,4c/,4mole, prepared by ammonolysis of aspartyl phosphate-c14 obtained enzymatically

Transcription

1 SYNTHETIC POLYNUCLEOTIDES AND THE AMINO ACID CODE, IX* BY ALBERT.J. WAHBA,t ROBERT S. MILLER, CARLOS BASILIO,t ROBERT S. GARDNER, PETER LENGYEL, AND JOSEPH F. SPEYER DEPARTMENT OF BIOCHEMISTRY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated by Severo Ochoa, Ma&, 1, 1963 Previous work led to the assignment of 41 out of 64 code triplets to the twenty amino acids.1'2 These results indicated extensive degeneracy of the amino acid code. Since it appeared that the triplet list was still incomplete, further work was carried out with polynucleotides (poly AC, UC, and UAC) varying widely in base composition. The results, reported in this paper, add 7 new triplets to the list and bring the number of code triplets to date to 48. Additional triplets of the indicated base composition have been assigned to the following amino acids: glutamine, isoleucine, and tyrosine, lulaic each; leucine and serine, 1U2C each; phenylalanine, 2U1C; and threonine, 1A2C. The present results are of interest for several reasons. Firstly, the list of code triplets now includes all 27 triplets of A, C, and U. Hence, any "nonsense" triplets will have to contain G. Secondly, degeneracy in the code appeared to obey a certain pattern whereby the code triplets for a given amino acid shared one doublet.2 The new triplets fit into this pattern, for the number of "shared doublets" increased from 12 to 17. In addition to the assignment of new code triplets the experiments reported in this paper have confirmed a number of previous assignments. Moreover, the good agreement of the assignment of one and the same code triplet to a given amino acid, based on results with polymers containing the same bases in widely different proportions, greatly strengthens the validity of the assumptions made and the methods used in our studies of the amino acid code. Preparations and Methods.-The polymers used in this work were prepared as previously described.3 Glutamine-C'4 of specific activity about 150uc/gmole was prepared from glutamic acid-c14 with purified glutamine synthetase kindly supplied by Dr. A. Meister, Tufts University School of Medicine. Glutamine was separated from glutamic acid by high voltage electrophoresis at ph 6.0 in a buffer consisting of pyridine: acetic acid: water (10:1:69). Asparagine-C 4 of specific activity 75,4c/,4mole, prepared by ammonolysis of aspartyl phosphate-c14 obtained enzymatically from aspartic acid-c14, was provided by Dr. Y. Kaziro of this laboratory. Both amino acids were diluted to a specific radioactivity of 1Ouc/uAmole before use. The source of C 14-labeled amino acids was as previously given. ' They were used at specific radioactivities between 1 and 10,c/,4mole. The incubations were as previously described.3' 4 The precipitating reagents used to determine the incorporation of radioactive amino acids into protein-like products in experiments with the various polymers are described in a footnote to Table 1. The procedure with 20% trichloroacetic acid as the precipitating reagent [experiments with poly UC (4.3:5.7), UC (1:5), AC (1:5), and UAC (1:1:4], which deviated in some respects from previous practice with C-rich polymers,2 was as follows. After incubation, 1 mg of carrier polyproline (Mann Research Laboratories, Inc., N. Y.) in 0.5 ml of water, and 10 ml of 20% trichloroacetic acid 880

