assignments for five of the remaining six amino acids, namely alanine, asparagine,

Transcription

1 VOL. 48, 1962 BIOCHEMISTRY: SPEYER ET AL. 441 these two, of the known sialic acids, were used in these studies; NAN-aldolase = the enzyme that. cleaves NAN and NGN to pyruvate and the corresponding N-acyl-D-mannosamine; CMP- NAN or CMP-NGN = compounds containing cytidylic acid linked to the corresponding sialic acid. l Comb, D. G., F. Shimizu, and S. Roseman, J. Am. Chem. Soc., 81, 5513 (1959). 2 Barry, G. T., and W. F. Goebel, Nature (London), 179, 206 (1957); Barry, G. T., J. Exptl. Med., 107, (1958). 3 Svennerholm, L., Biochim. et Biophys. Acta, 24, (1957). 4Aminoff, D., Biochem. J., 81, (1961 ). 6 As previously noted,' the CMP-sialic acids are extremely labile compounds, particularly at acid ph. The freshly prepared nucleotides contained no detectable quantities of free CMP or the sialic acids, but some hydrolysis occurred on storage for a week as the ammonium salts in the dry state at - 15 ; small amounts of the hydrolytic products were then detected on paper chromatography. 6 Comb, D. G., and S. Roseman, J. Biol. Chem., 235, (1960). Crystalline NAN and several sialic acid glycosides have been assigned anomeric configurations solely on the basis of optical rotation studies and application of Hudson's rules of isorotation. It has not yet been established that the rules of isorotation are actually applicable to these complex sugars (Roger Jeanloz, personal communication). For this reason, the anomeric configuration of the CMP-sialic acid glycosidic bonds will not be assigned at this time. As is the case with other sialic acid glycosides, the CMP-sialic acids ar- assumed to possess pyranose ring structures. 8 Markham, R., and others, in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1957), vol Warren, L., Biochim. et Biophy8. Acta, 44, (1960). 10 A manuscript (Jourdian, G. W., and S. Roseman) is being submitted to the J. Biol. Chem. describing the chemical preparation of the N-glycolylhexosamines and their chromatographic and electrophoretic behavior. These techniques can be used to characterize any of the following compounds; D-glucosamine, D-galactosamine, D-mannosamine, their N-acetyl, and their N-glycolyl derivatives. 11 Roseman, S., G. W. Jourdian, D. Watson, and R. Rood, these PROCEEDINGS, 47, (1961); Warren, L., and H. Felsenfeld, Biochem. Biophys. Res. Comm., 5, (1961). 12 Leloir, L. F., in Transactions of the Third Conference on Polysaccharides in Biology, ed. G. Springer (New York: Josiah Macy, Jr., Foundation Publications, 1957), pp Berg, P., and G. Newton, J. Biol. Chem., 222, (1956). 14 Berg, P., J. Biol. Chem., 233, (1958). 15 Whelan, W. J., Ann. Rept. on Progr. (Chem. Soc. London), 54, (1957). 16 Jourdian, G. W., F. Shimizu, and S. Roseman, Federation Proc., 20, 161 (1961). 17 Telep, G., and R. Ehrlich, Anal. Chem., 30, (1958). SYNTHETIC POLYNUCLEOTIDES AND THE AMINO ACID CODE, IV* BY JOSEPH F. SPEYER, PETER LENGYEL, CARLOS BASILIO, t AND SEVERO OCHOA DEPARTMENT OF BIOCHEMISTRY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated January 30, 1962 Triplet code letter assignments (of as yet unknown sequence) for fourteen amino acids have been made on the basis of experiments with a cell-free Escherichia coli system and synthetic polynucleotides.'-3 Continuation of this work has led to assignments for five of the remaining six amino acids, namely alanine, asparagine, aspartic acid, glutamic acid, and methionine. An experimental value for the code

2 D 442 BIOCHEMISTRY: SPEYER ET AL. PROC. N. A. S. : 2 000Q v. letter of glutamine is still unavailable but a 1U 1C 1G letter for this amino v 9 9 -v acid can be predicted from replace- > > >> P > ; > >> 9 N * ment data of Tsugita4 in nitrous acid,t 00 co.l.mutants of tobacco mosaic virus. In s.ng,:the voo meantime,... our assumption, for do5v- simplicity's sake, of a triplet code _Cq.000 found experimental support in the elegant genetic experiments of Crick, N 0 :: 0. Barnett, Brenner, and Watts-Tobin.5. 0~~~~~~~~ In presenting the experimental X =, : : fl~~~c evidence for the last assignments it : will be desirable to make a general o N survey of all of our results and to coma pare them in detail with the available z ><, '3s0 amino acid replacement data in.t C nitrous acid mutants of tobacco 0 mosaic virus (Tsugita,4 6Tsugita and -G0o U5.