Viral Proteins Formed in a Cell-Free RABBIT Reticulocyte System Programmed with RNA from a Temperature-Sensitive Mutant of Sindbis Virus

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Copyrighti 1974 AmericanSocietyforMicrobiology PrintedinU.S.A.

Viral

Proteins Formed

in

a

Cell-Free

RABBIT Reticulocyte

System

Programmed with

RNA from

a

Temperature-Sensitive

Mutant

of

Sindbis

Virus

RANIERI CANCEDDA,I RICHARD SWANSON, AND MILTON J. SCHLESINGER

Department of Microbiology, Divisionof Biologyand Biomedical Sciences, Washington University, St.Louis,Missouri63110

Received for publication 29April 1974

Viral messengerRNA was isolated from BHK cells infected with a

tempera-ture-sensitive mutant of Sindbis virus and was further purified using an

oligo(dT) column. Addition of this mRNA cell-freeextracts from rabbit reticulo-cytesledtoformation ofdiscrete authentic viral capsid protein when thereaction

wasperformedat 29 C. However,thissameprotein-synthesizingsystemfailedto

make discrete viralcapsid when incubated with the viral RNAat39C. Instead, larger-molecular-weight polypeptides that contained the viral capsid peptide sequenceswere produced. The inabilityto makeaseparateviral capside protein in vitroatelevatedtemperaturesby the mRNA fromthismutantexactlymimics

the phenotype of this ts mutant in viral-infected cells. Three mechanisms are

discussed that might accountforatemperature-sensitive release of capsid. One of these is based on a model in which there are multiple sites for initiation of translation ofpolypeptides ona polycistronic viral mRNA.

Among a variety of temperature-sensitive

mutantsofSindbisvirusthat have been charac-terized are a group that fails to make virion proteins atthe nonpermissivetemperature even though viral RNA formation appears normal (3). Extracts of cells infected with these mu-tants at 40 C show the presence of a large polypeptideofmolecular weight -130,000 that

is not detectable at lower temperatures or in

cellsinfected with wild-typevirusatthehigher temperature (18). Tryptic peptide fingerprints of this large protein clearly indicated that it

contained those amino acidsequences that are

normallyfound inthe three individual Sindbis

virion proteins (20). Thus, the mutational de-fect prevented the normal processing of the

Sindbis'viral protein. Primarily onthe basisof

thephenotypeof thiskindof temperature-sensi-tive mutant, investigators studying Sindbis

virus formation have postulated that the

nor-mal pathway of synthesis of virion proteins consists of translation from a polycistronic

mRNA in which initiation ofsynthesis occurs only atthe 5' terminus of thenucleic acid and

the individual proteins arise by a

post-transla-tional proteolytic cleavage (16, 18). Otherdata from analysis ofRNA and proteins in Sindbis

IPresentaddress: 2ndFacultyofMedicine,2nd Institute

ofBiochemistry, UniversityofNaples,Naples,Italy.

virus-infected cellsgenerallyareconsistent with

this model if one presumesthat theproteolytic

cleavagesoccurduring translationof theprotein onthepolyribosome. Furthermore, there is now

substantial evidence that this model describes

the mechanism for viral protein synthesis in

cells infected withpicornaviruses (5, 10, 11, 23).

We initiated studiesontheinvitroformation of Sindbis viral proteins in cell-free extracts

programmed with viral RNAs obtained from

Sindbis-infected BHK cells in order to obtain evidence for posttranslation proteolysis and to

gain additional information about Sindbis viral protein synthesis. Thus far, wehaveshown that the addition of a 26S species of viral RNA to

rabbit reticulocyte extracts leads to formation of the viral capsid protein, but therewas little

or no evidence for an involvement ofa

proteo-lyticenzyme in theprocess(6, 7). Furthermore,

there were only limited amountsoftranslation

other than the capsid cistron, although varying

the components of the rabbit reticulocyte sys-tem aswellasthesourceof the viral RNA does

allow for larger-molecular-weight proteins to

form. We expected that in vitro translation of themRNAfromatemperature-sensitive mutant

unable to make capsid or discrete

envelope

protein at the nonpermissive temperature

would provide further insight into our under-standingofSindbis viral protein

synthesis.

