Reprinted tram the Proceedings of the NATIONAL ACADEMY OP SCIENCEX
      Vol. 47, No. 10, BP. 1.588-1602.  October, 1961.

9"HE' DEPENDENCE OF CELL- FREE PROTEIN SYNTHESIS IN E. COLI
     lrPON Nil TURALLY OCCURRING OR SYNTHETIC
             POLYRIRONUCLEOTIDES

BY MARSHALL W. ~IHENBEE~G ANI) J. HEINHICH MATTHAEI*

NATIONAL ISSTITUTES OF HEALTH, BETHESDA, MARYLAND

Comnunicated by Joseph E. Smadel, August 3, 1961

A stable cell-free system has been obtained from E. coli which incorporates
C14-valine int)o protein at, a rapid rate.  It was shown that this apparent protein
synthesis was energy-dependent, was st'imulated by a mixture of L-amino acids,
and was markedly inhibited by RNAane, puromycin, and chloramphenico1.l  The
present, communication describes a novel characteristic of the system, that is, a
requirement for templat'e RNA? needed for amino acid incorporation even in the



1590          BIOCHEMISTRY-: XIRENBERG A.VD MATTHAEI    PROC. N. A. S.

RNAase (Worthington Biochemical Company) at, 35" for 60 min.  RiXAase was destroyed by
four phenol extrart#ions performed as given above.  After the last phenol extraction, the samples
were dialyzed against standard buffer minus mercaptoethanol.  RNA samples were treated with
trypsin by incubation with 20 rg per ml of twice recrystallized trypsin (Worthington Biochemical
Company) at 35' for 60 min.  The solution was treated four times with phenol and was dialyzed
in the same manner.

The radioactive amino acids used, their source, and t,heir respective specific activities are as
follow-s: U-C14-glycinr, U-C14-L-isoleucine, U-C14-L-tyrosine, LT-C1l-L-leucine, U-C14-L-proline,
L-histidine-2(ring)-C14, U-C'4-L-phenklalanine, U-Cl4-L-threonine, L-methionine (methyl-C'4) ,
U-C~4-L-nrgininej and U-C14-L-lysine obtained from Nurlear-Chicago Corporation, 5.8, 6.2, 5.95,
6.25, 10.5, 3.96, 10.3, 3.9, 6.5, 5.8, 8.3 mC/mIVI, respectively; C14-L-aspartic acid, C14-Lglu-
tamic acid, C14-L-a1anine, obtained from Volk, 1.04, 1.18,0.75 m C/m&l, respectively; C)-L-trypto-
phan-3 Cl", obtained from New England Nuclear Corporation, 2.5 mC/mM; SZS-L-cystine obtained
from the Ahhott Lahorat,orics . , 2.4 mC/mM; U-C14-L-serine obtained from the Kuclcnr-Chicago
Corporation, 0.2 mC/mM.  Other materials and methods used in this study- are described in the
accompanying paper.'  All assays were performed in duplicate.
Results.---Stimulation by ribosomal RNA:  In bhe previous paper,l it was shown
that DWAase markedly decreased amino acid incorporation in this system after
20 min.  For t.he purpose of this invest,igat,ion, 30,000 X g supernatant fluid frac-
tions previously incubatfed with DXAase and other components of the reaction
m&m-es (Incubated-S-30 fractions) were used for many of the experiments.

Figure 1 shows that incorporation of C14-L-valine into protein by Incubated-
S-30 frac%ion was stimulated by t,he addition of purified E. coli soluble RKA.
Maximal stimulation was obt,ained with approximately 1 mg soluble RSA. III
some experiment's, increasing t'he concentration S-fold did not further st#imulate t,he
q&em.  Soluble RXA was added to all reaction mixtures unless ot,herwise specified.
Figure 2 demonstrates that E. coli ribosomal RKA preparations markedly stimu-
                            2oo r- ~-- --T-~---- 7----T-

        I    1     1         I
0   05    10    1.5    20   2.5 30
        MG. SOLUBLE RNA

FIG 1:-- Stimulation of amino acid incor-
porat,ion int,o protein by E. coli soluble RNA.
Composition of reaction mistures is specified
in Table 1.  Samples were incubated at 35"
for 20 min.  Reaction mixtures contained 4.4
mg. of Incubated-S-30 protein.