~.~ ~.:. Fraenkel-Conrat,7 and Wittmann8). :0 z opreparations ; and Methods.-These a; C) to,e,, 0 have been the same as in.p.work" previous I except for the - following E-4, C changes: (1) during preincubation. all twenty C2amino acidswerepres-. b=<0r~tqent at 0.045,mole/ml each, (2) in 0 wo<> s '.S;C boo the incubation mixture nineteen C12 z O c~s H XOUY0SXQ amino acids and one C'4 amino acid Z X.e.. were present at 0.2,g4mole/ml each. -4 Results.-Experiments with alanine, ;,= O asparagine, aspartic acid, glutamic.o -I acid, and methionine: The results of z a^> several experiments with these amino mi:o: 7! o. s 0;, <, 00:= acids are recorded in Table 1. ~o" n n O fl or <,<,Alanine incorporation was stimulated z 20 o; z only by poly UCG. A slight stimula- 000 >t -,, tion by this polymer of alanine in- CD * c C c 0O0 cc 4- corporation had previously been observed but not recorded as it :+z :; :~appeared too small and inconsistent. 0: :c::: *O The average phe/ala incorporation N E 0 +0~ ratio in two experiments was 31 (Table 2). Since the calculated fre-... * quency ratio of UUU to 1U 1C 1G 4. t triplets in poly UCG (6:1:1) is 36,9 2t= 3.-2 this suggests 1U 1C 1G as the code 4 - C = E- E-, z H > >.E40 letter. Incorporation of asparagine 0 S "'was stimulated by poly UA, poly

3 VOL. 48, 1962 BIOCHEMISTRY: SPEYER ET AL. 443 UAC and poly UAG. Asparagine had not been available to us until recently and had not previously been tried with these polymers. The average incorporation ratio phe/aspn with poly UA, UAC, and UAG, was 15, 15, and 20, respectively (Table 2). The calculated frequency ratios of UUU to 1U 2A in TABLE 2 CODE LETTER ASSIGNMENTS* Polynucleotides Amino UC UA UG UAC UCG UAG Code letter acid (5:1) (5:1) (5:1) (6:1:1) (6:1:1) (6:1:1) (unknown sequence) Ala U 1C 1G Arg U 1C 1G AspN U 2A (1U 1A 1C) Asp U 1A 1G Cys U 1G Glu U 1A IG GluN U 1C 1Gt Gly U 2G His U 1A 1C Ileu U 1A Leu U 1C (2U 1A, 2U 1G) Lys U 2A Met U 1A 1G Pro U 2C Ser U 1C Thr t U 1A 1C (1U 2C) Try U 2G Tyr U 1A Val U 1G * The experimental values given are ratios of stimulation of phenylalanine incorporation to that of the amino acid in question by different polynucleotides and are in all cases averages of two or more experiments from this and previous papers of this series. Code letters in parentheses indicate other possible code letters for a given amino acid (degenerate code). t No experimental value available. Code letter predicted from glutamine-*valine replacement in HNO2 mutant of tobacco mosaic virus (Tsugita4).2. ' * In previous experiments2 the phenylalanine/threonine ratio with poly UC (5:1) was 17. In more recent experiments, stimulation of threonine incorporation by this polymer was negligible in one and nil in another. This will be reinvestigated. poly UA (5:1), UAC (6:1:1), and UAG (6:1:1) is 25, 36, and 36 respectively. This suggests 1U 2A and 1U 1A 1C (poly UAC was twice as effective as expected from its 1U 2A content alone) as code letters for asparagine. Since the code may be degenerate,5 the existence of more than one code letter for a given amino acid is not improbable. Aspartic acid poses no problems as its incorporation was stimulated only by poly UAG and the phe/asp incorporation ratio, 41 (Table 2), was not far from the calculated value, i.e. 36, for a IU 1A 1G letter. Glutamic acid behaved similarly except that stimulation of its incorporation by poly UAG was smaller and the phe/glu ratio of 64 was almost twice as high as theory for a 1U 1A 1G letter. However, no other polymer stimulated incorporation of this amino acid and the code letter 1U 1A 1G may tentatively be assigned to it. This assignment is in line with amino acid replacement data (cf. Table 5). The code letter assignment 1A 1U 1G is amply justified for methionine. Its incorporation was stimulated only by poly UAG and the phe/met ratio of 23 (Table 2), although lower than the theoretical 36, is not too far off. Repetition of experiments with other amino acids: Since the modifications in methods introduced here and in the preceding paper3 considerably increased the effect of synthetic polynucleotides on the incorporation of amino acids we reinvestigated certain previously described code triplet assignments.

