Reprinted trom the Proceedings of the National ACADEMY OF Scrences Vol. 47, No. 10, pp. 1588-1602. October, 1961. PHE DEPENDENCE OF CELL- FREE PROTEIN SYNTHESIS IN E. COLI UPON NATURALLY OCCURRING OR SYNTHETIC POLY RIBON UCLEOTIDES By MarsHAauu W. NinENBERG AND J. Heinricn Marruarr* NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Joseph E. Smadel, August 3, 1961 A stable cell-free system has been obtained from E. coli which incorporates C™-valine into protein at a rapid rate. It was shown that this apparent protein synthesis was energy-dependent, was stimulated by a mixture of L-amino acids, and was markedly inhibited by RNAase, puromyein, and chloramphenicol.! The present communication describes a novel characteristic of the system, that is, a requirement for template RNA, needed for amino acid incorporation even in the Vout. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1589 presence of soluble RNA and ribosomes. It will also be shown that the amino acid incorporation stimulated by the addition of template RNA has many proper- ties expected of de novo protein synthesis. Naturally occurring RNA as well as a synthetic polynucleotide were active in this system. The synthetic polynucleo- tide appears to contain the code for the synthesis of a “protein” containing only one amino acid. Part of these data have been presented in preliminary reports.? 3 Methods and Materials ‘The preparation of enzyine extracts was modified in certain respects from the procedure previously presented.) &. cold W3100 cells harvested in early log phase were washed and were disrupted by grinding with alumina (twice the weight of washed cells) at 5° for 5 min as described previously.2. The alumina was extracted with an equivalent weight of buffer containing 0.01 Tris(hydroxy methyl jaminomethane, pH 7.8, 0.01 M Inagnesium acetate, 0.06 Af KCI, 0.006 M mercaptoethanol (stundard buffer). Alumina and intact cells were re- moved by centrifugation at 20,000 x g for 20 min. The supernatant fluid was decauted, and 3 wg DNAase per ml (Worthington Biochemical Co.) were added, rapidly reducing the viscosity of the suspension, which was then ceutriftuged again at 20,000 x g for 20 min. ‘The supernatant fluid was aspirated and was centrifuged ut 20,000 X g for 30 niin to clear the extract of remaining debris. The liquid layer was aspirated (8-30) and was centrifuged at 105,000 x gy for 2 hr to sediment the ribosomes. The supernatant. solution (8-100) was aspirated, and the solution just above the pellet was decanted and discurded. The ribosomes were washed by resuspension in the standard buffer and centrifugation again at 105,000 X g for 2 hr. Supernatant fluid was discarded and the ribosomes were suspended in standard buffer (W-Rib). Fractions S-30, 8-100, and W-Rib were dialyzed against 60 volumes of standard buffer overnight at 5° and were divided into aliquots for storage at — 15°. In some cuses, fresh S-30 was incubated for 40 min at 35°. The reaction mixture components in ymoles per mi were as follows: 80 Tris, pH 7.8: 8 magnesium acetate; 50 KCl: 9 mercapto- ethanol; 0.075 each of 20 amino acids; 2.5 ATP, K salt; 2.5 PHP, K salt; 15 «gz PEP kinase (Boehringen & Sons, Manuheim, Germany). After incubation, the reaction mixture was dialyzed at 5° for 10 hr against 60 volumes of standard buffer, changed once during the course of dialysis. The incubated S-30 fraction was stored in aliquots at —15° until needed ( Lneubated-S-30). RNA fractions were prepared by phenol extraction using freshly distilled phenol. Ribosomal RNA was prepared from fresh, washed ribosomes obtained by the method given above. In later RNA preparations, « 0.2% solution of sodium dodecyl sulfate recrystallized by the method of Crestfield ef ai.4 was udded to the suspension of ribosomes before phenol treatinent. The suspen- sion was shaken at room temperature for 5 min. Higher yields of RNA appeared to be obtained when the sodium dodecyl sulfate step was used: however, good RNA preparations were also obtained when this step was omitted. An equal volume of HoO-saturated phenol was added to ribosomes suspended in standard buffer after treatment with sodium dodecv! sulfate, and the suspension was shaken vigorously at room temperature for 8 10 tin, The aqueous phase was aspirated from the phenol phase after centrifugation at 1450 x ¢ for 15 min. The aqueous layer was extracted (wo nore times in the same manner, using ?