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MATERIALS AND METHODS

Chemicals. Pactamycin was generously provided by D.Rekosh, MassachusettsInstitute ofTechnology. Itwaspreparedasamillimolarsolution inmillimolar acetic acid anddilutedtogiveafinalconcentrationof

3x 10-1 M in the reaction mixture. Guanidinewas an

ultrapurepreparationfromSchwartz-Mann (Orange-burg, N.Y.); ureasolutionswereshakenwith Amber-lite MB-3(Mallinckrodt Chemicals, St. Louis, Mo.) (10 g/liter of4Murea) beforeuse.Allotherreagents, including [35S ]methionine and [3HJuridine, were identicaltothose describedpreviously (6).

Virus and cell preparations. The ts-2 Sindbis

virus mutant wasprovided by Boyce Burge,

Massa-chusetts Institute of Technology. Virions were iso-lated fromculture fluidsofBHK-infectedcellsgrown in roller bottles at 30C. Purification of virus and isolation and purification of RNA from virus and viral-infected cells were identical to the methods described (6, 7), except that infected cells were harvested after 13.5 h at 30 C. A profile of the electrophoretic mobilityinanacrylamide-agarose gel

of themRNAused in this work ispresentedinFig.1.

Invitroproteinsynthesissystemandisolation of products.All in vitroexperimentswith RNA from the

ts-2 virus and viral-infectedcellswereperformed with

the cell-free system II prepared from lysed rabbit

reticulocytes. Details of thispreparation, the compo-nentsof thereactionmixture, and the analysisof the proteins formed are described elsewhere (6). For preparation of the tryptic peptide fingerprints, sam-ples from the reaction mixtures were precipitated directlywith 9 volumes ofcoldacetoneafterreduction andalkylation, andnodialysiswasperformed.

RESULTS

Effect of temperature on in vitro protein

synthesisby viral RNAs. RNAspartially

puri-fied from BHKcells infected with either the ts-2 Sindbis virus mutantorwild-type virus

stimu-12,000

1100

78,000 as18

50

x~~~~~~~~~~~~0u

In

GELSLICE NUMBER

FIG. 1. Partiallypurifiedpreparation of viral RNA

from BHK cells infected with ts-2at 30C.

Electro-phoresis was performed on a 1.8% polyacrylamide,

0.5% agarose gel (24). Symbols: -, 'H counts per minuteof viral RNA;...1Ccountsperminuteof18S

and28Sribosqmal marker RNA.

lated the incorporationof [35S]methionine into

protein incell-freeextractsfromrabbit reticulo-cytes(Table 1).Stimulationby added RNAwas

observed inreactionsincubated at 29and39 C, andtheamountof35S-labeled protein formedat

the two temperatures was not significantly

different. For reactions containing the ts-2

RNA, however, thepattern ofproteins revealed

in sodium dodecyl sulfate

(SDS)-polyacryl-amide slab gels formedatthetwotemperatures was noticeably different (Fig. 2). We have shown previously that band II is identical to

virion capsid protein and noted that bands I

and III contain capsid peptides (6). Both the gel autoradiogram and densitometric tracings of reactions at 39 C containing ts-2 RNA showed a large decrease in the amount of

capsid and asignificant increase in two

higher-molecular-weight bands (Fig. 2aand b) and in

material that stayedatthetop ofthe5%spacer gel. Some variations were also observed in

capsid polypeptides formed by wild-type viral RNA withmoreofthe smallerproteins appear-ing. The shift to larger-molecular-weight

pro-teinsin incubations at 39 C with the ts-2 RNA was confirmed by an analysis of the reaction mixtures after rate-zonal centrifugations in a urea-sucrose gradient (Fig. 3).

Analysis of protein formed by ts-2 RNA at 29and39 C.Despite the marked disappearance of capsid polypeptides from the 39-C reaction mixturecontainingts-2RNA,wecould show by

apeptidefingerprintofatrypticdigestionofthe total reaction mixturethat thecapsidcistron of

ts-2 RNA was translated (Fig. 4). In fact, the fingerprints fromthe29- and39-Cmixtures are indistinguishable and both show nine peptides that are absent from the fingerprint of the reaction mixture containing no viral RNA. Of the nine, sevencanbe relatedtopeptidesofthe isolated viral capsid (Fig.4C). Thetwo intense spots in fingerprints A, B, and D ofFig. 4 are

TABLE 1. Stimulationof35Sincorporationinto

protein

35Sincorporatedinto

Speciesof RNA gaddedto protein(X

10'3

SpeciesofRNA reactiona counts/minm)

29C 39C

Wild-type viral 13 123.2 134.8 ts-2viral ... 15 113.7 102.3

None 63.0 56.0

aTotal volume of reactionmixture was 50

Mliters,

andreaction was stopped after 60min. Components of reaction mixture are noted in ref. 6.