2 VOL. 49, 1963 BIOCHEMISTRY: WAHBA ET AL. 881 were added to each tube. The pre- f cipitate was collected by centrifuga- I tion, suspended in 5 ml of 20% trichloroacetic acid, heated in a boiling waterbath for 15 min and the mixture > % cooled to room temperature. The precipitate, collected by centrifugation, ' E. :: was evenly suspended in 0.5 ml of t 4< N ammonium hydroxide, quantita- c tively transferred to a planchette, in a flow window counter. Results.-Effect of various polynucle- E _ f =r4 ~ 0 C -,vq otides on amino acid -. incorporation O Previous work with the E. coli system D had shown that UC copolymers pro- _ 03 -,O - t- r.i0 C moted the incorporation into acid-in- x : o O) c: V -O soluble products of only four amino.d* acids, namely, leucine, phenylalanine, 0 proline,andserine. Fromexperiments : :e ::i -.a with poly UC (5: 1), five of the eight triplets in this polynucleotide weree assigned as follows: leucine 2U1C, & :q C. :- phenylalanine 3U, proline 1U2C and 3C, and serine 2U1C. One 2U1C and > f o two 1U2C triplets remainedunassigned. o : 0 Lo The possibility that these triplets z ;D could be additional code letters for z as 0 some of the above amino acids was E- 0.`5D 60. tested with use of UC copolymers of v widely different base composition, S... LO namely, UC (5:1), UC (4.3:5.7), and ; v *; *: UC (1:5). The results of these exper- ko iments (Table 1) led to the assignment :: ' :,, ; E of the remaining triplets to leucine ; (1U2C), phenylalanine (2U1C) and 'O serine (1U2C). >0X X4 A 0 Of the eight triplets in AC copoly-... mers, seven had been assigned as a rz result of experiments",2 with 0 poly A, OD poly AC (5:1), and poly C, namely, lysine (3A), proline (3C), asparagine, glutamine, and threonine (2A1C each), and histidine and proline (1A2C each). One 1A2C triplet remained unassigned. A In order to see whether this was a meaningful triplet, experiments were & * S E

3 882 BIOCHEMISTRY: WAHBA ET AL. PROC. N. A. S. carried out with A-rich and C-rich AC copolymers, namely, poly AC (5:1) and AC (1: 5). The results (Table 1) led to the assignment of the remaining 1A2C triplet to threonine. Three of the six lulaic triplets were previously assigned to asparagine, histidine, and threonine (see Table 5 of reference given under footnote 2). The possibility that the remaining three triplets of this series might be letters of the amino acid code was investigated with C-rich and U-rich UAC copolymers, namely, poly UAC (1:1:4), which had not been previously used, and the previously used poly UAC (6:1:1). The results (Table 1) led to the assignment of one lulaic triplet each to glutamine, isoleucine, and tyrosine. Basis for additional code triplet assignments: The basis for the new assignments is the same as in previous papers. The data leading to these assignments are given in Tables 2, 3, and 4, for the UC, AC, and UAC copolymers, respectively. In most cases the stimulation of the incorporation of an amino acid by a certain polymer (last column of tables) is given as per cent of the stimulation of the amino acid whose incorporation is promoted to the greatest extent by this polymer. The amino acid in question is phenylalanine for U-rich polymers, proline for C-rich polymers, and lysine for A-rich polymers."1 2 The per cent incorporation of the amino acid is then TABLE 2 DATA FOR ADDITIONAL CODE TRIPLET ASSIGNMENTS (COPOLYMERS OF U AND C) - Calculated triplet frequency -- Sum of calculated Amino acid Amino acid 3U 2U1C lu2c 3C triplet frequencies incorporation Poly UC (5: 1) Leucine Phenylalanine Proline Serine ?3.6 Poly UC (4.7:5.3) Leucine Phenylalanine Proline Serine Poly UC (1:5) Leucine Phenylalanine Proline Serine TABLE 3 DATA FOR ADDITIONAL CODE TRIPLET ASSIGNMENTS (COPOLYMERS OF A AND C) --Calculated triplet frequency - Sum of calculated Amino acid Amino acid 3A 2A1C la2c 3C triplet frequencies incorporation Poly AC (5: 1) Asparagine Glutamine Histidine Lysine Proline Threonine Poly AC (1:5) 'Asparagine "Glutamine 'Histidine r Lysine VProline [ Threonine