/» volume of HyO-saturated phenol in each case. The final aqueous phase was chilled to 5° and NaCl was added to a final concentration of 0.16. Pwo volumes of ethyl alcohol at —20° were added with stirring to precipitate the RNA. The suspension was centrifuged at 20,000 X yg for 15 min and the supernatant sulution was de- canted and discarded. The RNA pellet. was dissolved in minimal concentrations of standard buffer (minus mercaptoethanol) by gentle homogenization in a glass Potter-Elvehjent homogenizer (usually the volume of buffer used was about ‘/s the volume of the original ribosome suspension), The opalescent solution of RNA was dialyzed for 18 br against. 100 volumes of standard buffer (minus mercaptoethanol) at 5°. The dialyzing buffer was changed once. After dialysis, the RNA solution was centrifuged at 20,000 X g for 15 min and the pellet was discarded. The RNA solution, which contained less than 1% protein, was divided into aliquots and was stored at ~ 15° until needed. Soluble RNA was prepared from 105,000 x g supernatant solution by the phenol extraction method described above. Soluble RNA wus ulso stored at — 15°. Atkali-degraded RNA was prepared by incubating RNA samples with 0.3 44 KOH at 35° for 18 hr. The solutions then were neutralized and dialyzed against standard buffer (minus mercaptoethanol). RNAase- digested sunples of RNA were prepared by incubating RNA with 2 pg per ml of crystalline 1590 BIOCHEMISTRY: NIRENBERG AND MATTHAEI Proc. N. A. 8. RNaAase (Worthington Biochemical Company) at 35° for 60 min. RNAase was destroyed by four phenol extractions 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 yg 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 their respective specific activities are as follows: U-C™-glycine, U-C14-L-isoleucine, U-C14-L-tyrosine, U-C14-L-leucine, U-C!L-proline, L-histidine-2(ring)-C'4, U-C14-L-phenylalanine, U-C1*-L-threonine, L-methionine (methyl-C¥) , U-C!“-L-arginine, and U-C'-L-lysine obtained from Nuclear-Chicago Corporation, 5.8, 6.2, 5.95, 6.25, 10.5, 3.96, 10.3, 3.9, 6.5, 5.8, 8.38 mC/mM, respectively; C1-L-aspartic acid, C'Lglu- tamic acid, C4-L-alanine, obtained from Volk, 1.04, 1.18, 0.75 m C/mM, respectively ; D-L-trypto- phan-3 C14", obtained from New England Nuclear Corporation, 2.5 mC/mM; §-L-cystine obtained from the Abbott Laboratories, 2.4 mC/mM; U-C"-L-serine obtained from the Nuclear-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 the previous paper,! it was shown that DNAase markedly decreased amino acid incorporation in this system after 20 min. For the purpose of this investigation, 30,000 X g supernatant fluid frac- tions previously incubated with DNAase and other components of the reaction mixtures (Incubated-S-30 fractions) were used for many of the experiments. Figure 1 shows that incorporation of C"™-L-valine into protein by Incubated- 8-30 fraction was stimulated by the addition of purified E. coli soluble RNA. Maximal stimulation was obtained with approximately 1 mg soluble RNA. In some expcriments, increasing the concentration 5-fold did not further stimulate the system. Soluble RNA was added to all reaction mixtures unless otherwise specified. Figure 2 demonstrates that E. coli ribosomal RNA preparations markedly stimu- 200 1 1 T 200; T T T TT T T 175 oh 1 : COUNTS /MINUTE/MG. PROTEIN COUNTS/ MINUTE/M6, PROTEIN 100 4 75 50 J QO ° ask ! ! l 25: I o Ft ae en i 0 10 20 3.0 L l | 1 i MG. RIBOSOMAL RNA o O5 10 16 20. 28 30 . . . Lye Fic. 2.—Stimulation of amino acid incor- MG. SOLUBLE RNA poration into protein by E. coli ribosomal Fie 1--—Stimulation of amino acid incor- poration into protein by #. cold soluble RNA. Composition of reaction mixtures is specified in Table 1. Samples were incubated at 35° for 20 min. Reaction mixtures contained 4.4 mg. of Incubated-S-30 protein. RNA in the presence of soluble RNA. Com- position of reaction mixtures is specified in Table 1. Samples were incubated at 35° for 20 min. Reaction mixtures contained 4.4 mg of Incubated-S-30 protein and 1.0 mg £. col? soluble RNA. Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1591 24mg. RNA 200 0.60 mg. RNA 0.30 mg. RNA | 0.12mg RNA 4 Minus RNA | COUNTS/MINUTE /mg PROTEIN po 0 10 20 30 40 50 60 70 80 90 MINUTES Fic. 3.