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RNA codons recognized by transfer RNA from amphibian embryos and adults
DEVELOPMENTAL
RNA
BIOLOGY
19,
l-11
(1969)
Codons Recognized by Transfer RNA Amphibian Embryos and Adults RICHARD
MARSHALL
AND MARSHALL
Laboratory of Biochemical Genetics, National Institutes of Health, Accepted
from
NIFXNBERG
National Heart Institute, Bethesda, Maryland
March 28, 1968
INTRODUCTION
Many studies indicate that the genetic code is largely universal. However, the fidelity of translation can be altered in viva by extragenie suppressors, and in vitro by altering components or conditions required for protein synthesis (for reviews, cf. Gorini and Beckwith, 1966; Nirenberg, 1965). Thus, cells sometimes may differ in specificity of codon translation. Theoretically, a change in tRNA1 concentration, structure, or function could differentially regulate rates of mRNA translation. To determine whether responses of AA-tRNA to synonym RNA codons change during embryonic development, codon recognition by AA-tRNA from amphibian embryos and adult liver were compared. Nucleotide sequences and template activities of RNA codons were determined by stimulating the binding to ribosomes of AA-tRNA from neurula of Xenopus laevis, the South African clawed toad, with trinucleotides of known sequence. The data were compared to responses of AA-tRNA from adult Xenopus Zaevis liver and muscle (Marshall et al., 1967) and Escherichia coli (Nirenberg et al., 1965; Still et al., 1965). Responses of neurula and adult liver AA-tRNA were found to be similar. However, both neurula and adult Xenopus AA-tRNA were *The following abbreviations and symbols are used: tRNA, transfer RNA; AA-tRNA, aminoacyl-tRNA; mRNA, messenger RNA; U, uracil; C, cytosine; A. adenine; G, guanine; ATP, adenosine triphosphate; UCA represents a trinucleoside diphosphate with S’-terminal hydroxyl attached to U and 2’, 3’-terminal hydroxyls attached to A ( UpCpA); codon, codeword; Arg-, arginine, Asp-, aspartic acid; Glu-, glutamic acid; Gly-, glycine; Ile-, isoleucine; Lys-, lysine; Met-, methionine; Phe-, phenylalanine; Ser-, serine; Thr-, threonine; Tyr-, tyrosine; Val-, valine-tRNA; A,,, absorbancy at 260 mp; DEAE, diethylaminoethylcellulose. @ 1969 by Academic
Press, Inc.
2
MARSHALL
shown to differ markedly to certain trinucleotides.
AND
NIRENBERG
from E. coli AA-tRNA
METHODS
AND
in relative response
MATERIALS
Adult specimens of Xenopus laevis were obtained from Mr. Jay Cook, Cockeysville, Maryland. Methods described by Brown and Littna (1964) were used to obtain breeding adults, eggs, and to rear the embryos to the neurula stage [stages 16-20 of Nieuwkoop and Faber ( 1956) 1. N eurula were treated with papain and then washed as described by Dawid (1965) to remove jelly and adherent bacteria. Part of the preparation was removed to assess the possible presence of bacteria; the remaining embryos then were frozen and stored in a liquid-nitrogen refrigerator until a sufficient quantity had been obtained for tRNA isolation. Bacterial contamination was assessed by macerating 5 gm of Xenopus neuda in 5 ml of water, and culturing portions on blood agar plates and in thioglycolate broth for 3 weeks at 4’C, 24°C and 37°C. Less than 500 bacteria were detected per 0.5 gm of neurula (wet weight). Hence, bacterial contaminants were insignificant. Transfer RNA was prepared from neurula embryos by the methods of Brunngraber (1962) and Brown and Littna ( 1964), slightly modified. A typical preparation was as follows: 50 gm of frozen neurula was thawed rapidly in a solution (150 ml) containing 75 ml of phenol saturated with H,O and 75 ml of 1 M NaC1 and homogenized for 2 minutes in a Waring blendor at 4°C. The suspension was shaken mechanically for 30 minutes and centrifuged at 15,060 g for 20 minutes at 24°C. The aqueous phase was removed and one-tenth volume of 20% potassium acetate and three volumes of absolute ethanol were added. The suspension was stored for 3 hours at -20°C; the precipitate then was collected by centrifugation and subsequently dissolved in 30 ml of 0.1 &I Tris-HCl, pH 7.5. Solutions of recrystallized sodium dodecyl sulfate and MgCI, were added so that the final concentrations were 0.5% sodium dodecyl sulfate and 0.03 M MgCI,. Treatment with sodium dodecyl sulfate and MgCl, removes components from tRNA preparations which precipitate during the assay of AA-tRNA binding to ribosomes. Thirty-five milliliters of phenol saturated with H,O was added; the suspension was shaken mechanically for 20 minutes and then centrifuged for 20 minutes at 20,000 g at 24°C. The aqueous