° Measured on 5Mlitersofreaction mixture.

14,1974

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SCHLESINGER

WILD a I

TYPE

su

--i4 _5 z

v : I f!

a-I

I_

aa*t_

-, 4 4 6

AI II II

II

-o

ii

II

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l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I l l~~~~~~~~~~~~~~~~~~~~~~~~I

<

=

X< _ 1z, X f

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CF TL'. KEP

SPACER OEL RUINN'NOOGEL

G--FIG. 2. Patternof proteinsformed invitro with wild-type andts-2 viralRNAs.Samples aredescribedin

Table 1.Insetshows autoradiogram of slab gel electropherogram.Samples 1 and2,No addedRNA;3and4,

wild-type viral RNA;5and6, ts-2viral RNA. Samples1,3,and5wereincubatedat29C;samples2, 4,and 6

were incubated at 39C. Autoradiogram was scanned in a Gilford gel-scanning device to provide the

densitometertracingsshown.Upperpanel:...Sample3,wildtype,29C;-,sample4,wild-type,39C.Lower

panel:. Sample5, ts-2, 29C;-,sample6, ts-2, 39C.

believed to be unincorporated [35S]methionine and methionine sulfoxide. The reaction

mix-tures were not dialyzed before acetone

precipi-tation and digestion, and not all the

unincor-porated [35S ]methionine was removed by this

procedure.

Characteristics of the ts-2 mutation. The

data from the experiments described above

suggest thatat leastoneapparent defect in the

translation ofts-2 RNA is an inability to form discrete capsid polypeptides. One possible

ex-planation for this is that the ts-2 capsid is

abnormalinstructureandat39 Caggregates to

higher molecular-weight forms.To testthis, we

treated the reaction mixtures with 5 M guani-dine hydrochloride immediatelyafter the reac-tion and displayed the products on a

sucrose-ureagradient.Theresultswereidenticaltothat

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noted in Fig. 3. Wealso used slab gels contain-ing 4M urea and0.1% SDS forelectrophoresis

as well as heating samples to 100 C in SDS before loading in the gels. In no casecould we

observe the capsid or the capsid-like protein bands in the 39-C reaction mixtures with ts-2

RNA.

The ts-2 mutation doesnot affect the initia-tion ofprotein synthesis onthe basis ofresults with pactamycin, an inhibitor of initiation of polypeptide chains in eukaryoticcells(8).

Addi-tion ofpactamycin 2minafter initiationat29C

followed byanimmediate shiftto39 Cgavethe

same amount of inhibition as noted when the

incubation was contained at 29 C (Table 2). A slab gel electropherogram of these reaction mixtures showed that capsid was absent from the 39-C incubation but present after a 29-C

incubation. Thus, the ts-2 defect was still ex-pressed even though initiation of synthesis had been inhibited at39C. The initiation of synthe-sis by ts-2 RNAat 39 C did not inhibit subse-quent translation at 29 C (Table 2), and a slab gel electropherogram showed a strong capsid protein band.

Evidence for a protease activity. In an experiment designed to look for an unusual

aggregation of the ts-2 polypeptides at higher

temperatures, areaction mixture that had been incubated for 60 min at 29 C was treated with RNaseto stop synthesis and placed at39 C for

an additional 60min. An SDS-polyacrylamide

gel pattern of this reaction mixture showed a disappearance of the faster-moving capsid-related band ILL (Fig. 5), suggesting aprotease activity in therabbit reticulocyte cell-free sys-tem. The protease seemed to be relatively specific in that capsid and the lower-molecular-weight band appeared stable. Wearecurrently

investigatingthisproteaseactivitytodetermine whether it is viralspecificand whether it playsa meaningful role in the Sindbis virus protein formation invitro.