—Dependence of C1-L-valine incorporation into protein upon ribosomal RNA. The composition of the reaction mixtures and the incubation conditions are presented in Table 1. Reaction mixtures contained 0.98 mg of E. cold soluble RNA and 4.4 mg of Incubated-S-30-protein. lated incorporation of C™-valine into protein even though maximally stimulating concentrations of soluble RNA were present in the reaction mixtures. A linear relationship between the concentration of ribosomal RNA and C1-valine incorpora- tion into protein was obtained when low concentrations of ribosomal RNA were used. Increasing the soluble RNA concentration up to 3-fold did not replace the effect observed when ribosomal RNA was added. The effect of ribosomal RNA in stimulating incorporation of Ch-valine into protein is presented m more detail in Figure 3. In the absence of ribosomal RNA. incorporation of C1-valine into protein by the incubated-S-30 fraction was quite low when compared with 8-30 (not incubated before storage at. — 15°) and stopped almost completely after 30 min. At low concentrations of ribosomal RNA, maxi- mum amino acid incorporation into protein was proportional to the amount of ribosomal RNA added, suggesting stoichiometric rather than catalytic action of ribosomal RNA. Total incorporation of C*-valine into protein was increased more than 3-fold by ribosomal RNA in this experiment even in the presence of maximally stimulating concentrations of soluble RNA. Ribosomal RNA may be added at any time during the course of the reaction, and, after further incubation, an increase in incorporation of C4-valine into protein will result. Characteristics of amine acid incorporation stimulated by ribosomal RNA: In Table 1 are presented the characteristics of C'-L-valine incorporation into protein 1592 BIOCHEMISTRY: NIRENBERG AND MATTHAE!S Proc. N. ALS, TABLE | CHARACTERISTICS OF C!LL-Varmng INCORPORATION INTO PROTEIN Experi- ment no, Addition Counts/min/mg protein I — Ribosomal RNA 42 + oe ad 904 + “ “+ 0.15 zmole Chloramphenicol 58 + “ “+ 0.20 umole Puromycin 7 4- “ “ deproteinized at zero time 8 2 — Ribosomal RNA 35 + a ce 10L + “ “« — ATP, PEP, PEP kinase 7 + “ “+ 10 wg RN Aase 6 + “ "+ 10 ng DN Aase 110 + Boiled Ribosomal RNA 127 + Ribosomal RNA, deproteinized at zero time 8 3 — Ribosomal RNA 34 _ « ¢ — 201. amino acids 21 + “ “ 99 + “ — 20-L-amino acids 52 The reaction mixtures contained the following in wmole/inl: 100 Tristhydroxymethyl) aminomethane. pH 7.8; 10 magnesium acetate: 50 KCl; 6.0 mercaptoethanol: 1.0 ATP; 5.0 phosphoenolpyruvate, K_ salt; 20 zg phosphoenolpyruvate kinase, crystalline; 0.05 each of 20 L-amino acids minns valine: 0.03 each of GTP, CTP, and UTP; 0.015 C'-L-valine (~70,000 counts); 3.1 mg. E. coli ribosomal RNA where indicated, and 1.0 mg E. coli soluble RNA; 3.2, 3.2, and 1.4 mg of incubated-S-30 protein were present in Experiments 1, 2, and 3, respectively. In addition 4.4 mg protein of W-Rib were added in Experiment 3. Total volume was 1.0 ml. Samples were incubated at 35° for 20 nin, were deproteinized with 10 per cent trichloroacetic acid, and the precipitates were washed and counted by the method of Siekevitz.2° stimulated by the addition of ribosomal RNA. Amino acid incorporation was strongly inhibited by 0.15 umoles of chloramphenicol and 0.20 umoles/ml reaction mixture of puromycin. Furthermore, the incorporation was completely dependent upon the addition of ATP and an ATP-generating system and was totally inhibited by 10 ug/ml RNAase. Equivalent amounts of DNAase had no effect upon the incorporation stimulated by the addition of ribosomal RNA. Placing a ribosomal RNA preparation in a boiling water bath for 10 min did not destroy its C'-valine Incorporation activity; instead, a slight increase in activity was consistently ob- served. However, when these RNA preparations were placed in a boiling water bath, a copious, white precipitate resulted. Upon cooling the suspension in an ice bath, the precipitate immediately dissolved. The data of Table | also demonstrate that the incorporation of amino acids into protein in the presence of ribosomal RNA was further stimulated by the addition of a mixture of 20 L-amino acids, suggesting cell-free protein synthesis. C- and N-terminal analyses of the ribosomal RNA-dependent product of the reaction were performed with carboxypeptidase and 1-fluoro-2,4-dinitrobenzene respectively (Dr. Frank Tictze kindly performed these. analyses). Four per cent of the radioactivity was released from the C-terminal end and 1% was associated with the N-terminal end. The remainder of the C'-label was internal. Similar results were obtained when reactions were performed using 8-30 enzyme fractions which had not been treated with DNAase. Protein precipitates isolated from re- action mixtures after incubation were completely hydrolyzed with HCl, and the C'Jabel incorporated into protein was demonstrated to be valine by paper chro- matography. Many of the experiments presented in this paper were performed with enzyme fractions prepared with DNAase added to reduce their viscosity. Ribosomal Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1593 RNA also stimulated C-valine incorporation when enzyme extracts prepared in the absence of DNAase were used. To be effective in stimulating amino acid incorporation into protein, the ribo- somal RNA required ihe presence of washed ribosomes. The data of Table 2 show TABLE 2 ‘Tue INEFFECTIVENESS OF RiposomaL RNA In Stimunating C'}-L-VaLine INCORPORATION INTO PROTEIN IN THE PRESENCE OF RIBosomEs oR 105,000 X g SUPERNATANT SoLUTIONS ALONE Additions Counts/min Complete 51 « + 2.1 mg Ribosomal RNA 202 “ — Ribosomes 17 “ — Ribosomes + 2.1 mg Ribosomal RNA 20 “ — Supernatant solution 36 “ — Supernatant solution + 2.1 mg Ribosomal RNA 45 “ Deproteinized at zero time 25 The components of the reaction mixtures and the incubation conditions are presented in Table 1. 