EMBRYOSIC
AND
ADULT
AMPHIBIAN
TRANSFER
RNA
3
layer was aspirated and diluted lo-fold with 0.1 M Tris-HCI, pH 7.5, and applied to a 2-ml column of DEAE (chloride form) which had previously been washed with 0.1 h1 Tris-HCI, pH 7.5. Transfer RNA was eluted with 1.0 M NaCl in 0.1 hl Tris-HCl, pH 7.5 and then precipitated with one-tenth volume of 20% potassium acetate and 3 volumes of absolute ethanol. The suspension was stored overnight at -20°C. The precipitate was collected by centrifugation, dissolved in H,O, and stored in a liquid nitrogen refrigerator. j units) of tRNA were obtained from 50 Approximately 3 mg (72 A 2,0 gm of Xenopus neurula, wet weight. A solution containing 1.0 mg of RNA per milliliter of H,O was assumed to be equivalent to 24 A,,, units in a cuvette with a l-cm light path, with the use of a Zeiss spectrophotometer. Aminoacyl-tRNA synthetases were prepared from Xenopus neurula or adult Xenopus liver by the method of Moldave (1963) modified as described previously ( Marshall et nl., 1967). Aminoacyl-tRNA synthetases from neurula were used for the acylation of neurula tRNA; AA-tRNA synthetases from adult liver were used for the acylation of adult liver tRNA. Each tRNA preparation was acylated with one radioactive and 19 unlabeled amino acids. Trinucleotide synthesis, isolation, purity, and nucleotide sequence analyses are described elsewhere (Leder et nl., 1965; Bernfield 1965, 1966; Nirenberg and Leder, 1964; Bernfield and Nirenberg, 1965). The presence of an ultraviolet-absorbing contaminant comprising 2% of the total would be detected by the methods employed. An unidentified contaminant (10%) was found in the GGU preparation; no contaminants were detected in other trinucleotide preparations. Formation of AA-tRNA-ribosomes-codon complexes were assayed as described previously (Nirenberg and Leder, 1964). Each reaction contained the following in a final volume of 55 ~1: 0.05 h1 Tris-acetate, pH 7.2; 0.05 hl potassium acetate; 0.02 hl magnesium acetate unless otherwise indicated): E. coli \$73100 ribosomes, washed three times (Nirenberg, 1963); “H- or ‘“C-labeled AA-tRNA, as indicated in Table 1; and oligonucleotides and trinucleotides as specified. Ribosomes were washed on Millipore filters and dried; radioactivity was determined in a liquid-scintillation counter (Packard Instrument Company) with a counting efficiency of 70-80% for carbon-14 and lO-15% for tritium. All assays were performed in duplicate.
4
MARSHALL
RESULTS
AND
AND
NIRENBERG
DISCUSSION
Amounts of AA-tRNA and ribosomes added to reactions are shown in Table 1. Aminoacyl-tRNA synthetase preparations from Xenopus neurula and adult Xenopus liver were used to catalyze the acylation of tRNA from neurula and adult liver, respectively. Escherichia coli ribosomes were used for binding studies so that responses of AA-tRNA to trinucleotide codons could be investigated under uniform conditions. Xenopus neurula and adult liver AA-tRNA preparations appear to bind to E. coli ribosomes as efficiently as E. coli AA-tRNA. TABLE AA-tRNA
AND
RIBOSOMES
1 ADDED
Components
TO REACTIONS added to each reaction
W- or aH-AA-tRNA Radioactivea amino acid
Am units
K-or ,H-AA (~WmJles)
QH-Argb t’4GAspc
0.16 0.14
5.5
1.5
4.9
2.0
DL-~-~H-G~u~
0.29
2-3H-Glyb +C-Ilec DL-&~-~H-L~s~ I.-W-Metb 9H-Phec QH-Serd L-W-Thrb 1,-3,5-~H-Tyr~ +C-Vap
0.34 0.25 0.10 0.10 0.10 0.24 0.27 0.15 0.12
1.0 7.5 2.9 2.5 4.3 1.4 7.2 5.4 1.6 4.6
2.5 2.5 2.0 0.25 2.0 2.0 2.0 2.0 1.5 2.0
E. c~Ii~mix”s”es,
2
a All isotopes uniformly labeled except where specified. b Nuclear Chicago Corporation. c New England Nuclear Corporation. d Schwarz BioResearch Corporation.
Trinucleotide specificity and activity in stimulating binding of Xenopus neurula AA-tRNA to ribosomes is shown in Table 2. For comparative purposes, previously reported responses of adult Xenopus liver AA-tRNA (Marshall et al., 1967) and E. coli AA-tRNA - (Nirenberg et al., 1965; So11et al., 1965) are shown also. Relative responses of AA-tRNA to synonym codons can be compared by reading down rather than across columns in Table 2. Usually it is more convenient to compare the relative responses of a preparation of AA-tRNA to synonym codons with those of another AA-tRNA preparation rather
TABLE TEMPLATE
SPECIFICITY
2
OF TRINUCLEOTIDES
FOR
AiwxoAcYL-tItNh”
14c- OR 3H-LABELED
A prrmoles W- or 3H-AA-tRNA Addition
Arg-tRNA (378 cpm//Lpmole) CGU CGC CGA CGG AGG AGA UGG None (ppmoles) Ile-tRNA (363 cpm/Fpmole) AUU AT’C AUh UUA, L4A, UAG iSone (ppmoles) Lys-tRPTA (1035 cpm/ppmole) AAA A4G None &moles) Met-tRNA (318 cpm/ppmole) ,4UG GCG UUG CUG AUA None (ppmoles) Ser-tKNA (180 cpm/ppmole) UCIJ UCC UCA UCG AGU AGC UUU None (ppmoles) Thr-tRNA (202 cpm/ppmole) ACU ACC ACA ACG uu1: Sone (ppmoles)