FIG. 2.-St,imulation of amino acid incor-
poration into protein by E. coli ribosomal
RNA in the presence of soluble RNA.  Com-
position of reaction mixtures is specified in
Table 1.  Samples were incubated at 35" fo1
20 min.  Reaction mixt,ures contained 4.4 mg
of Incubated-S-30 protein and 1.0 mg E. coli
soluble RKA.


10  20  30  40  50  60  70  80  90
     MINUTES

FIc:. X.--Uependencr of C'4-I,-valiue inwrpwatioll iuto protriu upon ribosomul 1tSh. `l'ht~
composition of the react,ion mixtures and thr incubation wutlitions :tw prrwntrtl in Tahlr I
Reaction misturrs contained 0.98 my of E. co/i soluk)le RNA :tud 4.4 mg of Inc~ut):~tc~ti-S-:~~-l)~~t(~ill.

lated incorporation of C14-valine into protein even though maximully stimulating
concentrations of soluble RKA were present in t,hc reaction miktures.  A linrar
relationship between t,he concent&ion of ribonomul K SA and C14-valine incorporu-
t,ion into protein was obtained when low ~mcent rations of ribosomul RKA were
used.  Increasing the soluble RNA concentration rip to :&fold did noi rt~pl:~~ tht>
effect observed when ribosonial 1iS.A \vas aticird.

The effect of ribusomul KKA ill st~irnulatiug illc,olpolatioli of ("`-valinc illto
prot,ein is prc,srlltcd in more detail in Figure 3.  In t'hr absence of ribosomal RS.l.
incorporation of Cl"-saline into protein by thca inctlbatcad-S-80 fruchu n-as quite
low when compared with S-:30 (not, incubated before, storagv at, - 15") a11d stopped
almostj complet,ely after 30 min.  At. low concent,rations of ribosoniul KS-A, mxsi-
mum amino acid incorporation into protein was proportional to the amount, of
ribosomal RNA added, suggesting stoichiomctric rather t(han cat,alytk a&on of
ribosomal RNA. Totma incorporation of C14-vsline int'o prot,ein was incrcuscd
more t,hnn 3-fold by riboaomal RKA in this experimrlit even in thr prescncc of
maximally stimulat~ing conc+entrations of soluble RNA.  Ribosomal RNA mlq be
added at any time during t,he course of the reaction. and, aftrr frlrthrr in(*llh;l.tioll,
ali increase in incorporation of CIJ-valinr into prokin will rrsult.

Characteristics of arni,rw ucid incorporation stimulated by ribosomal RNA: In
Table 1 are prcsmkd the rharactjerist,ics of C14-T,-valinr incorporzat,ion illto protein


stimulated by t,hP addit,iotl of rihosomal RNA.  Amino acid incorporation was
strongly inhibited hy 0.15 pmoles of rhloramphenicol and 0.20 ~moles,/ml reaction
mixture of purnmyrin.  Flu-thwmorc, the incorporation was complet,cly dependent
llpon the addition of AT1 and an ATP-gwerating system and was totally inhibited
by IO Kg/ml R?;Aascl.  l~~(]~tival~nt amountIs of DYAase had no effect upon the
inwrporation stinull:~tcd by the addition of rihosomal RNA.  Placing a ribosomal
li?;X prcparatiou in a boiling water bath for 10 min did not) destroy its Cl"-valinc
iil(~ol,poratiolI acLt,i\,it,y; instead, a slight incwaw in activity was consistrntly ob-
~crwd.  HOWYW, wh(an thwc RX.4 prrparat#ions were placed in a boiling water
bath, a cwpiolw, whit,cL prwipit,ate rwultrd.  Upon cooling t,h(h suspension in an
ice bath, the prwipitatc immrdiatply dissolvrd.

The data of Table I also demonstrate that, t,he incorporation of amino acids into
protein in the presence of ribosomal RNA was further stimulated by the addit,ion of
a mixt8ure of 20 I,-amino acids, suggesting cell-free protein synthesis.