0.86 and 3,: mg protein were present in the ribosome (W-Rib) and 105,000 & g supernatant (8-100) fractions, respectively. that both ribosomes and 105,000 X g supernatant solution were necessary foi ribosomal RNA-dependent amino acid incorporation. No incorporation of aminc acids into protein occurred when the 105,000 X g supernatant solution alone was added to ribosomal RNA preparations, demonstrating that ribosomal RNA prepara- tions were not contaminated with intact ribosomes. This conclusion also was substantiated by showing that the activities of ribosomal RNA preparations were not destroyed by boiling, although the activities of the ribosomes were destroyed by such treatment. The effect of ribosomal RNA upon the incorporation of seven different amino acids is presented in Table 3. The addition of ribosomal RNA increased the incorporation of every amino acid tested. The effect shown by ribosomal RNA was not observed when other polyanions were used, such as polyadenylic acid, highly polymerized salmon sperm DNA, or a high-molecular-weight polymer of glucose carboxylic acid (Table 4). Pretreat- ment of ribosomal RNA with trypsin did not affect its biological activity. How- ever, treatment of the ribosomal RNA with either RNAase or alkali resulted in a complete loss of stimulating activity. The active principle, therefore, appears to be RNA. The sedimentation characteristics of the ribosomal RNA preparations were examined in the Spinco Model E ultracentrifuge (Fig. 44). Particles having the characteristics of S-30, S-50, or S-70 ribosomes were not observed in these prepara- tions. The 8.7 of the first peak was 23, that of the second peak 16, and that of the third, small peak, 4. Pretreatment 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 suggested that the major component was high-molecular-weight RNA. Preliminary attempts at fractionation of the ribosomal RNA were performed by means of density-gradient centrifugation employing a linear sucrose gradient. The results of one such experiment are presented in Figure 5. Amino acid in- corporation activity of the RNA did not follow absorbancy at 260 my; instead, the activity seemed to be concentrated around fraction No. 5, which was approxi- mately one-third of the distance from the bottom of the tube. These results again 1594 BIOCHEMISTRY: NIRENBERG AND MATTHAEIL Proc, N. ALS, TABLE 3 SPECIFICITY oF Amino Actp LNCORPORATION STIMULATED BY Rinosomat RNA CtM-Amino Acid Addition Counts/min/mg protein C'*L-Valine Complete 25 “ “ + Ribosomal RNA 137 (OLL-Threonine “ 31 “ “ + Ribosomal RNA 121 C!L-Methionine “ 12] “ “ + Ribosomal RNA 177 C'-T-Arginine “ 49 “ “ + Ribosomal RNA 224 C'~L.-Phenylalanine “ 7 “ “ + Ribosomal RNA 147 (+DL-Lysine “ 36 “ “= + Ribosomal RNA 175 CUAL-Leucine “ 134 “ “ + Ribosomal RNA 272 « “ Deproteinized at. zero time 6 The composition of the reaction mixtures are presented in Table 1. The mixture of 20 L-amino acids ineluded all amino acids except the C!4amino acid added to one reaction mixture. Reaction mixtures contained 4.4 mg Incubated-8-30 protein. Famples were incubated at 35° for 60 min, 2.1 ing ribosomal RNA were added where indicated. TABLE 4 Riposomau RNA Conrrou EXPERIMENTS DESCRIBED IN TExt Iexperi- ment No. Addition Counts/min/mg protein I Complete 54 “ + 2.4 mg Ribosomal RNA 144 “ + 2.0 mg Polyadenylic acid 10 7 + 2.0 mg Salmon sperm DNA 4 “ + 2.0 mg Polyglucose carboxylic acid 49 “ + 2.4 mg Ribosomal RNA, deproteinized at zero time 7 2 Complete 39 “ + 2.0 mg Ribosomal RNA* 150 “ + 2.1 mg Ribosomal RNA preincubated with trypsin*® 166 “ + 2.0 mg Ribosomal RNA preincubated with RNAase*,} 47 “ Deproteinized at. zero time 8 3 Coniplete 20 ” + 1.2 mg Ribosomal RNA 82 ‘ + 1.2 mg Alkali degraded ribosomal RNA t 2) . Deproteinized at zero time 7 The composition of the reaetion mixtures and the incubation conditions are given in Table L. 44, 5.2, and 4.4 mg Incubated-8-30 protein were present in Experiments 1, 2, and 3, respectively. 2.4. 