sucrose gradient. Protein initiation factors are extracted from unwashed ribosomes and appear to be able to discriminate between messenger RNAs obtained from different stages of development. They promote formation of the 80 S complex only when messenger RNA is extracted from the same stage of development, providing a mechanism for control of protein synthesis by which ribosomes can select the messenger RNA to be translated. Two possibilities have been proposed to explain this phenomenon: (1) that a group of messenger RNAs from a given stage of development may have a specific sequence of nucleotides preceding the AUG codon. This sequence is recognized by a stage-specific element of the initiation machinery; (2) and or, the secondary structure of messenger RNA from a given stage of development may be specific and therefore recognized by a unique initiation factor. REFERENCES J. M., and CORY, S. (1970). Untranslated nucleotide sequence at the 5’-end of R17 bacteriophage RNA. Nature (London) 227, 570-574. BROWN, D. D., and LITTNA, E. (1964). RNA synthesis during the development of Xenopus laeuis, the South African clawed toad. J. Mol. Biol. 8, 669-687. BROWN, J. C., and SMITH, A. E. (1970). Initiator codons in eukaryotes. Nature (London) 226, 610-612. CASKEY, C. T., FLEDFIELD, B., and WEISSBACH, H. (1967). Formylation of guinea pig liver methionyl-sRNA. Arch. Biochem. Biophys. 120, 119-123. DE WACHTER, R., and FIERS, W. (1969). Sequences at the 5’-terminus of bacteriophage Qfl RNA. Nature (London) 221, 233-235. ADAMS,

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DE WACHTER, R., VERHASSEL, J.-P., and FIERS, W. (1968). The S’terminal end group of the RNA bacteriophage MS2. Biochim. Biophys. Actu 157, 195-197. GROSS, P. R., MALKIN, L. I., and MOYER, W. A. (1964). Templates for the first proteins of embryonic development. Proc. Nat. Acad. Sci. Cr. S. 51, 407-414. HENSHAW, E. C. (1968). Messenger RNA in rat liver polyribosomes: evidence that it exists as ribonucleoprotein particles. J. Mol. Viol. 36, 401-411. HEYWOOD, S. M. (1970). Formation of the initiation complex using muscle messenger RNAs. Nature (London) 225, 696-698. HOGAN, B. L., and KORNER, A. (1968). The role of ribosomal subunits and 80-S monomers in polysome formation in an ascites tumor cell. Biochim. Biophys. Acta 169, 139-149. HOUSMAN, D., JACOBS-LORENA, M., RAJBHANDARY, U. L., and LODISH, H. F. (1970). Initiation of haemoglobin synthesis by methionyl-tRNA. Nature (London) 227, 913-918. Hsu, W. T., and WEISS, S. B. (1969). Selective translation of T,-infected Escherichiu coli. Proc. Nat. Acad. Sci. U. S. 64, 345-351. ILAN, J. (1968). Amino acid incorporation and aminoacyl transfer in an insect pupal system. J. Biol. Chem. 243, 5859-5866. ILAN, J. (1969a). Studies on template-active ribonucleic acid from nuclei of insect pupae. Biochemistry 8, 4825-4831. ILAN, J. (1969b). The role of the adenine-uridine-guanine codon in the initiation of polypeptide synthesis by a eukaryotic organism. J. CeEE Biol. 43, 57a. ILAN, J., and ILAN, J. (1970). A possible role of the AUG codon in the initiation of polypeptide synthesis in a eukaryotic organism. Biochim. Biophys. Acta 224, 614-619. ILAN, J., and LIPMANN, F. (1966). A cell-free protein synthesis system from pupae of Tenebrio molitor. Acta Biochim. Polon. 