C- and S-trrminal analyses of the rihosomal RNA-dependent product, of t&he
rcac:t,ion mere perfomwd with carboxypeptidase and I-fluoro-Z,&dinit,robenzene
rrspwti\~cl~~ (Dr. Frank Tict,zr kindly performed these analyses).  Four per cent of
thr radioactivity was rclrascd from the C-t,crminal end and 1% was associat,ed
\vith the S-trrminal end. The remainder of the Cl"-label was int,rrnal. Similar
rcwtlts wcr(' obtaiwd whw rractions were performed using S-30 enzyme fractions
which had not, twcw troa,tcd with DKAasc. Protein preripitatrs isolat,ed from re-
action mixtlu'cs after incubation were compltt8ely hydrolyzed wit,h I-ICI, and the
Cl"-label incmorporatcd into protein was drmon&ated to be valine by paper chro-
matography.

Many of t'hc experiment,s presented in this paper were performed with enzyme
frn.at,ions prr>parcd with I)FAase added tjo reducr their viscosity. Ribosomal


RNA also stimulated Cl*-valine incorporation when wzyme extracts prepared
in the absence of DNAase were used.
To be effective in stimulating amino acid incorporat8ion into protein, the ribo-
somal RNA required the presence of washed ribosomes.  The data of Table 2 show

                         TABLE 2

THE INEFFECTIVENESS UF RIB~s~MAL RKA IN STIMULATING C1CL-V.4~1~~ I~~Y~IWORATIOX INTO
PROTEIN IN THE PRESENCE OF RIBOSOMES OR 105,000 X g SVFERNATANT SOI,~.TI~SS AI.ONE
                              Additions                    Counts/nlin
    Complete                                            51
         l`   + 2.1 mg Ribosomal RNA                         202
         I,    - Ribosomrs
         "

"
`,
"

- Ribosomes + 2.1 mg Ribosomal RNA
- Supernatant solution
- Gupernatant solution + 2.1 mg Rihosomal RNA
Deproteinised at zero time

that both ribosomes and 105,000 X g supernatant solut'ion were necessary fat
ribosomal RNA-dependent amino acid incorporation.  ?;o incorporation of amine
acids into protein occurred when the 105,000 X g suprrnatant solution alone waf:
added t,o ribosomal RNA preparations, demonstrat)ing that, ribosomal RKA prepara-
tions were not contaminated with intact ribosomes.  This conclusion also was
substantiated by showing that the acbivities of ribosomal RXA prcxparat,ions were
not destroyed by boiling, although t,he activit,ies of t,he ribosonws wrrr drstroyed
by such treatment.

The effect of ribosomal RXA upon the incorporation of seven differcut amino
acids is present#ed in Table 3. The addition of rihosomal R?;A incwascd th(x
incorporation of every amino arid test)ed.

The effect shown by ribosomal RXA was not observed when ot#hcr polyanions
were used, such as polyadenylic acid, highly polymerized salmon sperm I>SA, or a
high-molecular-weightj polymer of glucose carboxylic acid (Table 4). Pretreat-
ment of ribosomal RNA with trypsin did not affect its biological activity.  How-
ever, treatment of t'he ribosomal RNA with eit,her RKAase or alkali rcsult,ed in a
complete loss of stimulating activity.  The act,ive principle, therefore, appears to
hr RiSA.

The sedimcntat.ion charac:teri&cs of thr ribosomal RNA preparations were
exwlitwd in the Spinco Model E ultraccnt,rifuge (Fig. 49).  Particles having the
charactrristics of S-30, S-50, or S-70 ribosomes were not observed in these prepara-
tions. The SE of the first peak was 23, that, of the second peak 16, and that of
the third, small peak, 4.  Pret'reatment with trypsin did not affect, the S$ values
of the peaks appreciably (Fig. 4C) ; however, treatment, with RNAase completely
destroyed the peaks (Fig. 4B), confirming the ancillary evidence which had suggest,ed
t,hat the major component was high-molecular-weight RNA.