0.98. and 0 me &. col? soluble RNA were present in Experiments 1, 2, and 3, respectively. * Ribosomal RNA preparations were deproteinized by phenol extraction after enzymatic digestion as specified under Methods and Materials. ft mg Ribosomal RNA refers to RNA concentration hefore digestion. demonstrate that the activity was not associated with a soluble RNA fraction, present in maximum concentration in fraction No. 11, near the top of the tube. In addition, all amino acid incorporation analyses were performed in the presence of added soluble RNA, and the addition of more soluble RNA would not stimulate C™-L-valine incorporation into protein. Effects of RN A obtained from different species: The data of Table 5 demonstrate that; RNA from different. sources stimulates C™-valine incorporation into protein. Yeast ribosomal RNA prepared by the method of Crestfield et al.4 was considerably more effective in stimulating incorporation than equivalent. amounts of EF. coli ribosomal RNA. Yeast ribosomal RNA prepared hy this method has little or no amino acid acceptor activity and has a molecular weight of about 29,000.7 Tobacco mosai¢ virus RNA prepared by phenol extraction and having a molecular weight of Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAKEI 1595 TABLE 5 STIMULATION oF AMINO Acrp IncoRPORATION BY RNA FRACTIONS PREPARED FROM DIFFERENT SPECIES Additions Counts/min/mg protein None 42 + 0.5 mg E. coli ribosomal RNA 75 + 0.5 mg Yeast ribosomal RNA 430 + 0.5 mg Tobacco mosaic virus RNA 872 + 0.5 mg Ehrlich ascites tumor microsomal RNA 65 The components of the reaction mixtures and the inenbation conditions are presented in Table 1. Reaction samples cantained 1.9 mg Incuhated-S-30 protein. approximately 1,700,000} stimulated amino acid incorporation strongly. Marked stimulation due to tobacco mosaic virus RNA was observed also with HE. cola en- zyme extracts which had not been treated with DNAase. More complete details of this work will be presented in a later publication. Stimulation of amino acid incorporation by synthetic polynucleotides: The data of Figure 6 show that the addition of 10 ug of polyuridylic acidt per ml of reaction mixture resulted in a remarkable stimulation of C'-L-phenylalanine incorporation. Phenylalanine incorporation was almost completely dependent upon the addition of polyuridylic acid, and incorporation proceeded, after a slight lag period, at a linear rate for approximately 30 min. The data of Table 6 demonstrate that no other polynucleotide tested could re- place polyuridylic acid. The absolute specificity of polyuridylic acid was con- TABLE 6 POLYNUCLEOTIDE SPECIFICITY FOR PHENYLALANINE INCORPORATION Experi- ment no. Additions Counts/min/mg protein 1 None 44 + 10 wg Polyuridylic acid 39, 800 + 10 ug Polyadenylic acid 50 + 10 pg Polycytidylic acid 38 + 10 pg Polyinosinic acid 57 + 10 wg Polyadenylic-uridylic acid (2/1 ratio) 53 + 10 yg Polyuridylic acid + 20 ug polyadenylic acid 60 Deproteinized at zero time 17 2 None 75 + 10 we UMP 81 + 10 wg UDP 77 4 10 pe UTP 72 Deproteinized at zero time 6 Components of the reaction mixtures are presented iu Table 1, Reaction mixtures contained 2.3 mg Incubated- $-30 protein. 0.02 zmoles U-C!-L-phenylalanine (~125,000 counts/minute) was added to each reaetion mixture. Samples were incubated at 35° for 60 min. firmed by demonstrating that randomly mixed polymers of adenylic and uridylic acidt (Poly A-U, 2/1 ratio and 4/1 ratio) were inactive in this system. A solution of polyuridylic acid and polyadenylic acid (which forms triple-stranded helices) had no activity whatsoever, suggesting that single-strandedness is a necessary requisite for activity. Experiment 2 in Table 6 demonstrates that UMP, UDP, or UTP were unable to stimulate phenylalanine incorporation. The data of Table 7 demonstrate that both ribosomes and 100,000 X g super- natant solution, as well as ATP and an ATP-generating system, were required for the polyuridylic acid~dependent incorporation of phenylalanine. Incorpora- tion was inhibited by puromycin, chloramphenicol, and RNAase. The incorpora- 1596 BIOCHEMISTRY: NIRENBERG AND MATTHAEI Proc. N. ALS. Fig. 4.—-E. coli ribosomal RNA preparations. (4) Untreated (above). (2) digested with RNA- aase; (C’) digested with trypsin. Preparation and digestion of samples presented under Methods and Materials. 