Reprinted from the Proceedings of the NaTronaL ACADEMY OF SCIENCES Vol. 48, No. 1, pp. 104-109. January, 1962. AN INTERMEDIATE IN THE BIOSYNTHESIS OF POLYPHENYLALANINE DIRECTED BY SYNTHETIC TEMPLATE RNA By Marsuatu W,. Nirenserc, J. Heinrich MarrHar,* AND OLIVER W. JONES NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Richard B. Roberts, November 24, 1961 We have recently found that, simple, synthetically prepared polyribonucleotides such as polyuridylict acid and polycytidylic acid function as RNA templates in a cell-free protein-synthesizing system prepared from £. coli. 2 In this system, poly U contains the information for the synthesis of polyphenylalanine; therefore, the code for phenylalanine is one or more uridylic acid residues. Poly U was much more effective in increasing the rate of cell-free protein synthesis than naturally occurring informational RNA,? possibly because the synthesis of a protein containing only one amino acid was faster than the synthesis of a protein containing 20 amino acids. We are using this model system currently to study the enzymatic sequence of protein synthesis. Although sRNA and amino acid—activating enzymes have been studied exten- sively, there is controversy concerning their relationship to protein synthesis.*—¢ Vou. 48, 1962 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES 105 One purpose of this investigation was to determine whether sRNA is an intermediate in the synthesis of polyphenylalanine directed by a synthetic RNA template. Our results demonstrate that the incorporation of phenylalanine into sRNA and its subsequent transfer from sRNA are steps in the synthesis of polyphenylalanine. Methods.—-Dialyzed extracts of E. coli containing ribosomes and 100,000 x g supernatant solu- tions were obtained as described perviously.2. These extracts correspond to the previously described 8-30 fractions.2. Ribosomes were sedimented by centrifuging 8-30 extracts at 105,000 x gfor 2 hrat 3°. The supernatant solution was aspirated and will be referred to hereafter as 100,000 X g supernatant solution. The ribosomes were resuspended in 0.01 Jf Tris (hydroxy- methyl)aminomethane, pH 7.8, 0.01 17 magnesium acetate, 0.06 Af KCI, and were centrifuged again at 105,000 x g for 2 hr. The supernatant solution was decanted and was discarded. The ribosomes were washed two more times in the same manner by resuspension and centrifugation. Both 100,000 x g supernatant solutions and washed ribosomes were stored in small aliquots under liquid nitrogen. Thawed preparations were not refrozen and reused. sRNA was purified from #. colt 100,000 X g supernatant solutions by phenol extraction. sRNA was charged with C'-phenylalanine enzymatically, and the C!-aminoacylsRNA was purified by the method described by von Khrenstein and Lipmann.‘ The specific radioactivity of the C-aminoacyl-sRNA varied from 800 to 36,000 counts/min/ng sRNA. The optical density of img of sSRNA in H2O at 260 my was assumed to be 24.4 The RNAase-digested aminoacylsRNA described in Table 2 was prepared by incubating Cl4- phenylalanine-sRNA with L yg crystalline RNAase (Worthington Biochemical Corporation) per ml at 37° for 1 hr. The RNAase was removed by 5 consecutive phenol extractions using equal volumes of phenol. The aqueous phase was then dialyzed overnight against 1,000 volumes of HO. The alkali-degraded C'-phenylalanine-sRNA described in Table 2 was prepared by incubating aminoacyl-sRNA in 0.3 47 KOH at 35° for 18 hr. The solution was then neutralized and was dialyzed against 1,000 volumes of H.O. Also, C'-phenylalanine-sRNA was incubated in 0.4 M glycine buffers, pH 11.0 at 37° for Lhr. The solution was then dialyzed against 1,000 volumes of H.0. Radioactive protein precipitates were washed and counted as before? by a modification of the method of Siekevitz.?’ Protein concentrations were determined by a micro modification of the method of Lowry. All assays reported in this paper were performed in duplicate. Materials —U-C''-L-phenylalanine was obtained from the Nuclear-Chicago Company and had a specific radioactivity of 5-10 mC/mmole. The purified transfer enzyme (after DEAE column chromatography, purified about 10-fold’) and some C1phenylalanine-sRNA used were the gener- ous gift of Daniel Nathans and Fritz Lipmann. Polynucleotide phosphorylase was used to synthesize poly U. The molecular weight of the poly U was between 30,000 and 50,000. Results-—Components of reaction mixtures are given in Table 1. The data pre- sented in Table 1, Experiment 1, demonstrate that C'-phenylalanine was incor- porated into protein only when poly U was added to the reaction mixture. When 2 ymoles of unlabeled, C'’-phenylalanine were added to a reaction mixture contain- ing 0.019 umoles of C!*-phenylalanine, the incorporation of C!phenylalanine into protein was markedly reduced as was expected, due to dilution of the tracer. C'-phenylalanine-sRNA was prepared by incubating sRNA with C'phenyl- alanine and nonpurified phenylalanine activating enzyme present in 100,000 X g supernatant solutions. These extracts catalyzed a phenylalanine-dependent exchange of labeled pyrophosphate into ATP. Other amino acid-activating en- zymes, stimulating a similar exchange, were also present. In Experiment 2, the tracer present in reaction mixtures was C!~phenylalanine- sRNA in place of free C'-phenylalanine. ATP, CTP, and UTP were omitted. In the absence of poly U, little C'phenylalanine was incoporated into protein. However, in the presence of poly U, approximately 60 per cent of the C'4-phenyl- 106 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES Proc. N. A.S. TABLE 1 Errscr or C12-PHENYLALANINE UPON C!PHENYLALANINE INCORPORATION INTO PROTEIN Experiment No. C1t-tracer Addition Counts/min 1 C'_L-Phenylalanine None 1,430 — Polyuridylie acid 68 + 2 »moles C!*-L-phenylalanine 93 — ATP, PEP, and PEP kinase 62 None, zero time AQ 2 44L-Phenylalanine-sRNA None 683 — Polyuridylic acid 9 + 2 ymoles C*-L-phenylalanine 603 — GTP, PEP, and PEP kinase 10 None, zero time 6 The reaction mixtures for Experiment 1 contained the following in wmoles/ml: 100 Tris (hydroxymethyl) - aminomethane, pH 7.8; 10 magnesium acetate; 50 KCl; 6 mercaptoethanol; 1 ATP; 5 phosphoenolpyruvate, K salt; 2 wg phosphoenolpyruvate kinase, crystalline; 0.03 each of GTP, CTP, and UTP; 10 ug polyuridylic acid: 0.019 wmoles of Cl4-L-phenylalanine, ~75,000 counts/min; and 0.46 and 1.04 mg of ribosomal and 100,000 x g supernatant solution protein, respectively, The reaction mixtures for Experiment 2 contained the following in zmoles/ml; 100 Tris (hydroxymethyl)- aminomethane, pH 7.8; 10 magnesium acetate; 50 KCl; 6 mercaptoethanol; 0.3 GTP; 5 phosphoenolpyruvate, K salt; 2 ng phosphoenolpyruvate kinase, crystalline; 10 wg polynridylic acid; 0.45 mg C1-phenylalanine-sRNA, ~1100 counts/min; and 0.46 and 1,04 mg of ribosomal and 100,000 X g supernatant solution protein, respectively, Total volume was 0.5 ml. Samples were incubated at 35° for 10 min, were deproteinized with 10 per cent tri- chloroacetic acid. alanine was transferred from sRNA to protein. The transfer required GTP and a GTP-generating system; however, with this dialyzed but unpurified system, ATP and an ATP-generating system was 70 per cent as effective as GTP in facilitating transfer. Addition of 2 wmoles of unlabeled C!*-L-phenylalanine did not decrease appreciably the transfer of C'-phenylalanine from sRNA to ribosomal protein, in contrast to the data of Experiment 1. These results demonstrate that C'-phenyl- alanine can be transferred undiluted from sR.NA to protein in the presence of a large pool of unlabeled, free phenylalanine and that poly U directs the transfer. Tn Figure i, the quantity of phenylalanine transferred from sRNA into ribosomal i rs rs es es tPOLY U COUNTS /MINUTE x 3 MINUTES Fic. 1.-Stimulation of C-pnenylalanine transfer from sRNA to protein by polyuridylic acid ©.5 ug polyuridylic acid added; a without polyuridylic acid. The components of the reaction mixtures are given in Table 1, Experiment 2. 0.17 mg of C'™phenylalanine-sRNA. 400 counts/ min, 0.46 mg ribosomal protein and 1.04 mg 100,000 X g supernatant fraction protein were added to each reaction mixture. protein is plotted against time in minutes. In the absence of poly U, little C! phenylalanine was incorporated. In the presence of poly U, C!-phenylalanine was transferred rapidly from sRNA to protein. Almost all of the C'phenylalanine was transferred during the first: five minutes of incubation. Some control experiments are presented in Table 2 demonstrating that C!