Preliminary attempts at, fractionat#ion of the ribosomal RNA were performed
by means of densit,y-gradient centrifugation employing a linear sucrose gradient.
The result,s of one such experiment' are presented in Figure 5. Amino acid in-
corporation activit#y of the RNA did not follow absorbancy at, 260 rnp; instead,
the activity seemed to be concentrated around fraction No. 5, which was approxi-
mately one-t.hird of the distance from the hott,om of the tube.  These result,s again


+ Ribosomal KKA

+ Ribosornal RNA

+ Rihosomxl RNA

+ Rihosornwl RN.4

+ Hihor;omal RNA

+ Ribosomal RNA
Ikproteinizrd at zero time


VOL. 47. 1961    HIOCHh'MIS7'RY: XTRENHERG ANI) MA'I'THAEI           1595

TABLE 5

STIMULATION OF AMINO Acm INCORPORATION BY RNA FRaCTIoNa PREPARED FROM I~TERE~-T
                           SPECIE5 L

                 .Additions                         Cormts/min/mg lxotein
  Eone
  + 0.5 mg E. coli ribosomal RNA            :i
  + 0.5 mg Yeast ribosomal RNA                           130
  + 0.5 mg Tobacco mosaic virus RNA                        872
  + 0.5 mg Ehrlich ascites t,unlor microsomsl RI\;A                   65

The cornl,onents of the reaction mivtrurs and thP incubation ronditi<rns BW prrwntcd in Tahlr 1.  Rrantion
snmpkn conkned 1.9 mg Incubated-P-30 protein.

approximately 1,700,OOOt stimulated amino acid incorporation &rongly.  Marked
stimulation due to t80bacco mosaic virus RNA was observed also with E. coli cn-
zyme extracts which had not been treated with D1;Aase.  More complet,e det,ails
of this work will bc prcsmt~cd in a later publicatSion.
Stimdation of amino acid incorporations b!g synthetic polynucleotidss:  The da t,a
of Figure 6 show t,hat, t#he addit,ion of 10 pg of polyuridylic acid1 per ml of reaction
mixture result,ed in a remarkable stimulation of Cl4-L-phenylalanine incorporation.
Phenylalanine incorporation was almost completely dependent upon the addition
of polyuridylic acid, and incorporation proceeded? aft'er a slight, lag period. at a
linear rate for approximat,ely 30 min.

The data of Tahlc 6 demonstrate that no other polynucleotide tested could re-
place polyuridylic arid. The a,hsolut,e specificity of polyuridylic acid was con-

TABLE 6

        POLYS~TCT,EOTII)F: SPECIFICITY FOR PHEN'~L.~I,ANINE IXCORPORATIOX

   Experl-
   ment
    no.                    Additions                  Counts/min/mg pwtrin
    1     None
       + 10 pg Polyuridylic acid          39, d1
       + 10 ~.rg Polyadenylic acid
      + 10 pg Polypgti$ylic acid            Ei
       + 10 pg Polymosmic acid                           57
       + 10 fig Polyadenvlic-uridylic acid (2/l ratio)
      + 10 pg Polyuridyiic acid + 20 pg polyadenylic acid     iii
      Deproteinized at zero time                           17
    2     None                                       75
       + 10 )Jg UMP                                  81
       + 10 1-(g UDP                                   77
       4 10 fig IiTP                                   72
       Ikproteinizc~d at zero time                             6

Comlmnents <)f thP wartion Inixtuws are Ixesenk! in Table 1.
S-30 I)rotrGn.                         Reaction mixtures wntsined 2.3 nrg Incuhated-
        0.02 ~lnolras U-Cl'-L-pIrenylalanine (-1?.5,000 counts/mim~w) was added to rach reachon mixtnrr.
SamtAes wew incubated at 3.5' for 00 min.

firmed by demonstrating that randomly mixed polymers of adenylic and uridylic:
acid$: (Poly A-U, 2/l ratio and 4,/l ratio) were inactive in this system.  A solution
of polyuridylic acid and polyadenylic acid (which forms triple-stranded hclices)
had no activity what'soever, suggestin g that' single-strandedness is a necessary
requisite for activity.  Experiment 2 in Table 6 demon&rates that UMP, I-D!?, or
UTP were unable to stimulate phenylalanine incorporation.