Xenopus lV3UUh
0.01 ivi Mgz+ 0.35 0.20 0.86 0.45 0.95 0.12 -0.09 (0.97) 0.76 0.26 0.07 (Do74) 0.01 M Mg2+ 0.38 0.4i (0.52) 0.01 ilf Mg*+ 3.07 0.36 0.10 0.05 0.02 (0.29)
bound to ribosomes~
AdultXmpus E. coli
0.01 1vf hlgz+ 0.6’2 0.27 1.10 0.60 1.25 0.02
0.02 M Mg2+ 0.90 0.47 1.09 0.20 0.10 0.12
(1. ‘70)
(1<7)
1.15 0.51 0.34
0.64 0.73 -0.01 -
(0.15) (0.59) 0.01 M Mg”+ 0.01 M Rlgz+ 1.00 0.87 0.62 0.07 (1.20) (0.70) 0.02 M ?r1gn+ 0.02 M R3g2+ 2.24 1.00 O.Sl 0.65 0.23 0.41 0.09 0.15 -0.02 -0.02 (0.26) (0.41)
1.84 0.16 1.30 0.24 0.90 0.86 0.03
2.46 0.48 2.27 0.36 1.40 1.29 -
1.27 0.54 1.56 1.09 0.21 0.26
(1.28)
(1.75)
(013,
2.67 1.77 2.55 1.03 o.oi (0 76)
2 9s 2.07 2.67 1.36 (0.66)
0.91 0.50 0.45 1.10
5
(0 63)
6
MARSHALL
TABLE
AND
NIRENBEBG
2 (Continued) a ppmoles IT- or 3H-AA-tRNA
Addition
Val-tRNA (309 cpm/ppmole) GUU GUC GUA GUG uuu None (Kpmoles) Asp-tRNA (261 cpm/MMmole) GAU GAC GAA GAG None (ppmoles) Glu-tRN.4 (1380 cpm/ppmole) GAU GAC GAA GAG None (ppmoles) Gly-tRNA (254 cpm/ppmole) GGU GGC GGA AGG None (/*/*moles) Phe-tRNB (1309 cpm/ppmole) UUTJ uuc UUA None (pfimoles) Tyr-tRNA (1040 cpm/ppmole) UAU UAC UAiz UAG None &moles) a The effects of trinucleotides to h’scherichia coli ribosomes. Xenopus liver (Marshall et al., et al., 1966; Still et al., 1965; components described under
bound to ribosomesh
Adult Xenopus liver
1.10 0.39 1.00 1.16 -0.02 (0.41)
0.89 0.40 1.10 0.96
E. coli
1.00 0.75 1.33 1.08 -
(0<3)
(0.30)
1.81 1.53 0.11 0.04 (0.17)
1.62 1 .52 0.11 0.03 (0.28)
1.29 1.32 0.01 0.02 (0.18)
0.03 0.03 0.22 0.06 (0.04)
0.09 0.03 0.69 0.56 (0.07)
0.05 0 0.30 0.46 (0.12)
0.33 0.88 -
1.28 2.46 -
0.69 1.48 3.38 -0.03 (1.02) 0.45 0.55 0.04 (0.27)
(0.42)
(3
.20)
0.83 0.75
1.29 1.59 -
(0.32)
(0 48)
0.18 0.13
0.81 0.56 -
(0<7)
(0<3)
upon the binding of AA-tRNA from Xenopus neurula For comparative purposes, previous results with 1967) and E. coli AA-tRNA are shown also (Nirenberg Brimacombe et al., 1965). Reactions contained the Materials and Methods and ribosomes and W- or
EMBRYOSIC
AND
ADULT
AMPHIBIAN
TRASSFER
RSA
7
than the ,+moles of each AA-tRNA preparation bound to ribosomes. Relative responses remain fairly constant under a variety of conditions. Both AA-tRNA and deacylated tRKA bind to ribosomes equally in response to codons (Nirenberg and Leder, 1964); therefore, the amount (ppmoles) of AA-tRNA bound to ribosomes depends upon the extent of acylation of tRNA. The extent of acylation of tRNA was shown in preliminary experiments to be limited only by the amount of tRNA present. However, some AA-tRNA’s are more stable than others, so AA-tRNA preparations are not equally acylated. Hence, relative responses of AA-tRNA to synonym codons can be compared with greater reproducibility than ,+moles of AA-tRNA bound to ribosomes. Similar nucleotide sequence-amino acid translations were obtained with AA-tRNA from each organism. Relative responses of Xenqus neurula AA-tRNA preparations to synonym trinucleotides resemble those obtained with adult liver AA-tRNA. However, relative responses of Xenopzts and E. coli AA-tRNA to certain trinucleotides were found to differ ( Marshall et al., 1967). For example, Arg-tRNA from Xenops neurula, adult liver, and E. coli bind to ribosomes in response to CGU, CGC, CGA, and CGG. However, the relative responses of amphibian Arg-tRNA to AGG and CGG are high compared to that of E. coli Arg-tRNA. AUU and AUC serve as active Ile-codons with tRNA from each organism. In addition, AUA stimulates binding of Ile-tRNA from liver and, to a lesser extent, Ile-tRNA from neurula; whereas, E. coli IletRNA does not respond detectably to AUA. Differences also are seen with Lys-tRNA. In reactions containing 0.01 Al Mg”, AAA and AAG stimulate binding to ribosomes of tRNA from Xenopus neurula and adult liver; under identical conditions, E. coli Lys-tRNA responds to AAA but only slightly to AAG. At 0.02 M Mg”, a more marked response of E. coli Lys-tRNA to AAG was observed. Codon recognition by Met-tRNA is of particular interest, for 3H-AA-tRSA as reported in Table 1. Reactions contained 0.150 i- 0.010 Az6,, unit,s of trinucleotide. Unless otherwise indirated, reactions for Senopzts Ar\-tRNh contained 0.0’2 JI Mg’+ and reactions for E. coli AA-tRKA contained 0.03 M &I@+. b A ppmoles was obtained by subtracting W- or 3H-AA-tRX\‘h bound to ribosomes without trinucleotides from that bound wit,h trinucleotides. The number of MMmoles of W-or 3H-A.k-tRXA bound to ribosomes in the absence of trinucleotides is enclosed within parentheses.