9.8 and 10.5 mg/ml RNA were present in Aand C. 1t.5mg/ml RNA was present in B (Continued on facing page) tion was not inhibited by addition of DNAase. Omitting a mixture of 19 L-amino acids did not, inhibit’ phenylalanine incorporation, suggesting that polyuridylic acid stimulated 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 the results given in Table 8, will be presented in a subsequent publication. Vou. 47, 196] BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1597 (Fig. 4—continued) before digestion. Photographs were taken in a model KE Spinco ultracentrifuge equipped with schlieren optics. The product. of the reaction was partially characterized and the results are pre- sented in Table 9. The physical characteristics of the product of the reaction resembled those of authentic poly-L-phenylalanine, for, unlike many other poly- peptides and proteins, both the product of the reaction and the polymer were resistant to hydrolysis by 6N HCl at 100° for 8 hr but were completely hydrolyzed by 12N HCl at 120-130° for 48 hr. Poly-L-phenylalanine is insoluble in most solvents” but is soluble in 33 per cent 1598 BIOCHEMISTRY: NIRENBERG AND MATVTHAEI Proc. N. ALS. TOT T T ] T TE A260 60 ba + 80 140 ~ + 70 QO 1.20 +60 2 2a © 1.00 + 50 G ~ < 800 140 = Zz 600 4305 mM 400 + 20 200 + 10 Oo 0 1 2 3 4 5 6 7 8 9 10 It 12 13 BOTTOM FRACTION NUMBER TOP Fic. 5.—Sucrose density-gradient centrifugation of ribosomal RNA. A linear gradient of su- crose concentration ranging from 20 per cent at the bottom to 5 per cent at the top of the tube was prepared.*5 The sucrose solutions (4.4 ml total volume) contained 0.01 1 Tris, pH 7.8, 0.01 .M Mg acetate and 0.06 17 KCI. 0.4 ml of ribosomal RNA (4.6 mg) was layered on top of each tube which was centrifuged at 38,000 & y for 4.5 hours at 3° in a swinging bucket rotor, Spinco type SW-39, using a Spinco Model J. ultracentrifuge. 0.30 ml fractions were collected after piercing the bottom of the tube.*4 0.025 ml aliquots diluted to 0.3 ml with H2O were used for A2® measurements. 0.25 ml aliquots were used for amino acid incorporation assays. Reaction mixtures contained the components pre- sented in Table 1. 0.7 mg of &. coli soluble RNA and 2.2 mg Incubated-8-30 protein were added. Control assays plus 0.25 ml 12.5 per cent sucrose in place of fractions gave 79 counts/min. This figure was subtracted from each value. Total volume was 0.7 ml. Samples were incubated at 35° for 20 min. 64,320 5 1 oper epee ep + POLY UY 56,000 - 4 48000 - 24,000 COUNTS/MINUTE/mg. PROTEIN 6,000 ° 0 ~* Is 30 45 60 75 30 MINUTES Fic. 6.—Stimulation of U-C!«-L-phenylalanine incorporation by polyuridylic acid. @ without polyuridylic acid; 410 ug polyuridylic acid added. The components of the reac- tion mixtures and the incubation conditions are given in Table 1. 0.024 umole U-C!“L phenylalanine (~600,000 counts/min) and 2.3 mg Incubated-S-30 protein were added /m\ of reaction mixture. Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEL 1599 TABLE 7 CHARACTERISTICS OF POLYURIDYLIC Acip-DEPENDENT PHENYLALANINE INCORPORATION Additions Counts/min/mg protein Minus polyuridylie acid 70 None 29,500 Minus 100,000 * y supernatant solution 106 Minus ribosomes 52 Minus ATP, PEP, and PEP kinase 83 + 0.02 umoles puromycin 7,100 + 0.31 wmoles chloramphenicol 12,550 + 6 pg RN Aase 120 + 6 ue DNAase 27,600 Minus amino acid mixture 31,700 Deproteinized at zero time 30 The components of the reaction mixtures are presented in Table 1. 10 ug of polyuridylic acid were added to all samples except the specified one. 2.3 mg of Incubated-S-30 protein were added to each reaction mixture except those in which ribosomes alone and 100,000 X g supernatant solution alone were tested. 0.7 mg W-Rib protein and 1.3 mg 5-100 protein were used respectively. 0.02 pmoles U-C!*L-phenylalanine, Sp. Act. = 10.3 mC/mM (~125,000 counts/minute) were added ta each reaction mixture. Samples were incubated at 35° for 60 min. TABLE 8 SpecIFICITY OF AMINO ACID INCORPORATION STIMULATED BY PoLyURipyLic ACID Experi- ment Counts/min/mg no. C!Lamino acids present Additions protein ] Phenylalanine Deproteinized at zero time 25 None 68 + 10 wg polyuridylic acid 38,300 2 Glycine, alanine, serine, Deproteinized at zero time 17 aspartic acid, glutamic None 20 acid + 10 wg polyuridylic acid 33 3 Leucine, isoleucine, threonine, Deproteinized at zero time 73 methionine, arginine, histidine, None 276 lysine, tyrosine, tryptophan, + 10 ug polvuridylic acid 899 proline, valine 4 Sa cysteine Deproteinized at zero time 6 None 95 + 10 wg polyuridylic acid 113 Components of the reaction mixtures are presented in Table 1. The unlabeled amino acid mixture was omitted. 