DISCUSSION

The expression of the ts-2 mutation by viral RNA in a cell-free protein synthesis system is shown by results presented heretobe remarka-bly similar to the phenotype of ts-2 virus-infected cells. In both cases, discrete viral

capsid protein virtually disappears at the

non-permissive temperature and larger, abnormal polypeptides accumulate. The largets-2protein

found in vivo contains aminoacidsequencesof

the viral capsid (20), and the tryptic peptide

fingerprints presented in this work show that

the polypeptides made in vitro from ts-2 RNA at 39 C also have capsid peptides. Thus, the

aE-u

n

FRACTION NUMBER Top

FIG. 3. Rate-zonalcentrifugationin aurea-sucrose

gradient of proteins formed in vitroby ts-2 viral RNA.

Arrowrefers to theposition ofE. coli

beta-galactosi-dasesubunit (molecularweight = 135,000). Symbols:

-, 29-C reactionmixture; ...39-C reactionmixture;

- -, endogenous reaction mixture at 39 C with no

addedRNA.

mutational defect in ts-2 blocks formation of a

discrete capsid protein. We can propose three

ways in which this might happen: (i) an amino

acid substitution resulting from a missense

rnutation distorts the growing polypeptide chain sothatthesite forproteolytic cleavageto

produce capsid protein is blocked; (ii) the mutation causes theprotease required for

cap-sid appearance to become temperature

sensi-tive; (iii) the mutation alters the viral mRNA structure so that the translation ofcapsid cis-tron becomes directly linked to the translation

ofthe viral envelope cistrons.

Our datapresented here and elsewhereonthe invitro formation ofSindbis viral proteins can

be interpreted to support each of the above proposals. Furthermore, the in vivo data that

has been accumulated in several laboratories

from experiments on Sindbis virus replication

are unable to clearly distinguish between these three hypotheses. We cite here the arguments

pro and con for the three possible modes of action ofthe ts-2mutation.

(i) A defective misfolded polypeptide chain

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A

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B

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4

6

9

0 #

6

8 J7

10

3

4

5

p0

S

0

C

2

4 i

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D

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FIG. 4. Tryptic peptide fingerprints. (A)Incubation with ts-2 viralRNA at29C; (B) incubation with ts-2

viral RNAat39C; (C) isolatedcapsidproteinbandfrominvitro reaction withwild-typeviral RNA incubated

at30C; (D)reactionmixture withnoadded viralRNA,incubationat39C.Thesameamountof[35S]peptides (-106counts/min) wasusedforfingerprints A, B,and D.Sampleswereappliedatthelowerleftcornerofthe

papers; electrophoresis was in theupwards direction at pH3.5 followed by descending chromatography in

butanol-acetic acid-water(2:0.5:2.5) (totheright in eachpanel). Seereference20foradditional details.

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TABLE 2. Effect ofpactamycinonin vitro translation of ts-2 RNAa

Incubation temp

RNA Pretreatment (C)

29 39

Wild type None 142.9 89.4

ts-2 None 132.6 102.1

ts-2 2.5minat39C 134.2 NDb

ts-2 Pactamycinat0min 7.9 ND ts-2 Pactamycinat2min 36.1 ND

at 29C

ts-2 Pactamycinat2min ND 34.8 at29C, then shift

to39 C

aFigures are 35S counts per minute incorporated

into protein after 60 min of incubation; they have been multipliedby10-3.

bND, Notdone.

thatcannqa 1eardtQjjrionproteins. This

modelexplains t invivodata

showing7that

a ts-2 protein cannot be chased into virion

pro-teinswhen infected cellsarelabeledat39C and shiftedto 29 C (18). It also canaccount forthe disappearance of the shorter capsid-related

polypeptides madeinvitro (bands0and1,Fig.

2) if weassumedthey areformedbya protease action.Furthermore,therearenumerous

exam-ples of missense mutations in which single

amino acidsubstitutionscanproduce profound

changes in a protein's conformation (19).

How-ever, a misfolded precursor polypeptide cannot

readily explain the ability of ts-2 mutants to complement Sindbis virus mutants with defec-tive envelope proteins (2). According to this model, a ts-2 viral RNA could not make a

discrete virion envelope protein at 39C, and thus a ts-2 mutant should not be able to

complementmutantswith alteredenvelope

pro-teins.

The model also predicts that, in vitro, the capsid anditsrelatedpolypeptidesarereleased

at the permissive temperature by a protease. Although we have presented data here for a proteaseactivity, ourearlierdata failedtoshow

aneffectoncapsid formation when the protease

inhibitors phenylmethane sulfonyl fluoride and

L-1-tosylamide-2-phenylethyl chloromethyl ke-tone (TPCK) were present in high concentra-tions (6).