8
MARSHALL
AND
N~RENI~ERG
N-formyl-Met-tRNA from E. coli may serve as an initiator of protein synthesis and, by selecting the first codon, may phase the reading of subsequent codons (Marcker and Sanger, 1964; Adams and Capecchi, 1966; Webster et al.,1966; Clark and Marcker, 1966; Sunderarajan and Thach, 1966; Nakamoto and Kolakofsky, 1966). Two fractions of Met-tRNA from E. coli have been separated by countercurrent distribution. The major peak of Met-tRNA can be converted to N-formylMet-tRNA and binds to ribosomes in response to AUG, GUG, and UUG; whereas the smaller peak of Met-tRNA does not accept formyl moieties and responds primarily to AUG (Clark and Marcker, 1966; Kellogg et al., 1966). Xenopus Met-tRNA responds well to AUG; relatively small responses to GUG and UUG were observed compared to those found with E. coZi Met-tRNA. Ser-tRNA preparations from Xenopus neurula, adult liver, and E. COG respond to UCU, UCC, UCA, UCG, AGU, and AGC. However, AGU and AGC were relatively more effective templates for Xenopus Ser-tRNA than for E. coli Ser-tRNA. Responses of amphibian Ser-tRNA to UCG were low compared to those found with E. coli Ser-tRNA. ACU, ACC, ACA, and ACG stimulate bacterial and amphibian Thr-tRNA binding to ribosomes; however, ACG is somewhat less active a template with Xenopus Thr-tRNA than with E. coli Thr-tRNA. ValtRNA preparations from each source respond similarly to GUU, GUC, GUA, and GUG. Responses of AA-tRNA from Xenopus neurula, liver and E. coli to the following codons were qualitatively similar: GAU and GAC, GAA and GAG, for glutamic acid; corresponding to aspartic acid; GGU, GGC, and GGA for glycine (Gly-codon GGG not tested); UUU and UUC, phenylalanine; and UAU and UAC, tyrosine. Recent results with purified AA-tRNA fractions show that mammalian and E. coli AA-tRNA preparations differ in the number and relative abundance of AA-tRNA species and also in codon-anticodon relationships corresponding to certain amino acids (Caskey et al., 1967). It should be emphasized that results obtained with fractionated species of AA-tRNA show that the amount of binding of unfractionated AA-tRNA to ribosomes usually is a function of the concentration of the corresponding species of AA-tRNA. Although no qualitative difference was detected between embryo and adult AA-tRNA for the twelve amino acids studied, quantitative
EMBRYONIC
AND
ADULT
AMPHIBIAN
TRANSFER
RNA
9
differences in response to certain codons were found. For example, differences were found between the responses of embryo and adult Ile-tRNA to AUA, and Glu-tRNA to GAG. However, further study is required to determine whether such differences are biologically significant. It should also be emphasized that the apparent absence of gross qualitative differences between embryonic and adult AA-tRNA by no means precludes the possibility that embryonic differentiation may be dependent upon changes in the codon recognition apparatus which may alter the rate of translation of certain codons. SUMMARY
Nucleotide sequences of thirty-seven RNA codons recognized by aminoacyl-transfer RNA from amphibian embryos were determined and were compared with codons recognized by transfer RNA from adult Xenopus liver and from Escherichia coli. The correspondence between RNA codons and amino acids, determined with aminoacyltRNA from embryos and adults, does not differ grossly for the twelve amino acids examined. However, both Xenopus embryo and adult aminoacyl-tRNA differ from E. coli aminoacyl-tRNA in relative response to certain synonym trinucleotides. It is a pleasure to thank Dr. Donald Brown for sharing his knowledge of Xenopus development with us throughout the course of this study. We are indebted to Theresa Caryk and Norma Zabriskie for excellent technical assistance. REFERENCES ADAMS, J., and CAPECCHI, M. ( 1966). N-Formylmethionyl-sRNA as the initiator of protein synthesis. PTOC. Natl. Acad. Sci. U.S. 55, 147-155. BERNFIELD, M. ( 1965 ). Ribonuclease and oligoribonucleotide synthesis. Synthetic activity of bovine pancreatic ribonuclease derivatives. J. Biol. Chem. 240, 4753-4762. Ribonuclease and oligoribonucleotide synthesis. II. BERNFIELD, 11. ( 1966). Synthesis of oligonucleotides of specific sequence. J. Biol. Chem. 241, 2014-2023. BERNFIELD, M., and NIRENBERG, M. (1965). RNA codewords and protein synthesis: The nucleotide sequences of multiple codewords for phenylalanine, serine, leucine, and proline. Science 147, 479-484. BmMAcohmE, R., TRUPIN, J., NIRENBERG, M., LEDER, P., BERNFIELD, ht., and JAOUNI, T. (1965). RNA codewords and protein synthesis. VIII. Nucleotide sequences of synonym codons for arginine, valine, cysteine, and alanine. Proc. Natl. Acad. Sci. U.S. 54, 954-960.