+- Vou. 48, 1962 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES 107 TABLE 2 AminoacyL, RNA Controu EXPERIMENTS Additions Counts/min C1+-phenylalanine-sRNA 704 C}*phenylalanine-sRNA treated with RN Aase* 2 14_phenylalanine-sRNA treated with 0.3 @ KOHt 9 C'-phenylalanine incubated at pH 11f 18 None, Zero time 4 The composition of the reaction mixtures is presented in Table i, Experiment 2. 1.04 mg protein in 100,000 X g supernatant solution, 0.46 mg ribosomal protein, and 0.45 mg Cu-phenylalanine sRNA containing ~1100 counts/min (before digestion) were added to each reaction mixture. *sRNA preparations were deproteinized by phenol extraction after RNAase treat- ment as specified in the Methods section. { Alkali treatment of C'*-phenylalanine-sRNA and incubation at pH 11 are described in the Metheds section. phenylalanine-sRNA has properties similar to those reported for aminoacyl-RNA. Treatment of C'*-phenylalanine-sRNA with RNAase or with 0.3 VN KOH destroyed its activity. Aminoacyl-sRNA is hydrolyzed to free amino acids and sRNA at pH 10.° Incubation of C'-phenyl-alanine-sRNA at pH 11 resulted in the hydrol- ysis of phenylalanine from sRNA as expected. In addition, our preparations of C'-phenyl-alanine-sRNA had a sedimentation value of 4.6 as determined by sucrose density-gradient centrifugation." The data of Table 3 demonstrate that both 100,000 X g supernatant fractions TABLE 3 REQUIREMENT FoR 100,000 < g SUPERNATANT SOLUTION AND RrposoMEs Additions Counts/min None 341 — 100,000 X g supernatant solution 14 — Ribosomes 5 None, zero time 7 The components of the reaction mixtures are presented in Table 1, Ex- periment 2. 0.46 and 1.04 mg protein were present in the ribosome and 105,000 X_g supernatant fractions, respectively. 1.0 meg of C!&phenyl- alanine-sRNA, ~800 counts/min, were added to each sample. and ribosomes were required for transfer of C'*-phenylalanine from sRNA to protein. Since the transfer enzyme is found in 100,000 X g supernatant solutions, it seemed likely that the requirement for this fraction could be replaced by purified transfer enzyme. The data of Table 4, Experiment 1, demonstrate that transfer enzyme TABLE 4 REPLACEMENT OF 100,000 X g SupERNATANT FRACTION wiTH TRANSFER ENZYME Experiment No. Addition Counts/min 1 None 404 ~— Transfer enzyme 10 — Polyuridylic acid 11 None, zero time 6 2 + GTP, + PEP, + PEP Kinase 566 ~ GTP, + PEP, + PEP Kinase 17 — GTP, + ATP, + PEP, + PEP Kinase 68 + GTP, + ATP, + PEP, + PEP Kinase 271 + GTP, — PEP, — PEP Kinase 17 + GTP, + ATP, — PEP, — PEP Kinase 13 + GTP, + PEP, + PEP Kinase, Zero Time 3 Components of the reaction mixtures are presented in Table 1, Experiment 2. 100,000 X g super- natant solution was omitted. 2.0 «moles of ATP/ml of reaction mixture were present where specified. 0.46 mg of ribosomal protein were present and 0.095 mg transfer enzyme protein were present except where specified. 0.30 mg C14phenylalanine-sRNA, ~800 counts/min, were added to each sample in Experiment 1 and 0.05 mg, ~1300 counts/min, were added to each sample in Experiment 2. In Ex- periment 2, for simplicity, the presence or absence of GTP, ATP, PEP, and PEP kinase are noted. 108 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES Pnroc. N. A.S. could replace 100,000 X g supernatant solution. The data of Experiment 2 show that GTP was necessary for the transfer, and that, in this purified system, ATP could not replace GTP effectively. The data of Tables 1, 3, and 4 show that C'- phenylalanine transfer from sRNA to protein required ribosomes, transfer enzyme, poly U, and GTP and a GTP-generating system. Discussion.—The data presented in this communication demonstrate that amino- acyl-sRNA is an intermediate in phenylalanine incoporation into protein mediated by poly U. Ina previous communication, we showed that the protein synthesized had unusual characteristics similar to authentic polyphenylalanine.? The initial steps in polyphenylalanine synthesis appear to be: phenylalanine- activating enzyme L-phenylalanine + ATP AMP-phenylalanine + P-P. (1) phenylalanine- activating enzyme AMP-phenylalanine + sRNA phenylalanine-sRNA + AMP. (2) Poly U ribosomes transfer enzyme GTP > 2 9 Phenylalanine-sRNA — —» > polyphenylalanine + sRNA. (3) The detailed mechanisms