4 VOL. 49, 1963 BIOCHEMISTRY: WAHBA ET AL. 883 matched to the calculated per cent frequency of IX one or several triplets relative to that of the triplet 0 00 or triplets (taken as 100) known to code for phenylalanine, proline, or lysine, as the case may be. The sum of the calculated relative frequencies of the pertinent triplets is given in the next to the last column of the tables. Matching of the values on the. Ad a. s s 0 same line of the last two columns is the basis for Ad a I triplet assignments. m UC triplets: Previous work with UC copolymers was carried out almost exclusively with poly UC f (5:1). Any effect of triplets other than those as-... signed to leucine (2U1C), phenylalanine (3U), and serine (2U1C), from experiments with this polymer, v > would be masked because of their lower frequency.., : Detection of such an effect should be possible, how- 4 ever, with use of poly UC (1:5). Table 2 shows C that this was indeed the case. The results with 0: 0 poly UC (4.3:5.7) are of interest. Since two triplets z out of the eight in the UC series can now be assigned z to each of four amino acids (leucine, phenylalanine, > > proline, serine), in the presence of this polymer, v :. which has a U: C ratio not far from unity, each of X the four amino acids should be incorporated approx- 4 0a imately to the same extent. As seen from Table 1, q cqn 9 ck c column 5, and from Table 2, this was essentially the v case. AC triplets: The data of Table 3 reveal that a z = new triplet (1A2C) may be assigned to threonine E-. from the results with a C-rich poly AC, namely, poly n v AC (1:5). The incorporation of threonine in the presence of this polymer (20.8% that of proline) was too high to be explained only in terms of its 2A1C triplet [2A1C previously assigned to threonine' : was confirmed in the present experiments with poly AC (5: 1) ], the frequency of which is only 3.3% of that of the two proline triplets (1A2C and 3C). v.) The remaining results in this table confirm and reinforce previously made assignments. UA C triplets: Experiments with the C-rich poly UAC (1: 1:4) (Tables 1 and 4) revealed the existence of two new lulaic triplets for isoleucine and tyrosine. As seen from Table 4 (last column) the incorporation of these two amino acids was 6.1 and X, 4.0% of that of proline, respectively. This is clearly.; = greater than the sum of calculated frequencies of E the previously detected 1U2A and 2U1A triplets for H H

5 884 BIOCHEMISTRY: WAHBA ET-AL. PROC. N. A. S isoleucine (2.0% of that of the proline triplets) and greater than the frequency of the previous 2U1A tyrosine triplet (1.0% of that of the proline triplets). Results with poly UAC (6:1:1) (Table 1) provide some basis for the assignment of a lulaic triplet to glutamine. This polymer promoted the incorporation of glutamine to the extent of 3.1% of that of phenylalanine. The polymer also contains one (2A1C) of the two triplets previously detected for glutamine.1 However, its frequency is only 0.4% of that of the combined (3U + 2U1C) phenylalanine triplets. The frequency of the lulaic triplet on the same basis is 2.4%. Therefore, the sum of the frequencies of these two triplets (0.4% %o = 2.8%) relative to that of those for phenylalanine closely matches the incorporation value (3.1%) of glutamine relative to that of phenylalanine. In view of the low incorporation, the lulaic assignment for glutamine is made only tentatively. Discussion and Conclusions.-The new triplets for glutamine, isoleucine, leucine, phenylalanine, serine, threonine, and tyrosine are listed in Table 5 along with the previously assigned ones. Table 6 lists the amino acids that can now be assigned to each of the twenty-seven triplets of the ACU series. TABLE 5 ADDITIONAL CODE TRIPLET ASSIGNMENTS Amino acid Additional code triplet Previously assigned code triplets Glutamine lulalc 2A1C, 1A2G Isoleucine lulalc 1U2A, 2U1A Leucine 1U2C 2U1A, 2U1C, 2U1G Phenylalanine 2U1C UUU Serine 1U2C 2U1C lalclg Threonine 1A2C lulaic, 2A1C, 2C1G Tyrosine lulalc 2U1A TABLE 6 SUMMARY OF CODE TRIPLETS OCCURRING IN COPOLYMERS OF A, C, AND U, AND OF AMINO ACIDS ASSIGNED TO THE TRIPLETS Triplet composition 3A 2A1C 2A1U 1A2C 1A1C1U 1A2U 3C 2C1U 1C2U 3U Number of triplets with this composition (Lys Asn Asn His Asn Ile Pro Leu Leu Phe Gln Ile Pro Gln Leu Pro Phe Amino acid Thr Leu Thr His Tyr Ser Ser Ile Thr Tyr Extensive degeneracy of the amino acid code and the indicatiolis of a uniform pattern of degeneracy, as suggested by the growing number of "shared doublets" referred to in the introduction, emerge as the most striking features of the code. Pronounced degeneracy reconciles the existence of a universal code with the wide divergence in base composition of the DNA of different organisms. It is conceivable that different organisms may utilize to a different extent the various triplets for a given amino acid according to the composition of their DNA. Recent experiments in this laboratory with a cell-free system from Alcaligenes faecalis (DNA with 66% GC) indicate that the amino acid code triplets thus far established are the same as for E. coli (DNA with 52% GC).5