10
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AND
NIRFJBERG
D., and LITTNA, E. (1964). RNA synthesis during the development of Xenopus kzeuis, the South African clawed toad. J. Mol. Biol. 8, 669-687. BROWN, D. and LITTNA, E. (1966). Synthesis and accumulation of low molecular weight RNA during embryogenesis of Xenopus Laevis. J. Mol. Biol. 20, 95-112. BRUNNGRABER, E. (1962). A simplified procedure for the preparation of soluble RNA from rat liver. Biochem. Biophys. Res. Commun. 8, l-3. CASKEY, C. T., BEAUDET, A., WILCOX, M., and NIRENBERG, M. (1967). Differences in RNA codon recognition as a function of cellular tRNA content. Federation PTOC. 26, 349. CLARK, B., and MARCKER, K. ( 1966). N-Formyl-methionyl-sribonucleic acid and chain initiation in protein synthesis: Polypeptide synthesis directed by a bacteriophage libonucleic acid in a cell-free system. Nature 211, 378-380. DAWID, I. (1965). Deoxyribonucleic acid in amphibian eggs. J. Mol. Biol. 12, 581-599. GORINI, L., and BECKWITH, J. (1966). Supp ression. Ann. Rev. Microbial. 20, 401422. KELLOGG, D., DOCTOR, B., LOEBEL, J., and NIRENBERG, M. (1966). RNA codons and protein synthesis. IX. Synonym codon recognition by multiple species of valine-, alanine-, and methionine-sRNA. Proc. Natl. Acad. Sci. U.S. 55, 912-919. LEDER, P., SINGER, M., and BRIMACOMBE, R. (1965). Synthesis of trinucleoside diphosphates with polynucleotide phosphorylase. Biochemistry 4, 1561-1567. MARCKER, K. and SANGER, F. (1964). N-Formyl-methionyl-sRNA. J. Mol. Biol. 8, 835940. MARSHALL, R., CASKEY, C. T., and NIRENBERG, M. (1967). Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA. Science 155, 820-826. MOLDAVE, K. ( 1963). The preparation of C4-aminoacyl soluble-RNA. Methods Enzymol. 6, 757-761. NAKAMOTO, T., and KOLAKOFSKY, D. ( 1966). A possible mechanism for initiation of protein synthesis. PTOC. Natl. Acad. Sci. U.S. 55, 606-613. NIE~~KOOP, P. P., and FABER, J. (1956). “Normal Table of Xenopus Laevis ( Daudin) .” North-Holland Publ., Amsterdam. NIRENBERG, M. ( 1963). Cell-free protein synthesis directed by messenger RNA Methods Enzymol. 6, 17-23. NIRENBERG, M. ( 1965). Protein synthesis and the RNA code. Harvey Lectures Ser. 59, 155-185. NIRENBERG, M., and LEDER, P. (1964). RNA codewords and protein synthesis. I. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science 145, 1399-1407. NIRENBERG, M., LEDER, P., BERNFIELD, M., BRIMACOMBE, R., TRUPIN, J., ROTTMAN, F., and O'NEAL, C. (1965). RNA co d ewords and protein synthesis VII. On the general nature of the RNA code. Proc. Natl. Acad. Sci. U.S. 53, 11611168. SBLL, D., OHTSUKO, E., JONES, D., LOHRMANN, R., HAYATSU, H., NISHIMURA, S., and KHORANA, H. (1965). Studies on polynucleotides. XLIX. Stimulation of the binding of aminoacyl-sRNA’s to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids. PTOC. Natl. Acad. Sci. U.S. 54, 13781385. BROWN,
EMBRYONIC
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T., and THACH, R. ( 1966). Role of the formylmethionine codon AUG in phasing translation of synthetic messenger RNA. J. Mol. Biol. 19, 74-90. WEBSTER, R., ENGELHARDT, D., and ZINDER, N. ( 1966). In uitro protein synthesis; chain initiation. Proc. Natl. Acad. Sci. U.S. 55, 155-161. SUNDARARAJAN,
RNA
BIOLOGY
19,
l-11
(1969)
Codons Recognized by Transfer RNA Amphibian Embryos and Adults RICHARD
MARSHALL
AND MARSHALL
Laboratory of Biochemical Genetics, National Institutes of Health, Accepted
from
NIFXNBERG
National Heart Institute, Bethesda, Maryland
March 28, 1968
INTRODUCTION
Many studies indicate that the genetic code is largely universal. However, the fidelity of translation can be altered in viva by extragenie suppressors, and in vitro by altering components or conditions required for protein synthesis (for reviews, cf. Gorini and Beckwith, 1966; Nirenberg, 1965). Thus, cells sometimes may differ in specificity of codon translation. Theoretically, a change in tRNA1 concentration, structure, or function could differentially regulate rates of mRNA translation. To determine whether responses of AA-tRNA to synonym RNA codons change during embryonic development, codon recognition by AA-tRNA from amphibian embryos and adult liver were compared. Nucleotide sequences and template activities of RNA codons were determined by stimulating the binding to ribosomes of AA-tRNA from neurula of Xenopus laevis, the South African clawed toad, with trinucleotides of known sequence. The data were compared to responses of AA-tRNA from adult Xenopus Zaevis liver and muscle (Marshall et al., 1967) and Escherichia coli (Nirenberg et al., 1965; Still et al., 1965). Responses of neurula and adult liver AA-tRNA were found to be similar. However, both neurula and adult Xenopus AA-tRNA were *The following abbreviations and symbols are used: tRNA, transfer RNA; AA-tRNA, aminoacyl-tRNA; mRNA, messenger RNA; U, uracil; C, cytosine; A. adenine; G, guanine; ATP, adenosine triphosphate; UCA represents a trinucleoside diphosphate with S’-terminal hydroxyl attached to U and 2’, 3’-terminal hydroxyls attached to A ( UpCpA); codon, codeword; Arg-, arginine, Asp-, aspartic acid; Glu-, glutamic acid; Gly-, glycine; Ile-, isoleucine; Lys-, lysine; Met-, methionine; Phe-, phenylalanine; Ser-, serine; Thr-, threonine; Tyr-, tyrosine; Val-, valine-tRNA; A,,, absorbancy at 260 mp; DEAE, diethylaminoethylcellulose. @ 1969 by Academic
Press, Inc.