4 444 BIOCHEMISTRY: SPEYER ET AL. PROC. N. A. S. The results, shown in Table 1, agree with the earlier data in the case of histidine, lysine, proline, serine, tyrosine and valine. In the case of threonine, in contrast with previous results,2 poly UC (5: 1) was relatively ineffective. On the other hand, stimulation of the incorporation of this amino acid by poly UAC (6:1:1) was high, relative to stimulation of phenylalanine incorporation, in the present as well as in the former experiments. A phe/thr ratio of 11 was obtained (Table 2) whereas the expected ratio for a 1U 1A ic letter is 36. Since our sample of threonine-c"4 had a very low specific radioactivity, repetition of these experiments with a sample of higher specific activity is clearly desirable. For the present, 1U IA ic may tentatively be taken as a code letter for threonine with 1U 2C as a possible additional letter for this amino acid. There is some support for this assumption in the fact that, in recent experiments, poly CU (5:1) promoted threonine incorporation although to a lesser extent than that of proline for which a 1U 2C code letter is definitely established. In preliminary experiments it has been found that inosinic acid (I) can replace G. Thus, like poly UG (5:1), poly UI (5:1) stimulated the incorporation of phenylalanine, cysteine, glycine, tryptophan, and valine. The phe/cys and phe/val ratio was close to 5 (theory for 2U 1I letters); the phe/gly and phe/try ratio was about 25 (theory for 1U 2I letters). This is as expected since hypoxanthine resembles guanine in its hydrogen bonding properties. Discussion.-Survey of code letter assignments: With the experimental results reported in the preceding section, code letter assignments are now available for 19 amino acids. A directly determined code letter for glutamine is still lacking.3 However, a 1U IC 1G letter may he predicted for this amino acid as previously discussed.2 Table 2 summarizes the experimental basis for our code letter assignments. As previously explained,'-3 the assignments are based on a comparison of the ratio of stimulation by a given polymer of the incorporation of phenylalanine to that of another amino acid with the calculated frequency ratio of UUU triplets to other triplets in this polymer. For ease of comparison of these two ratios the calculated UUU to other triplet frequency ratios are given in Table 3. It may be seen that in most cases the assignment is made on the basis of a very close agreement of the phenylalanine to other amino acid incorporation ratio with one of the calculated UUU to other triplet ratios for one or more of the polymers. This statement is definitely borne out in the case of the following amino acids (cf. Tables 2 and 3): Alanine, arginine, aspartic acid, cysteine, glycine, histidine, isoleucine, serine, tryptophan, tyrosine, and valine. The agreement is not as good with the remaining amino acids. These give phenylalanine to other amino acid incorporation ratios either higher (glutamic acid, lysine) or lower (asparagine, histidine, methionine, proline, threonine) than corresponds to the code letter assignment made. Since the calculated UUU to other triplet ratios are based on a random distribution of nucleotides in the synthetic copolymers,0' 11 a slight deviation from randomness could explain the lack of perfect agreement in some cases. In spite of this we feel that the data taken as a whole bear out the proposed code letter assignments. If we take glutamic acid and methionine as examples of too high and too low ratios, respectively, it is clear that their code letters must contain 1U 1A 1G since their incorporation was stimulated only by poly UAG of the six different copolymers tested.