The data of Table 7 demon&rate that both ribosomes and 100,000 X g super-
nstant solution, as well as ATP and an ATP-generating syst#em, were required
for the polyuridylic acid-dependent incorporation of phenylalanine. Incorpora-
tion was inhibited by puromycin, chloramphenicol, and RNAase.  The incorpora-


FIG. 4.--E. coli ribosomal RNA preparations.  (A) Untreated (above).  (U) digested with RSA-
aasc; (C) digested with t,rypsin. Preparation and digestion of samples presented under Methods a.nd
Matwinls.  9.8.and 10.5 mg/ml RNA vxw present in A and C.  1 I .5 mg/ml R?TA was present in R
                                (Continued on facing page)
tion was not, inhibited by addition of DNAase.  Omit'ting a mixture of 19 L-amino
acids did not inhihit phenylalanine incorporation, suggeskg that' polyuridylic
acid stimulat,ed the incorporation of L-phenylalanine alone.  This conclusion was
substantiated by the data presented in Table 8.  Polyuridylic acid had little effect
in stimulating the incorporation of 17 other radioactive amino.acids.  Each labeled
amino acid was tested individually, and these data, corroborating t#he results
given in Table 8, will be presented in a subsequent publication.


VW. 47, 1961    HiOCHEiWi87'R~: NIRENHERG AND MATTHAEI          1597

(big. 4-continued)
before digest,ion.
schlieren opt,ics.  Phot,oyra.phs wrr 1.s ken in :I, model P; Spinro ultJr;lrent,rifnge quipped wit,h

Thp prodwl, of t,he rea,&orr was partially charwterized and the rwults arc' pre-
scntcd in `I'ahk !J.  The phy&al charact.erist,irs of the product, of t#hr reaction
resembled t,hose of autShent,ic* poly-T,-phenylalanine, for, unlike many other poly-
peptides and proteins, hot,h t,he product of the reaction and the polymer were
resistant t,o hydrolysis by AN HCl at 100" for 8 hr hut, were romplet,+ hydrolyzed
hy 12N HCl at 120-130" for 48 hr.

Poly-L-phenylalanine is insoluble in most solventszs but) is soluble in 33 per cent


1598

2
a

1.60

1.40

1.20

1.00

,800

,600

,400

,200

-d *260
  "14 R

L  `COUNTS/MINUTE     =A,

1 80 70 50 60 c s u-l 5

            I    I    I    I    I    I    I    I    I    I    I    I

        I   2 3 4 5 6   7 8 9   IO II   12 13

     BOTTOM       FRACTION NUMBER         TOP
Fro. 5.PYuc~rose density-gradient ccntrifugation of ribosomal RX-k.  :1 linear gradient of su-
crose conc'cutjration ranging from `0 per cent at the bottom to 5 per cent at the top of the tube was
prc~parc!d. *z  The sucrose solutions (1.4 ml total volumt~) contained 0.01 M Tris, pH i.8, 0.01 111 Mg
awt,ate and 0.06 .2f KU  0.4 ml of ribosomal RSh (4.6 rng) KW layered on top of each tube which
uxs wntrifuged at 38,000 X q for 4.5 hours at, 3" in a swinging bucket rotor, Rpinco type S\Y-39,
using a Spincn 1lotlrl T, 1lltr:rcrntrifngc~.  0.30 ml frwc+inns wcrc wllwted after piercing the bottom
of the tube.2'

0.015 ml aliquots diluted to 0.3 ml \vit,h H?() were used for ,Insfi measurements.  0.25 ml aliquots
~7 ere used for amino acid inrorporation assays.  Reaction mixtures contained t)hc components pre-
sented in Table 1,  0.7 my of E. co/i soluble RX.4 and 2.2 mg Incubatrd-H-30 protein were added.
Control assays plus 0.25 ml 12.5 per wnt sucro,~e in place of frwtions gave i9 counts/mix  This
figuw was subtracted from cxrh vnl\~cs.  Total vnIum(x was 0.T ml.  Samplw XWP incwhated at
35" for 20 min.