2
MARSHALL
shown to differ markedly to certain trinucleotides.
AND
NIRENBERG
from E. coli AA-tRNA
METHODS
AND
in relative response
MATERIALS
Adult specimens of Xenopus laevis were obtained from Mr. Jay Cook, Cockeysville, Maryland. Methods described by Brown and Littna (1964) were used to obtain breeding adults, eggs, and to rear the embryos to the neurula stage [stages 16-20 of Nieuwkoop and Faber ( 1956) 1. N eurula were treated with papain and then washed as described by Dawid (1965) to remove jelly and adherent bacteria. Part of the preparation was removed to assess the possible presence of bacteria; the remaining embryos then were frozen and stored in a liquid-nitrogen refrigerator until a sufficient quantity had been obtained for tRNA isolation. Bacterial contamination was assessed by macerating 5 gm of Xenopus neuda in 5 ml of water, and culturing portions on blood agar plates and in thioglycolate broth for 3 weeks at 4’C, 24°C and 37°C. Less than 500 bacteria were detected per 0.5 gm of neurula (wet weight). Hence, bacterial contaminants were insignificant. Transfer RNA was prepared from neurula embryos by the methods of Brunngraber (1962) and Brown and Littna ( 1964), slightly modified. A typical preparation was as follows: 50 gm of frozen neurula was thawed rapidly in a solution (150 ml) containing 75 ml of phenol saturated with H,O and 75 ml of 1 M NaC1 and homogenized for 2 minutes in a Waring blendor at 4°C. The suspension was shaken mechanically for 30 minutes and centrifuged at 15,060 g for 20 minutes at 24°C. The aqueous phase was removed and one-tenth volume of 20% potassium acetate and three volumes of absolute ethanol were added. The suspension was stored for 3 hours at -20°C; the precipitate then was collected by centrifugation and subsequently dissolved in 30 ml of 0.1 &I Tris-HCl, pH 7.5. Solutions of recrystallized sodium dodecyl sulfate and MgCI, were added so that the final concentrations were 0.5% sodium dodecyl sulfate and 0.03 M MgCI,. Treatment with sodium dodecyl sulfate and MgCl, removes components from tRNA preparations which precipitate during the assay of AA-tRNA binding to ribosomes. Thirty-five milliliters of phenol saturated with H,O was added; the suspension was shaken mechanically for 20 minutes and then centrifuged for 20 minutes at 20,000 g at 24°C. The aqueous
EMBRYOSIC
AND
ADULT
AMPHIBIAN
TRANSFER
RNA
3
layer was aspirated and diluted lo-fold with 0.1 M Tris-HCI, pH 7.5, and applied to a 2-ml column of DEAE (chloride form) which had previously been washed with 0.1 h1 Tris-HCI, pH 7.5. Transfer RNA was eluted with 1.0 M NaCl in 0.1 hl Tris-HCl, pH 7.5 and then precipitated with one-tenth volume of 20% potassium acetate and 3 volumes of absolute ethanol. The suspension was stored overnight at -20°C. The precipitate was collected by centrifugation, dissolved in H,O, and stored in a liquid nitrogen refrigerator. j units) of tRNA were obtained from 50 Approximately 3 mg (72 A 2,0 gm of Xenopus neurula, wet weight. A solution containing 1.0 mg of RNA per milliliter of H,O was assumed to be equivalent to 24 A,,, units in a cuvette with a l-cm light path, with the use of a Zeiss spectrophotometer. Aminoacyl-tRNA synthetases were prepared from Xenopus neurula or adult Xenopus liver by the method of Moldave (1963) modified as described previously ( Marshall et nl., 1967). Aminoacyl-tRNA synthetases from neurula were used for the acylation of neurula tRNA; AA-tRNA synthetases from adult liver were used for the acylation of adult liver tRNA. Each tRNA preparation was acylated with one radioactive and 19 unlabeled amino acids. Trinucleotide synthesis, isolation, purity, and nucleotide sequence analyses are described elsewhere (Leder et nl., 1965; Bernfield 1965, 1966; Nirenberg and Leder, 1964; Bernfield and Nirenberg, 1965). The presence of an ultraviolet-absorbing contaminant comprising 2% of the total would be detected by the methods employed. An unidentified contaminant (10%) was found in the GGU preparation; no contaminants were detected in other trinucleotide preparations. Formation of AA-tRNA-ribosomes-codon complexes were assayed as described previously (Nirenberg and Leder, 1964). Each reaction contained the following in a final volume of 55 ~1: 0.05 h1 Tris-acetate, pH 7.2; 0.05 hl potassium acetate; 0.02 hl magnesium acetate unless otherwise indicated): E. coli \$73100 ribosomes, washed three times (Nirenberg, 1963); “H- or ‘“C-labeled AA-tRNA, as indicated in Table 1; and oligonucleotides and trinucleotides as specified. Ribosomes were washed on Millipore filters and dried; radioactivity was determined in a liquid-scintillation counter (Packard Instrument Company) with a counting efficiency of 70-80% for carbon-14 and lO-15% for tritium. All assays were performed in duplicate.