5 VOL. 48, 1962 BIOCHEMISTRY: SPEYER ET AL. 445 From the data in Table 2, it appears that three amino acids, namely asparagine, leucine, and threonine, may have more than one code letter or, in other words, that the code for these amino acids is degenerate. The reasons for assigning more than one code letter to each asparagine, and threonine have been given in the preceding TABLE 3 FREQUENCY RATIOS OF UUU TO OTHER TRIPLETS IN SYNTHETIC POLYNUCLEOTIDES* Polynucleotide UC UA UG UAC UCG UAG Triplets (5:1) (5:1) (5:1) (6:1:1) (6:1:1) (6:1:1) 2U 1C U2C U 1A U 2A U 1G U 2G U 1A 1C U 1C 1G U 1A 1G Triplets without Ut * Calculated frequency ratios based on the composition of the polymers with assumption of complete randomness. The frequency ratios are for each one of the possible sequences for a given triplet, e.g. UUU/UUC or UUU/ UCU or UUU/CUU = 5 in case of the 2U 1C triplets. t In the polymers used, e.g. CCC in poly UC; or AAA, AAC, ACA, CAA, ACC, CAC, CCA, CCC in poly UAC, etc. section. As regards leucine it will be remembered that its incorporation into acid insoluble products was stimulated by most of the polynucleotides tested.1' 2 With a sample of leucine-c14 of high purity'2 we obtained essentially the same result (Table 2) except for a negligible stimulation by poly U. The phe/leu incorporation ratio of 5 with poly UC (5:1), as before, was exactly that expected for a 2U 1C code letter. The ratios of 7 and 8 obtained with poly UA (5:1) and poly UG (5:1), respectively, are not too far from 5, the expected value for a 2U 1A or a 2U 1G letter. Further, the ratio of 4 in the case of each poly UAC (6:1:1) and poly UCG (6:1:1) is close to 3, the expected value if stimulation of leucine incorporation by these polymers were caused by 2U 1C + 2U 1A triplets in poly UAC and by 2U 1C + 2U 1G triplets in poly UCG. The possibility must therefore be considered, as indicated in Table 2, that the results with leucine may be due to the existence of three code letters, namely 2U 1C, 2U 1A, and 2U IG, for this amino acid. A striking feature of the code triplets is that they all contain U. In fact, each of 8 triplets (those for cysteine, isoleucine, leucine, serine, tyrosine, and valine) out of the 23 listed in Table 2 contains two U residues and the phenylalanine triplet contains three. Since out of 41 = 64 triplets, 37 contain U (cf. Table 3) and only 24 of these are accounted for, there remain 13 triplets some, or all, of which might be "nonsense." How many of the 27 non-u triplets are "nonsense" cannot be decided by our present methodology which, due to limited sensitivity (cf. Table 3), can only be used for the detection of U-containing code letters. However, the code might be degenerate to a greater extent than is apparent from our present data and there could be additional code letters consisting of triplets without U. With copolymers of similar composition to those used by us, Martin et al.'3 have recently reported results for 15 amino acids largely in agreement with ours,

6 446 BIOCHEMISTRY: SPEYER ET AL. PRoc. N. A. S. Distribution of code letters among various triplets: It is interesting to note from Table 4 that in no case does the number of code letters of the same composition exceed the number of possible sequences in the corresponding triplet group. Had this occurred, some of the assignments would have been undoubtedly in error. In Table 4 the triplets of the 2U 1A and 1U 2A and those of the 1A 1U 1C and 1U 1A 1G series have been paired so that every triplet in each of the two groups is TABLE 4 DISTRIBUTION OF CODE LETTER AMONG VARIOUS TRIPLETS* Amino UUU 2U1C 1U2C 2U1A 1U2A 2U1G 1U2G lulciglulaic lulaig acid UUC UCC UUA AAU UUG UGG UCG UAC AUG UCU CUC UAU AUA UGU GUG UGC UCA AGU CUU CCU AUU UAA GUU GGU CUG AUC UAG CGU ACU UGA GUC CUA GAU GCU CAU GUA Ala Arg + AspN + (+) Asp + Cys...