FIG. 6.--Ytimulat,ion of U-C14-L-phen$alanine incorporation by polyuridylic acid. o
without polyuridylic acid; A 10 fig polyurldylic acid added.  The components of the reac-
tion mixtures and the incubation conditions are given in Table 1.  0.024 pmole U-C14-L
phenylalanine (~500,000 count,s/min) and 2.3 mg Incubated-S-30 protein were added/ml
of reaction mixture.


Minus 100,000 X q siqwrnnt~ant solution
Minus rihosomes
Minus ATP. PEP, and PEP kinaar
+ 0.0`2 pmoles puromyciu
$ E.;i guts chloramphrnicol
        I mr
+,fi pg DXAaw
Mmus ammo acid mixture

29,500
  106
  52
  83
7,100
12,550
  120
27,600

Deprotrinizrd st, arro t,imft                    31,700
                                          30

The components of the reaction mixtures are presented in Table 1.
samples except the specifirad one.                    10 eg of polyuridylic acid were added to all
               2.3 mg of Incubated-S-30 protein MWY added to each reaction mixture except
those in which ribouomcs alone and 100,000 X g supernatant solution alone were tested.
1.3 me S-100 protein RCI'C usrd rrspectivclp.                       0.7 mg W-Rib protein and
                        0.02 gmulps IT-C"I,-phen?-talanine, Sp. Act. = 10.3 mC,`mM
(-125,000 rount?, ininntp) MPIC ndrlrd +U rarh wartion mistlirc.  Samples WPW inc,>hxtrd at .iT," for ti0 Illin.

                          TARI,b; 8

I    Phenylalxnine

2   (;lycine, alanine, serine!
    aspartic arid, glntamlc
      acid
3   I,eucine, isoleucine, threonine,
    methlonine, arginine, histidme,
    lysinc, tyrosine, t,ryptophnn,
    proline, valine
4    S3Gyst,eine

Deprot,einixed at zero t.ime
NO&

+ 10 pg polyuridylic acid
Deprot,einized at zero time
None

+ 10 pg polyuridylic acid
Deproteinised at zero time
SOiF

+ 10 pg polyuridylic acid

Depruteirrizetl at, zero t,imr
None

+ 10 pg pol>wridplic arid

  25
  68
38 ) a00

17
20
33

73
2Tfi

9;
113

Components of the rettctiun Inixtores arr presented in Table 1.
0.015 pM of each labeled amino acid -`as used.           The unlabeled amino acid misturr was olrlitted.

in the Melhods and Moferials section.       The specific activities of the labeled amino acids are present
reaction mixture.           2.3 mg of protein of preinrubatrd S-30 enzyme fraction were added to each
          All samples were incubated at 35' for 30 min.

TABLE 9

COMPARTSOS OF CHARACTERISTICS OF PRODUCT OF REACTIOX AND POI.Y-L-PHENVLALANINE
          Treatment                Product of reaction        Poly-L-phenylalaninP
6 A' HCI for 8 hours at 100"
12 .V HCl for 18 hours at 120~1:~O"    Partially hydrolyzed

Estrwtiou \\ith :3X , ITl3r in glacial   Complrtf4y h~drol~wtl   I'arti:tll,y hydrol,wcd
                                CompMcly hytirol,wtl

  arctic :wi(l                   Soluhlr              SOlUl,l~~
l':stractjion* with the following sol-
  vcnt.s: H?O, lwnzcne, nitrohcnzcne,
  chloroform,   ?;,i\;-dimethylform-
  amide, ethanol, petroleum ether,
  concentrated phosphoric arid, gla-
  cial acetic arid, dioxanr, phenol,
acetone, ethyl acetate! pyridinr,
  acet,ophenone, formic acid         Insoluble            Insoluble
* The product was said to be insoluble if 10.002 gm of product was soluble in 100 ml of solvent at 24O.  Eutrar-
tions were performed by adding 0.5 mg of authentic jloly-L-phen~lalanine and the ClLprodnct of a reaction rnixtnw
(1800 counts/min) to 5.0 ml of solvent.
wntrifugui.              The suspensions were viyorously shaken for 30 min at 21' and were
      The precipitntw wew plated and t,heir radioactivity was determinpd.