4
MARSHALL
RESULTS
AND
AND
NIRENBERG
DISCUSSION
Amounts of AA-tRNA and ribosomes added to reactions are shown in Table 1. Aminoacyl-tRNA synthetase preparations from Xenopus neurula and adult Xenopus liver were used to catalyze the acylation of tRNA from neurula and adult liver, respectively. Escherichia coli ribosomes were used for binding studies so that responses of AA-tRNA to trinucleotide codons could be investigated under uniform conditions. Xenopus neurula and adult liver AA-tRNA preparations appear to bind to E. coli ribosomes as efficiently as E. coli AA-tRNA. TABLE AA-tRNA
AND
RIBOSOMES
1 ADDED
Components
TO REACTIONS added to each reaction
W- or aH-AA-tRNA Radioactivea amino acid
Am units
K-or ,H-AA (~WmJles)
QH-Argb t’4GAspc
0.16 0.14
5.5
1.5
4.9
2.0
DL-~-~H-G~u~
0.29
2-3H-Glyb +C-Ilec DL-&~-~H-L~s~ I.-W-Metb 9H-Phec QH-Serd L-W-Thrb 1,-3,5-~H-Tyr~ +C-Vap
0.34 0.25 0.10 0.10 0.10 0.24 0.27 0.15 0.12
1.0 7.5 2.9 2.5 4.3 1.4 7.2 5.4 1.6 4.6
2.5 2.5 2.0 0.25 2.0 2.0 2.0 2.0 1.5 2.0
E. c~Ii~mix”s”es,
2
a All isotopes uniformly labeled except where specified. b Nuclear Chicago Corporation. c New England Nuclear Corporation. d Schwarz BioResearch Corporation.
Trinucleotide specificity and activity in stimulating binding of Xenopus neurula AA-tRNA to ribosomes is shown in Table 2. For comparative purposes, previously reported responses of adult Xenopus liver AA-tRNA (Marshall et al., 1967) and E. coli AA-tRNA - (Nirenberg et al., 1965; So11et al., 1965) are shown also. Relative responses of AA-tRNA to synonym codons can be compared by reading down rather than across columns in Table 2. Usually it is more convenient to compare the relative responses of a preparation of AA-tRNA to synonym codons with those of another AA-tRNA preparation rather
TABLE TEMPLATE
SPECIFICITY
2
OF TRINUCLEOTIDES
FOR
AiwxoAcYL-tItNh”
14c- OR 3H-LABELED
A prrmoles W- or 3H-AA-tRNA Addition
Arg-tRNA (378 cpm//Lpmole) CGU CGC CGA CGG AGG AGA UGG None (ppmoles) Ile-tRNA (363 cpm/Fpmole) AUU AT’C AUh UUA, L4A, UAG iSone (ppmoles) Lys-tRPTA (1035 cpm/ppmole) AAA A4G None &moles) Met-tRNA (318 cpm/ppmole) ,4UG GCG UUG CUG AUA None (ppmoles) Ser-tKNA (180 cpm/ppmole) UCIJ UCC UCA UCG AGU AGC UUU None (ppmoles) Thr-tRNA (202 cpm/ppmole) ACU ACC ACA ACG uu1: Sone (ppmoles)
Xenopus lV3UUh
0.01 ivi Mgz+ 0.35 0.20 0.86 0.45 0.95 0.12 -0.09 (0.97) 0.76 0.26 0.07 (Do74) 0.01 M Mg2+ 0.38 0.4i (0.52) 0.01 ilf Mg*+ 3.07 0.36 0.10 0.05 0.02 (0.29)
bound to ribosomes~
AdultXmpus E. coli
0.01 1vf hlgz+ 0.6’2 0.27 1.10 0.60 1.25 0.02
0.02 M Mg2+ 0.90 0.47 1.09 0.20 0.10 0.12
(1. ‘70)
(1<7)
1.15 0.51 0.34
0.64 0.73 -0.01 -
(0.15) (0.59) 0.01 M Mg”+ 0.01 M Rlgz+ 1.00 0.87 0.62 0.07 (1.20) (0.70) 0.02 M ?r1gn+ 0.02 M R3g2+ 2.24 1.00 O.Sl 0.65 0.23 0.41 0.09 0.15 -0.02 -0.02 (0.26) (0.41)
1.84 0.16 1.30 0.24 0.90 0.86 0.03
2.46 0.48 2.27 0.36 1.40 1.29 -
1.27 0.54 1.56 1.09 0.21 0.26
(1.28)
(1.75)
(013,
2.67 1.77 2.55 1.03 o.oi (0 76)
2 9s 2.07 2.67 1.36 (0.66)
0.91 0.50 0.45 1.10
5
(0 63)
6
MARSHALL
TABLE
AND
NIRENBEBG
2 (Continued) a ppmoles IT- or 3H-AA-tRNA
Addition
Val-tRNA (309 cpm/ppmole) GUU GUC GUA GUG uuu None (Kpmoles) Asp-tRNA (261 cpm/MMmole) GAU GAC GAA GAG None (ppmoles) Glu-tRN.4 (1380 cpm/ppmole) GAU GAC GAA GAG None (ppmoles) Gly-tRNA (254 cpm/ppmole) GGU GGC GGA AGG None (/*/*moles) Phe-tRNB (1309 cpm/ppmole) UUTJ uuc UUA None (pfimoles) Tyr-tRNA (1040 cpm/ppmole) UAU UAC UAiz UAG None &moles) a The effects of trinucleotides to h’scherichia coli ribosomes. Xenopus liver (Marshall et al., et al., 1966; Still et al., 1965; components described under
bound to ribosomesh
Adult Xenopus liver
1.10 0.39 1.00 1.16 -0.02 (0.41)
0.89 0.40 1.10 0.96
E. coli
1.00 0.75 1.33 1.08 -
(0<3)
(0.30)
1.81 1.53 0.11 0.04 (0.17)