of the steps involved in reaction (3) are under investiga- tion. It should be noted that our data do not preclude the possibility of alternative routes of synthesis of polyphenylalanine. When poly A and poly U are mixed, doubly- and triply-stranded RNA is formed (U-A and U-U-A).!2 13) We have shown previously that poly U in the doubly- and triply-stranded state was completely inactive as template RNA.2 Further experiments have corroborated and extended these findings and will be published in a separate communication. These data strongly suggest that the portion of the RNA molecule which functions as a template for protein synthesis is single-stranded. Simple predictions may be made concerning the primary and secondary structures of the hypothetical “template-recognition portion” of phenylalanine-sRNA. Since a sequence of one or more uridylic acid residues in poly U is the code for phenylalanine in this system, it is probable that phenylalanine-sRNA contains a complementary sequence of one or more adenylic acid residues which base-pair with the template. It is also probable that the portion of sRNA recognizing the template is single-stranded. The genetic code may not be universal ; lt may differ from species to species. Since sRNA may be a cofactor which functions as an “adaptor” carrying an amino acid to its proper place on template RNA, a variant sRNA base-pairing with a different code letter of template RNA would substitute one amino acid for another during protein synthesis.‘ Although E. coli sRNA can be used for the cell-free synthesis of rabbit hemoglobin,*: 4 it is possible that in species other than £. cold, poly U may be either meaningless or may serve as a template for a different amino acid.t Changes in the code could occur at different stages in the translation of information from DNA to the finished protein, for example, at the level of the DNA or RNA templates, at the level of sRNA, or at the level of amino acid—activating Vou. 48, 1962 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES 109 enzymes. We arc using the poly U system to determine whether the code for phenylalanine is the same in different species. Summary. —Phenylalanine-soluble RNA was shown to be an intermediate in the cell-free synthesis of polyphenylalanine directed by a synthetic template RNA, polyuridylic acid. We wish to thank Mrs. Linda Greenhouse for her excellent help in performing some of the analyses. * NATO postdoctgral fellow. } The following abbreviations are used: Polyuridylic acid, poly U; polycytidylie acid, poly C; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; sRNA, soluble ribonucleic acid; RN Aase, ribonuclease; ATP, adenosine triphosphate; UTP, uridine triphosphate; CTP, cytidine triphos- phate; and GTP, guanosine triphosphate; PEP, phosphoenolpyruvate; and PEP kinase, phos- phoenolpyruvate kinase. ¢ That this may be the case is suggested by preliminary experiments performed in collaboration with Harry Gelboin showing that poly U does not stimulate incorporation of C!phenylalanine in a rat liver amino acid—incorporating system. Similar results have been obtained by S. Ochoa (personal communication). Nirenberg, M. W., and J. H. Matthaei, V International Congress of Biochemistry, Moscow, August 1961. 2 Nirenberg, M. W., and J. H. Matthaei, these ProcEepines, 47, 1588 (1961). 5 Beljanski, M., Biochim. et Biophys. Acta, 41, 104 (1960). ‘ Khrenstein, G. von, and F. Lipmann, these Procerpinas, 47, 941 (1961). 5 Aronson, A. I, E. T. Bolton, R. J. Britten, D. B. Cowie, J. D. Ducrksen, B. J. MeCarthy, Ix. MeQuillen, and R. B. Roberts, Carnegie Institute of Washington, Fearbook (1959-1960), p. 229. § Nisman, B., and H. Fukuhara, C. &. Acad. Sci. (Paris), 248, 2036 (1959). 7 Siekevitz, P., J. Biol. Chem., 195, 549 (1952). § Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, zbid., 193, 265 (1951). ° Nathans, D., and F. Lipmann, these Procenpinas, 47, 497 (1961). 1 Hecht, L. I, M. LL. Stephenson, and P. C. Zamecnik, ibid., 45, 505 (1959). 1 We thank Robert Martin for performing these determinations for us. 2 Rich, A., and D. R. Davies, J. Am. Chem. Soc., 78, 3548 (1956). 13 Felsenfeld, G., and Rich, A., Biochim. et Biophys. Acta, 26, 457 (1957). 1 Benzer, 8., and B. Weisblum, these ProceEpinGs, 47, 1149 (1961).