6 VOL. 49, 1963 BIOCHEMISTRY: SOBELL, TOMITA, AND RICH 885 Note added in proof: The assignment of 1A2G as one of the glutamine triplets' has been made questionable by the finding that, in the presence of an excess of cold glutamic acid, there was little or no stimulation of the incorporation of glutamine by poly AG (5: 1). This suggests that the previously observed stimulation may have been due to the occurrence of deamidation of glutamine to glutamic acid, the incorporation of which is stimulated by this polymer. * 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 Jane Coffin Childs Fund for Medical Research. The abbreviations used in this paper are the same as in previous papers of this series. t Fellow of the Jane Coffin Childs Fund for Medical Research. t International Postdoctoral Fellow of the National Institutes of Health, U.S. Public Health Service. Permanent address: Instituto de Qufmica Fisiologica y Patol6gica, Universidad de Chile, Santiago, Chile. I Gardner, R. S., A. J. Wahba, C. Basilio, R. S. Miller, P. Lengyel, and J. F. Speyer, these PROCEEDINGS, 48, 2087 (1962). 2 Wahba, A. J., R. S. Gardner, C. Basilio, R. S. Miller, J. F. Speyer, and P. Lengyel, these PROCEEDINGS, 49, 116 (1963). 3 Lengyel, P., J. F. Speyer, and S. Ochoa, these PROCEEDINGS, 47, 1936 (1961). 4Speyer, J. F.. P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 441 (1962). 6 Protass, J., et al., to be published. THE CRYSTAL STRUCTURE OF AN INTERMOLECULAR COMPLEX CONTAINING A GUANINE AND A CYTOSINE DERIVATIVE* BY HENRY M. SOBELL,t KEN-ICHI TOMITA, AND ALEXANDER RICH DEPARTMENT OF BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY Communicated by William N. Lipscomb, April 19, 1963 The molecular basis for the specificity of the nucleic acids is believed to reside in the detailed system of hydrogen bonds formed between the purines and the pyrimidines. This bonding is specific in that adenine pairs to thymine and guanine to cytosine, thereby accounting for the complementarity which is found in the structure of DNA. X-ray diffraction studies have shown that DNA has a doublestranded helical structure in agreement with the hypothesis of Watson and Crick. However, it is not possible to determine the precise manner in which the purine and pyrimidine bases come together solely from the X-ray fiber diffraction pattern. This is because the sequence of bases in DNA is irregular, and therefore they do not give rise to the coherent X-ray scattering which is necessary to determine atomic coordinates of individual bases. Most of our knowledge concerning the molecular structure of purines and pyrimidines comes from X-ray diffraction studies of model compounds.2 These studies, however, have concentrated largely on the structure of individual molecules and this provides only indirect information regarding the assumptions concerning hydrogen bonding between different purines and pyrimidines. Here we present some results of a single crystal X-ray diffraction analysis in which we demonstrate that guanine and cytosine derivatives form a hydrogenbonded complex in the crystalline state. In Watson and Crick's original description of the DNA molecule, they postulated that the adenine-thymine pair, as well as the guanine-cytosine pair, were each held together by two hydrogen bonds. Three years later Pauling and Corey3 extensively