HBr in glacial acetic wid.$ The product, of the reaction had the same apparent
solubility as authentic poly-L-phenylalanine.  The product of the reaction was
purified by means of its unusual solubility behavior.  Reaction mixtures were de-
proteinized after incubation, and precipitated proteins were washed in t,he usual


manner according to the method of 8iekevitz.22  Dried protein pellets containing
added carrier poly-L-phenylalanine were then rxtract,ed with 33 per cent HBr
in glacial acetic acid, and the large arnouut, of insoluble material was discarded.
Polyphetlyla,larliltr was then prrc:ipitat,rd from solut,iull by the addiGon of Hz0
and was washed several times with H20.  Sevrnty per wnt of the total amount of
C'4-L-phellylalallille incorporated into protein due tv thr additive of polyuridylic
acid could be rrwwrrd by this procedure.  Cornplet,e hydrolysis of the purified
rea&on product, with 12N HCl followed by paper electrophoresis** demonstrated
that, the react,ion product contained C14-phenylalanine. No other radioactive
spots were found.
Discussiott .-- -111 t,his il~vrstigut,ion, WC have dcw~oustrat~etl that template RNA is
a requirrmt~r~t for wll-fwr amit wid it ~wrporatiol~.  .\dtiit,ioll of solut)lr~ KKA could
not replace template RKA iu this s~yystem.  In addit,ion, t,he density-gradient
cent~rifugation rxprrirnrlltjs showed tJhat t,hr ac:t,ive fra&iwls itI the riboaomal RNA
pwpawtious srdirurl~tjrcl much faslt~r tjhail sohlblr KXA.  lt should 1~ noted t,hat
ribosomal RNA is clualitwtively difl'rrrtlt from soluble RNA, siuw bases such a$
pseudouruc*il, mrt~hylwt~rd gu:~IIilws! rtv., fo1111t1 ill solut~l~~ RNA., :LW lrot present iu
ribosonial l< NA."
`Thr hulk of the H.NA in our ribosomal K&A fractions may be inactive as tem-
plates, for t~obacw' mosaic: virus RNA wad 20 t,imes as at+ive in stimulatiug amino
acid iIl~`Orp~Jl'at~hl ;ts equivalent amounts of E. coli ribosomal RXA.  In addition,
preliminary frac:t,iouation of rihosorual .KNA indicat,rd t#hat, only a portion of the
total RKA was active.
It should tw rmphasixrd that ribosomal RNA could not subst,itutr for ribosomes,
indicat,ing that, l~ihJSoillW were not, assenlhled from thr added RNA i7L toto.  The
function of ribosornal RNA remains an wigma, although at letlst part of the total
RKA is t8hought t#u serve as templates for protein synthesis and has been termed
"nwssrugrr" R?4C'A." -I4  Alternatively, a pwt of thp RX?;,4 rr~ay be essential for
t,he syllthwis of a&w rihowmes from smaller ribosomal partic:les.15-21
Ribosonial l< SAk m:~y hr an aggregat,e of subunit,s which can dissociat,e after
i$Yqw" t.rt:ut llle11t~.6 h  I'hPuol C&T&CJil of &. f:Oll' l'it)OsoIiw j&h two types of
1iXA molecules with s'," of %:3 and 16 (Fig. 4j, eyuivulrut. 10 Ill(J~W&lI' \vrights of
l,OOO,OOO and 560,000, respectiveIy.Y~ II'  Thew RNA specirn can be degraded by
boiling to products having sedimwtation aoefIicients of 13.1,8,8, and 4.4, correspond-
ing to molecular wcighta of 288,000, 144,WO, and 29,000.  Although the Sedimenta-
Ii011 riistribut,ions of t,he latter preparat,ions suggest, a high degree of honlogeneit,y
;LIUVU~ the tuolecuk,s of each class, thesr obServati(JnS do not eliminate the possi-
bility t'hat the subunits are linkrd to one another viu covalent bonds."  Yreliminary
ctvidrnce illdiwtcs t,hat the subunits n~ay be act,ive in our system, since t,he super-
natantI solution obtained after boiling E. coli ribosomal R?;A for 10 min and centri-
fugation at 10+5,000 X y for 60 min was active.  Examination of boiled ribosomal
RNA with the spinco Model E ult,racentrifuge showed a dispersed peak with a
sedimentat'ion corficient of 4-8. This may be the same material found in the
sucrow density-gradient experiment (using non-boiled RNA preparations), \vherc? a
small pwk of activity somrwhsrt hwviw t,hatl Soluhl~ HN.4 was usually noted
(Fig. 5).
In our system, at' low concentrations of ribosomal RKA, amino acid incorporation