(ii A

teMDerature-sensitiv'-

uiral

-coded

p)ro-ea A ts-2 mutationin aproteasecan explain e in vivocomplementation noted above, and

it can also explain why none of the shorter capsid-related polypeptides appear at 39C in vitro. Butthis model predictsthat thisprotease

is aproduct of the in vitrosynthesisat29 C. We

have noevidencethat the protease observedin our system is viral coded. Furthermore, the reactiondemonstratingthisproteolytic activity

was carried out at 39 C, the nonpermissive

i.

;.4.

-4,

J

Iik

1. *:

FIG. 5. Effect ofincubation at 39C onts-2 prod-uctsformed at 29C. After60min at29C, reaction mixtureswereprovided with 5,ilitersofRNase A (50

I.g/ml)and incubated60minat39C. (1) Wild-type

RNA at 29C; (2) ts-2 RNA at 29C; (3) wild-type RNAat29 C +60minat39 C; (4) ts-2 RNA at 29C +60minat39C.Equal amounts of 3"S radioactivity wereadded to each slot of the gel.

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CANCEDDA, SWANSON,

temperature for ts-2. The appearance of the cant change in the levels of viral envelope

different capsid-related polypeptides in vitro proteins (24).

(bands I and III, Fig. 2) was affected mostly by But the suppression of normal termination by the relative efficiency of the protein-synthesiz- ts-2fails to account forcomplementationforthe ing system with longer polypeptides formed same reasons citedabove, unlessone proposed

when moresoluble cellularenzymes wereadded that a low level ofinternal initiation occurs at

(7). This result is not easily explainable by a the envelope proteincistrons of the ts-2 mRNA protease model. at39 C.Toexplain the disappearanceinvitro of

(iii) in the the lower-molecular-weight capsid-related

pro-tr iNtire.

lthugmolst

oft Temper- teins by ts-2 RNA at 39 C, we would suggest re-sensitive mutations examined lead to al- that these latter proteins all terminate in the

terations in protein structure, there is at least same manner but that they initiatetranslation

one report ofatemperature-sensitive mutation within the cistron. There is good evidence that

t in an RNA. A single base changein the tyrosyl initiations within a cistron can occur in bacte-tRNA of Escherichia coli affects the function of rial mRNA (14). Noevidenceisavailable thata

thetRNA athightemperature(22). The postu- similartype of reaction occurs inmRNA'sfrom lated change here in the viral mRNA would eukaryotic cells; however, Morrison and Lodish suppress a normal termination signal and per- (15) showed that extracts from eukaryotic cells mit continued translation. The major criticism can recognize the initiation sequences of inter-of this model is that it challenges the basic nal cistrons in a polycistronic bacteriophage

mechanism proposed for production of Sindbis RNA.

virion proteins because it presumes thatcapsid This modelis themostsuitableforexplaining

protein translation is independent ofthetrans- the unusual and remarkable ability of the

tlation of envelope proteins. According to this cell-free protein-synthesizing systems de-model, ribosomes could load at the initiation scribed here and in other reports

(6,

7) to sitesof at leasttwocistronsinthepolycistronic selectively form large amounts of

capsid.

We

mRNA in a manner analogous to that demon- hadsuggestedthat formation of

capsid

invitro

strated for the cistrons in E. coli operons (25) was aresultofa

specific

termination of transla-and inthe cistrons of the E. coli RNA bacterio- tion,andweproposehere thatthissame

mecha-phages (12, 13). nism operates in the viral-infected cell. The

j Amodelproposingindependenttranslationof presence of internal initiation sites

containing

Sindbis viral RNA cistrons can explain several RNA regions with

secondary

structure could pieces of data that are inconsistent with the also explain why the 26S Sindbis RNA maybe single-site initiation mechanism forviral RNA found in a "33S" form and why this

species

translation. For example, itcan accountforthe makes

larger

size

polypeptides

invitro than does non-stoichiometric levels of the virion proteins the26SRNA

(7).