+ Glu.. + GluNt + Gly + His Ieu + Leu +.. (+).. (+) Lys Met Phe +.. Pro Ser.. + Thr.... (4) Try Tyr Val * Individual sequences are given under the nucleotide composition of each triplet. Plus signs in parentheses denote possible additional code letters for a given amino acid. t Predicted. next to its complementary. Since only three triplets out of six appear to be utilized in each of the 1U 1A 1C and 1U 1A 1G series, it is possible that only one triplet in each complementary pair is a code letter. If so, there would be no complementary code letters in this group. Some exclusion of complementarity occurs in the 2U 1A and 1U 2A series as only two positions are occupied by code letters in the latter. The more or less complete elimination of complementary triplets in the code letters would have the advantage of restricting hydrogen bonding (both intra- and intermolecular) in messenger RNA an occurrence that could interfere with its function. Correlation with amino acid replacement data in nitrous acid mutants of tobacco mosaic virus: With the availability of experimentally determined code letters for 19 amino acids and of new data on amino acid replacements in nitrous acid mutants of tobacco mosaic virus6-8 it is of interest to examine the latter in the light of our code letter assignments. This has been done in Table 5. In recording agreement or lack of agreement, on the last column of the table, it was assumed that the only well established changes brought about by treatment of RNA with nitrous acid are (1) conversion of C to U, and (2) conversion of A to G.8 On this basis 11 out of 16

7 VOL. 48, 1962 BIOCHEMISTRY: SPEYEP ET AL. 447 replacements (or approximately two-thirds of the replacements thus far observed) can be explained by our code letter assignments. However, it is possible that base changes other than C-s+U and A--G occur as a result of nitrous acid treatment. TABLE 5 AMINO ACID REPLACEMENTS IN HNO2 MUTANTS OF TOBACCO MOSAIC VIRUSt Times Replacement observed Reference Code letter change Agreement** Asp*-)Ser 4 Tsugita & Fraenkel-Conrat,7 UAG, UAA(UAC)UUC Wittmann8 Asp*--.A1a 6 Tsugita,4 Wittmann8 UAC--UGC Asp*-G1y 2 Wittmann8 UAG--UGG + Arg--*Gly 5 Tsugita6 UCG--UGG Glu--Gly 1 Tsugital UAG-s.UGG Glu*--Gly 2 Wittmann8 UAG-sUGG + GluN-s.Val 1 Tsugita4 (UCG)j-UUG Glu*--)Val 2 Wittmann8 (UCG)--UUG Ileu-i-Val 1 Wittmann8 UUA-JUUG Leu-,Phe 1 Wittmanns UUC--UUU Pro--Leu 2 Tsugita,4 Wittmann8 UCC-JUC Pro--Ser 3 Wittmann8 UCO-JUUC + Ser--Leu 1 Wittmann8 UUC--UUC(UUA, UUG) Ser-oPhe 3 Tsugita,4 Wittmann8 UUCUUU Thr-Ser 1 Tsugita4 UCC-UUC Thr--Ileu 7 Wittmann8 UAC--UAU + Thr--Met 3 Wittmann8 UAC(UCC)-..UAG Tyr-,Phe 1 TsugitaW UUA--UUU t Asp* may be either aspartic acid or asparagine, glu*, either glutamic acid or glutamine. I Letter for glun not available experimentally; predicted from this replacement. ** + indicates that the reported replacement is in agreement with the code letter assignments in this paper on the basis that either C is converted to U or A converted to G by nitrous acid treatment of tobacco mosaic virus RNA; - indicates lack of agreement on this basis. Moreover, some of the replacements observed only once might be the result of spontaneous mutation. Lack of agreement in those replacements observed two or more times, e.g., asp*--o-ser, arg--gly, and thr-.)met (Table 5), is more disturbing. One possibility is that these replacements might reflect the occurrence of additional, non-u letters as considered elsewhere in this paper. The occurrence of undetected U-letters is another possibility. Thus, the asp*-soser and ser-dleu replacements (Table 5) might be explained if serine had a 1U 1C 1G in addition to the 2U 1C code letter. The small stimulation of serine incorporation by 1U 1C 1G triplets in poly UCG (6:1:1) would escape detection in the presence of a six times higher stimulation due to 2U 1C triplets in this polymer (2U ic: 1U 1C 1G frequency ratio = 6). The code letter changes would then be as follows: aspn (UAC)-.+ser (UGC); ser (UCG)->leu (UUG). Smith'4 has correlated single amino acid replacements in a number of human mutant hemoglobins with our code letters for 14 amino acids, under the restrictive assumption that each mutation involves replacement of only one base in a triplet, and found excellent agreement. From this comparison he was able to predict the code letters for four amino acids (alanine, asparagine, aspartic and glutamic acid) now reported in this paper. Universality of genetic code: From the agreement between the code letters derived from work on the E. coli system with amino acid replacements in mutants of tobacco mosaic virus and human hemoglobins, it would appear that there is but