13, 353-359. ILAN, J., ILAN, J., and QUASTEL, J. H. (1966). Effects of actinomycin D on nucleic acid metabolism and protein biosynthesis during metamorphosis of Tenebrio molitor L. Biochem. J. 100, 441-447. ILAN, J., ILAN, J., and PATEL, N. (1970). Mechanism of gene expression in Tenebrio molitor. Juvenile hormone determination of translational control through transfer ribonucleic acid and enzyme. J. Viol. Chem. 245, 12751281. JACKSON, R., and HUNTER, T. (1970). Role of methionine in the initiation of haemoglobin synthesis. Nature (London) 227, 672-676. JOKLIK, W. K., and BECKER, Y. (1965). Studies on the genesis of polyribosomes. I. Origin and significance of subribosomal particles. J. Mol. Biol. 13, 496-510. KABAT, D., and RICH, A. (1969). The ribosomal subunit-polyribosome cycle in protein synthesis of embryonic skeletal muscle. Biochemistry 8, 3742-3749. KAEMPFER, R. (1969). Ribosomal subunit exchange in the cytoplasm of a eukaryote. Nature (London) 222, 950-953. LIPMANN, F. (1969). Polypeptide chain elongation in protein biosynthesis. Science 164, 1024-1031. LODISH, H. F. (1969). Species specificity of polypeptide chain initiation. Nature (London) 224, 867-870. LODISH, H. F. (1970). Secondary structure of bacteriophage f2 ribonucleic acid and the initiation of in vitro protein biosynthesis. J. Mol. Biol. 50, 689-702. MARTIN, R. G., and AMES, B. N. (1961). A method for determining the sedimentation behaviour of enzymes: applications to protein mixtures. J. Viol. Chem. 236, 13721379.

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MARTIN, T. E., ROLLESTON, F. S., Low, R. B., and WOOL, I. G. (1969). Dissociation and reassociation of skeletal muscle ribosomes. J. Mol. Biol. 43, 135-149. PATEL, N., ILAN, J., and IL.AN, J., in preparation. PENMAN, S., SCHERRER, K., BECKER, Y., and DARNELL, J. E. (1963). Polysomes in normal and poliovirus-infected H&a cells and their relationship to messenger-RNA. Proc. Nat. Acad. Sci. U. S. 49, 654-662. PERRY, R. P., and KELLEY, D. E. (1968). Messenger RNA-protein complexes and newly synthesized ribosomal subunits: analysis of free particles and components of polyribosomes. J. Mol. Biol. 35, 37-59. POLLACK, Y., GRONER, Y., AVIV (GREENSHPAN), H., and REVEL, M. (1970). Role of initiation factor B(f3) in the preferential translation of T4 late messenger RNA in T4 infected E. coli. Fed. Eur. Biochem. Sot. 9, 218-221. PRICHARD, P. M., GILBERT, J. M., SHAFRITZ, D. A., and ANDERSON, W. F. (1970). Factors for the initiation of haemoglobin synthesis by rabbit reticulocyte ribosomes. Nature (London) 226, 511-514. REVEL, M., Avrv (GREENSHPAN), H., GRONER, Y., and POLLACK, Y. (1970). Fractionation of translation initiation factor B(f3) into cistron-specific species. Fed. Eur. Biochem. Sot. Lett. 9, 213-217. SMITH, A. E., and MARCKER, K. A. (1970). Cytoplasmic methionine transfer RNAs from eukaryotes. Nature (London) 216, 607-610. SPIRIN, A. S., and NEMER, M. (1965). Messenger RNA in early sea-urchin embryos: cytoplasmic particles. Science 150, 214-217. STEITZ, J. A. (1969). Polypeptide chain initiation: nucleotide sequences of the three ribosomal binding sites in bacteriophage RI7 RNA. Nature (London) 224, 957-964. TAKAEISHI, T., UKITA, T., and NISHMURA, S. (1968). Characterization of two species of methionine transfer ribonucleic acid from bakers’ yeast. J. Biol. Chem. 243, 57615769. TASCA, R. J., and HILLMAN, N. (1970). Effects of actinomycin D and cycloheximide on RNA and protein synthesis in cleavage stage mouse embryos. Nature (London) 225, 1022-1025. WIGLE, D. T., and DIXON, G. H. (1970). Transient incorporation of methionine at the N-terminus of protamine newly synthesized in trout testis cells. Nature (London) 227, 676-680. WILSON, 0. B., and DINTZIS, H. M. (1970). Protein chain initiation in rabbit reticulocytes. Proc. Nat. Acad. Sci. U. S. 66, 1282-1289.