Ina"33S"

form,

these

regions

formed in the infectedcell;there istwo tothree of

secondary

structurewould be convertedtoan

times more capsid piotein than

envelope

pro- extended

conformation,

thereby

leading

toa

de-teins in infected cells (21). This model also creased

mobility

ofthe 26S RNA in

polyacryl-explains the inability to accumulate a ts-2 amide

gels

and to a

greater

probability

for protein when amino acidanaloguesareaddedto

read-through

translation rather then

termina-Sindbis-infected cells

(18),

and it

explains

the tion and initiation ofnew

translation.

inabilitytoclearlyshowanaccumulationof ts-2 It is worth

noting again

that attempts to

protein when protease inhibitors wereaddedto

reproduce

the

experimental

results demonstrat-infected cells (16).Theonereport

claiming

that ing a

single

site of initiationon aviral

polycis-TPCK ledto increased amountsoflarger poly- tronic mRNA for

poliovirus

and

enceph-peptides actually showed very little increase in

alomyocarditis

virus have failed for Sindbis

larger-molecular-weight

protein

in contrast toa virus. These include theuseofprotease

inhibi-great decrease in theamount of virionproteins tors, amino acid

analogues,

and very short

(16). Ourexperiments withlow levels ofTPCK pulses of radioactive

isotopes

to infected cells. added for short periodsoftime before addition

And,

in contrast to Sindbis

virus,

the

picor-oflabel failed to show accumulation ofa ts-2 naviruses make

equimolar

amounts of viral protein whenanalyzed by

SDS-polyacrylamide

proteins encoded

by

the viral mRNA. Some slabgels (E.Duda,

unpublished data).

Amodel

experiments

with Semliki Forest

virus,

a

togavi-invoking separate sites of initiation for

capsid

rus

closely

related to

Sindbis,

have

suggested

andenvelope proteincanalso

explain

a50%loss

independent

sites oftranslation for virion pro-ofcapsidproteinincells infectedwith defective teins

(9),

although

larger-molecular-weight

pre-Sindbisviruseventhough therewas no

signifi-

cursors have been detected (4). These precur-670

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sors may be related to envelope proteins which were affecteddifferently fromcapsidwhen

can-avaninewassubstituted forargininein Semliki

Forest virus-infected cells (17). Finally, the in

vitrotranslationofSindbis viral RNAyieldsan authentic capsid protein in a cell-free system from ascites cells that produces

mainly

prema-turely terminated polypeptides from

enceph-alomyocarditis viral mRNA (1). It may well be

that thepolycistronic mRNAencodingSindbis

virus proteins functions

differently

than that of

thepicornavirusbecauseaportionof the mRNA codes forproteins thataredirectedintothe host cell membrane. Clearly, more intensive studies with the in vitrosystemand with inhibitors and analogues in vivo are needed to testthis model ofmultiple initiations on an animal virus

poly-cistronic mRNA.

Duringthepreparationof thismanuscript,we

learned that D. T. Simmons and J. H. Strauss

(J. Mol. Biol., in press) have also successfully translated in vitrothe 26S RNA isolated from cells infected with a temperature-sensitive

Sindbis mutant that is similar in its properties to the ts-2 mutant. Their results are similarto ours inthatcapsidproteinisnotformedat37 C but isfound at 27 C. However, theyreport the

presence of discrete

larger-molecular-weight

proteins equivalent in size to the B-1 and ts-2 proteinsin theirinvitro reactionproducts.The

former appeared at 27 C, whereas the latter appeared at 37 C. We have been unable to

detect these large proteinsasdiscrete bands in

polyacrylamidegels; instead, wefind a contin-uum ofradioactive material at the top ofour

slab gels. However, recent preliminary results suggest that thelargerdiscreteproteinsmade in

our in vitro reaction at 37Cusingts-226S RNA (Fig. 2a and b) contain capsid protein tryptic peptides.

ACKNOWLEDGMENTS

This research wassupported by a Public Health Service grant(CA14311-01) from the NationalCancer Institute and GB38657fromthe NationalScience Foundation.

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3. Burge, B. W., and E. R. Pfefferkorn. 1968. Functional defects of temperature-sensitive mutants of Sindbis virus.J.Mol.Biol.35:193-205.

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cleavagesin theformationofpoliovirusproteins. Proc.

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12. Jeppesen. P.G. N.,J. A. Steitz,R. F. Gesteland, andP.

F. Spahr. 1970. Gene orderin thebacteriophage R17

RNA: 5'-Aprotein-coat protein-synthetase-3'. Nature

(London)226:230-237.

13. Lodish,H.1970.Secondarystructureofbacteriophage f2 ribonucleic acid and the initiation ofin vitroprotein synthesis. J. Mol. Biol.50:689-702.

14. Miller,J. H.1974.GUG and UUGareinitiationcondons

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