8 448 BIOCHEMISTRY: SPEYER ET AL. PROC. N. A. S. one genetic code for all living things, i.e., that the code is universal. A more direct answer to this important question should soon be forthcoming. Summary.-Evidence is presented in this paper for the assignment of triplet code letters (of unspecified sequence) to the amino acids alanine, asparagine, aspartic acid, glutamic acid, and methionine. Nineteen out of twenty amino acids are now accounted for. An experimentally determined code letter for glutamine is not yet available but one has been predicted from amino acid replacement data in nitrous acid mutants of tobacco mosaic virus. Three amino acids, including asparagine, leucine, and threonine, appear to have more than one letter, indicating that their code may be degenerate. All of the present code letters contain uridylic acid residues. However, non-uridylic code letters could not have been detected by our method and the possibility that degeneracy of the code may be more extensive than is apparent at present, to include several non-uridylic code letters, should be kept in mind. The experimental basis for the code letter assignments, made in this and previous papers of this series, is discussed in detail and the results are compared with recent data on amino acid replacements in nitrous acid mutants of tobacco mosaic virus. We are greatly indebted to Dr. A. Tsugita, Virus Laboratory, University of California, Berkeley, for the preprint of a paper in press and for making unpublished replacement data available to us. We are also much indebted to Dr. H. G. Wittmann, Max-Planck-Institut fur Biologie, Tubingen, Germany, and Dr. F. H. C. Crick, Institute of Molecular Biology, Cambridge, England, for preprints of papers in press. * Aided by grants from the National Institute of Arthritis and Metabolic Diseases (Grant A-1845) of the U.S. Public Health Service and from the Rockefeller Foundation. The abbreviations used in this paper are the same as in previous papers of this series. The standard abbreviations are used for amino acids. t International Postdoctoral Fellow of the National Institutes of Health, U.S. Public Health Service. Permanent address: Instituto de Quimica Fisiol6gica y Pat6logica, Universidad de ChileY Santiago, Chile. I Lengyel, P., J. F. Speyer, and S. Ochoa, these PROCEEDINGS, 47, 1936 (1961). 2 Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 63 (1962). 3 Lengyel P., J. F. Speyer, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 282 (1962). 4 Tsugita, A., Protein, Nucleic Acid, Enzyme (Tokyo), 6, 385 (1961). 6 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, 192, 1227 (1961). 6 Tsugita, A., personal communication. 7 Tsugita, A., and H. Fraenkel-Conrat, J. Mol. Biol., in press. 8 Wittmann, H. G., Naturwissenschaften, 48, 729 (1961). 9 See Table 3 for calculated frequency ratios of UUU to other triplets in the various polynucleotides used in this work. 10 Heppel, L. A., P. J. Ortiz, and S. Ochoa, J. Biol. Chem., 229, 695 (1957). " Ortiz, P. J., and S. Ochoa, J. Biol. Chem., 234, 1208 (1959). 12 We are indebted to Dr. Mary L. Stephenson, Massachusetts General Hospital, for a sample of leucine-c14 prepared by Dr. R. B. Loftfield. 13 Martin, R. G., J. H. Matthaei, 0. W. Jones, and M. W. Nirenberg, Biochem. Biophys. Res. Comm., 6, 410 (1962). 14 Smith, E. L., these PROCEEDINGS (to appear, no. 4, 1962).