0.015 4M of each labeled amino acid was used. The specific activities of the labeled amino acids are present in the Methods and Materials section, 2.3 mg of protein of preincubated 8-30 enzyme fraction were added to each reaction mixture. All samples were incubated at 35° for 30 min. TABLE 9 ComPaRISON OF CHARACTERISTICS OF Propuct oF REAcTION AND Poiy-L-PHENYLALANINE Treatment Product of reaction Poly-L-phenylalanine 6 N HCI for 8 hours at 100° Partially hydrolyzed Partially hydrolyzed 12 N HCI for 48 hours at 120-130° Completely hydrolyzed Completely hydrolyzed Extraction with 336, HBr in glacial acetic acid Soluble Soluble Extraction* with the following sol- vents: HO, benzene, nitrobenzene, chloroform, N,N-dimethylform- amide, ethanol, petroleum ether, concentrated phosphoric acid, gla- cial acetic acid, dioxane, phenol, acetone, ethyl acetate, pyridine, acetophenone, formic acid Insoluble Insoluble * The product was said to be insoluble if <0.002 gm of product was suluble in 100 ml of solvent at 24°. Extrae- tions were performed by adding 0.5 mg of authentic poly-L-phenylalanine and the C)+-product of a reaction mixture (1800 counts/min) to 5.0 ml of solvent. The suspensions were vigorously shaken for 30 min at 24° and were centrifuged. The precipitates were plated and their radioactivity was determined, HBr in glacial acetic acid.§ 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 the usual 1600 BIOCHEMISTRY: NIRENBERG AND MATTHAEI Proc. N. ALS. manner according to the method of Siekevitz.22_ Dried protein pellets containing added carrier poly-L-phenylalanine were then extracted with 33 per cent HBr in glacial acetic acid, and the large amount of insoluble material was discarded. Polyphenylalanine was then precipitated from solution by the addition of H.O and was washed several times with H.O. Seventy per cent of the total amount of Ci-L-phenylalanine incorporated into proteim due to the addition of polyuridylic acid could be recovered by this procedure. Complete hydrolysis of the purified reaction product. with 12N HCl followed by paper electrophoresis** demonstrated that the reaction product contained C'-phenylalanine. No other radioactive spots were found. Discussion-- -Di this investigation, we have demonstrated that template RNA is a requirement for cell-free amino acid incorporation, Addition of soluble RNA could not replace template RNA in this system. In addition, the density-gradient centrifugation experiments showed that the active fractions in the ribosomal RNA preparations sedimented much faster than suluble RNA. 1t should be noted that ribosomal RNA is quatitatively different from soluble RNA, since bases such as pseudouracil, methylated guanines, ete., found in soluble RNA, are not present in ribosomal RNA. The bulk of the RNA in our ribosomal RNA fractions may be inactive as tem- plates, for tobacco mosaic virus RNA was 20 times as active in stimulating amino acid incorporation as equivalent amouuts of #. cold ribosomal RNA. In addition, preliminary fractionation of ribosomal RNA indicated that only a portion of the total RNA was active. It should be emphasized that ribosomal RNA could not substitute for ribosomes, indicating that ribusomes were not assembled from the added RNA in toto. The function of ribosomal RNA remains an enigma, although at least part of the total RNA is thought to serve as templates for protein synthesis and has been termed “messenger” RNA. Alternatively, a part of the RNA muy be essential for the synthesis of active ribosomes from smaller ribosomal purticles.~2! Ribosomal RNA may be an aggregate of subunits which can dissociate after proper treatment. > Phenol extraction of #. colt ribosomes yields two types of RNA molecules with SY of 23 and 16 (Fig. 4), eyuivalent to molecular weights of 1,000,000 and 560,000, respectively.* These RNA species cun be degraded by boiling to products having sedimentation coefficients of 13.1, 8.8, and 4.4, correspond- ing to molecular weights of 288,000, 144,000, and 29,000. Although the sedimenta- tion distributions of the latter preparations suggest a high degree of homogeneity among the molecules of each class, these observations do not eliminate the possi- bility that the subunits are linked to one another véa covalent bonds.’ Preliminary evidence indicates that the subunits may be active in our system, since the super- natant solution obtained after boiling FE. colt ribosomal RNA for 10 min and centri- fugation at 105,000 X g for 60 min was active. Examination of boiled ribosomal RNA with the Spinco Model E ultracentrifuge showed a dispersed peak with a sedimentation coefficient of 4-8. This may be the same material found in the sucrose density-gradient experiment (using non-builed RNA preparations), where a small peak of activity somewhat heavier than soluble RNA was usually noted (Fig. 5). In our system, at low concentrations of ribosomal RNA, amino acid incorporation VoL. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1601 into protein was proportional to the amount of ribosomal RNA added, suggesting a stoichiometric rather than a catalytic action of ribosomal RNA. In contrast, soluble RNA has been shown to act in a catalytic fashion." The results indicate that polyuridylic acid contains the information for the syn- thesis of a protein having many of the characteristics of poly-L-phenylalanine. This synthesis was very similar to the cell-free protein synthesis obtained when naturally-occurring template RNA was added, ie., both ribosomes and 100,000 X g supernatant 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 the singlet, triplet, etc., type has not yet been determined. Polyuridylic acid seemingly functions as a synthetic template or messenger RNA, and this stable, cell-free E. cold system may well synthesize any protein corresponding to meaningful information con- tained in added RNA. Summary.—aA stable, cell-free system has been obtained frum E. cold in which the amount of incorporation of amino acids into protein was dependent upon the addition of heat-stable template RNA preparations. Soluble RNA could not replace template RNA fractions. In addition, the amino acid incorporation re- quired both ribosomes and 105,000 X g supernatant solution. The correlation between the amount of incorporation and the amount of added RNA suggested stoichiometric rather than catalytic activity of the template RNA. The template RNA-dependent amino acid incorporation also required ATP and an ATP-generat- ing system, was stimulated by a complete mixture of L-amino acids, and was markedly inhibited by puromycin, chloramphenicol, and RNAase. Addition of a synthetic polynucleotide, polyuridylic acid, specifically resulted in the incorporation of L-phenylalanine into a protein resembling poly-L-phenylalanine. Polyuridylic acid appears to function as a synthetic template or messenger RNA. The impli- cations of these findings are briefly discussed. Note added in proof. —The ratio between uridylic acid units of the polymer required and mole- cules of L-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. As polyuridylic acid codes the incorporation of L-phenylalanine, polycytidylic acid t specifically mediates the in- corporation of l-proline inte a TCA-precipitable product. Complete data on these findings will he included in a subsequent publication. * Supported by a NATO Postdoctoral Research Fellowship. { Dr. Frankel-Conrat, personal communication. t We thank Drs. Leon A. Heppel and Maxine F. Singer for samples of these polvribonucleotides, and Dr. George Rushizky for TMV-RNA. § We thank Dr. Michael Sela for this information. ** We thank Drs. William Dreyer and Elwood Bynum for performing the high-voltage electro- phoretic analyses. ' Matthaei, J. H., and M. W. Nirenberg, these Proceeprnes, 47, 1580 (1961). ? Matthaei, J. H., and M. W. Nirenberg, Biochem. & Biophys. Res. Comm., 4, 404 (1961). 3 Matthaei, J. H., and M. W. Nirenberg, Fed. Prec., 20, 391 (1961). * Crestfield, A. M., K. C. Smith, and F. W. Allen, J. Béol. Chem., 216, 185 (1955). * Davis, F. F., A. F. Carlucei, and 1. F, Roubein, ¢bid., 234, 1525 (1959). § Hall, B. D., and P. Doty, J. Mol. Biol., 1, L11 (1959). 7 Osawa, S.. Biochim. Biophys. Acta, 43, 110 (1960). * Aronson, A. I., and B. J. MeCarthy, Biophys. J., 1, 215 (1961). 1602 BIOCHEMISTRY: NIRENBERG AND MATTHAET Proc. N. A. 8. ® Kurland, C. G., J. Mol. Biol., 2, 83 (1960). Littauer, U. Z., H. Eisenberg, Biochim. Biophys. Acta, 32, 320 (1959). 11 Hoagland, M. B., and L. T. Comly, these ProceEpines, 46, 1554 (1960). 12 Volkin, E., L. Astrachan, and J. L. Countryman, Virology, 6, 545 (1958). 18 Nomura, M., B. D. Hall, and 8. Spiegelman, J. Mol. Biol., 2, 306 (1960). 4 Hall, B. D., and S Spiegelman, these ProceEeprnes, 47, 137 (1961 ). 6 Bolton, E. T., B. H. Hoyen, and D. B. Ritter, in Microsomal Particles and Protein Synthesis, ed. R. B. Roberts (New York: Pergamon Press, 1958), p. 18. 16 Tissidres, A., J. D. Watson, D. Schlessinger, and B. R. Hollingworth, J. Mol. Biol., 1, 221 (1959). 1 Tissiéres, A., 1). Schlessinger, and F. Gros, these Proceepinas, 46, 1450 (1960). 18 McCarthy, B. J., and A. I. Aronson, Biophys. J., 1, 227 (1961). 19 Hershey, A. D., J. Gen. Physiol., 38, 145 (1954). 2 Siminoviteh, L., and A. F. Graham, Canad. J. Microbiol., 2, 585 (1956). 21 Davern, C. I., and M. Meselson, J. Mol. Brol., 2, 153 (1960). 22 Siekevitz, P., J. Biol. Chem., 195, 549 (1952). 23 Britten, R. J., and R. B. Roberts, Science, 131, 32 (1960). 24 Martin, R., and B. Ames, J. Biol. Chem., 236, 1372 (1961). % Bamford, C. H., A. Elliott, and W. E. Hanby, Synthetic Polypeptides (New York: Academic Press, 1956), p. 322.