into protein was proport,ional to the amount8 of ribosomal RXA added, suggest'ing a
stoichiometric rather than a catalytic action of ribosomal RNA. In contrast.
soluble RNA has been shown to act in a cat,alytic fashion."
The results indicate that polyuridylic acid contSains the information for tShe syn-
thesis of a protein having many of the charact'eristics of poly-L-phenylalanine.
This synthesis was very similar to the cell-free protein synthesis obtained when
naturally-occurring template RNA was added, i.e., both ribosomw and 100,000
X g supernat,ant solutions were required, and the incorporation was inhibited
by puromycin or chloramphenicol.  One or more uridylic acid residues therefore
appear to be the code for phenylalanine.  Whether the code is of t,he singlet, triplet,
etc., type has not, yet been determined.  Polyuridylic acid seerningly functions
as a synthetic template or messenger R,KA, and this stable, cell-free E. coli system
may well synthesizr uny protein wrrc3pontlillg tt) mwniligful information con-
tained in added RS,Y
Summwy.--A stable, cell-fwe system has been obtained from h'. coli in which
the arnount of incorporat,ion of amino acids into protein was dependent upon the
addition of heat-stable templat,e RNA preparations.  Soluble R?CA could not
replace template RNA fractions.  In addition, t,he amino acid incorporation rc-
quired both riboxomrn and 105,000 X g supernatant solution. The correlation
between the amount of incorporation and the amount, of added .RXA suggested
stoichiomet,ric rat,her t'han cat,alytic actjivity of the t~emplat~e RNA.  The t,emplate
RNA-dependent amino acid iworporation also required ATI' and an ATP-generat-
ing system, was st,imulated by a complete mixtuw of I,-ttmino acids. and was
markedly inhibited by puromycin, c:hloraI~lpllellicol, and K Nhasr.  A4ddition of a
synthetic polynwleot,ide, polyuridylic acid, specifically resulted in thr incorporutioll
of L-phenylalanine into a protein resembling poly-L-phcnylalanine.  l'olyuridylica
acid appenrs to fun&on as a synthetic template or messenger R?;A.  The impli-
cations of these findings are briefly discussed.

Note udded in. prouj. --The ratio between uridylic acid units of the polymer required and mole-
rules of I,-phenylalanine incorporated, in recent experiments, has approached the value of 1: 1.
Direct evidence for the number of uridylic acid residues forming the code for phenylalanine as well
as for the eventual stoichiometric action of the template is not, yet established.  4s polyuridylic
acid codes t,he incorporation of I,-pherlylalanine. l)olyq tidylica :rc,iti $ ,q)c~cific~:rlly mecliat t's the ill-
(~orporution of I,-proliue int,o :i `r(:A-prwil)il :ibl~ produ(,t  (`ornplcte Il:rt:t on thee findings \vill
lw included in :t sutwqucnt public2tion.
* Supported by :I NATO Postdoctoral Research Fellowship.
t Dr. Frankel-Conrat, personal communication.
1: We thank Drs. Leon A. Heppel and Maxine F. Singer For samples of these polyribonucleotides,
and Dr. George Rushizky for TMV-RNA.
5 We thank Dr. Michael Sela for this information.
** We thank Drs. William Drryrr and Elwood R~I~IIJII for performing t,hr high-voltage elwtro-
phoretic analyses.
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