1.62 1 .52 0.11 0.03 (0.28)
1.29 1.32 0.01 0.02 (0.18)
0.03 0.03 0.22 0.06 (0.04)
0.09 0.03 0.69 0.56 (0.07)
0.05 0 0.30 0.46 (0.12)
0.33 0.88 -
1.28 2.46 -
0.69 1.48 3.38 -0.03 (1.02) 0.45 0.55 0.04 (0.27)
(0.42)
(3
.20)
0.83 0.75
1.29 1.59 -
(0.32)
(0 48)
0.18 0.13
0.81 0.56 -
(0<7)
(0<3)
upon the binding of AA-tRNA from Xenopus neurula For comparative purposes, previous results with 1967) and E. coli AA-tRNA are shown also (Nirenberg Brimacombe et al., 1965). Reactions contained the Materials and Methods and ribosomes and W- or
EMBRYOSIC
AND
ADULT
AMPHIBIAN
TRASSFER
RSA
7
than the ,+moles of each AA-tRNA preparation bound to ribosomes. Relative responses remain fairly constant under a variety of conditions. Both AA-tRNA and deacylated tRKA bind to ribosomes equally in response to codons (Nirenberg and Leder, 1964); therefore, the amount (ppmoles) of AA-tRNA bound to ribosomes depends upon the extent of acylation of tRNA. The extent of acylation of tRNA was shown in preliminary experiments to be limited only by the amount of tRNA present. However, some AA-tRNA’s are more stable than others, so AA-tRNA preparations are not equally acylated. Hence, relative responses of AA-tRNA to synonym codons can be compared with greater reproducibility than ,+moles of AA-tRNA bound to ribosomes. Similar nucleotide sequence-amino acid translations were obtained with AA-tRNA from each organism. Relative responses of Xenqus neurula AA-tRNA preparations to synonym trinucleotides resemble those obtained with adult liver AA-tRNA. However, relative responses of Xenopzts and E. coli AA-tRNA to certain trinucleotides were found to differ ( Marshall et al., 1967). For example, Arg-tRNA from Xenops neurula, adult liver, and E. coli bind to ribosomes in response to CGU, CGC, CGA, and CGG. However, the relative responses of amphibian Arg-tRNA to AGG and CGG are high compared to that of E. coli Arg-tRNA. AUU and AUC serve as active Ile-codons with tRNA from each organism. In addition, AUA stimulates binding of Ile-tRNA from liver and, to a lesser extent, Ile-tRNA from neurula; whereas, E. coli IletRNA does not respond detectably to AUA. Differences also are seen with Lys-tRNA. In reactions containing 0.01 Al Mg”, AAA and AAG stimulate binding to ribosomes of tRNA from Xenopus neurula and adult liver; under identical conditions, E. coli Lys-tRNA responds to AAA but only slightly to AAG. At 0.02 M Mg”, a more marked response of E. coli Lys-tRNA to AAG was observed. Codon recognition by Met-tRNA is of particular interest, for 3H-AA-tRSA as reported in Table 1. Reactions contained 0.150 i- 0.010 Az6,, unit,s of trinucleotide. Unless otherwise indirated, reactions for Senopzts Ar\-tRNh contained 0.0’2 JI Mg’+ and reactions for E. coli AA-tRKA contained 0.03 M &I@+. b A ppmoles was obtained by subtracting W- or 3H-AA-tRX\‘h bound to ribosomes without trinucleotides from that bound wit,h trinucleotides. The number of MMmoles of W-or 3H-A.k-tRXA bound to ribosomes in the absence of trinucleotides is enclosed within parentheses.
8
MARSHALL
AND
N~RENI~ERG
N-formyl-Met-tRNA from E. coli may serve as an initiator of protein synthesis and, by selecting the first codon, may phase the reading of subsequent codons (Marcker and Sanger, 1964; Adams and Capecchi, 1966; Webster et al.,1966; Clark and Marcker, 1966; Sunderarajan and Thach, 1966; Nakamoto and Kolakofsky, 1966). Two fractions of Met-tRNA from E. coli have been separated by countercurrent distribution. The major peak of Met-tRNA can be converted to N-formylMet-tRNA and binds to ribosomes in response to AUG, GUG, and UUG; whereas the smaller peak of Met-tRNA does not accept formyl moieties and responds primarily to AUG (Clark and Marcker, 1966; Kellogg et al., 1966). Xenopus Met-tRNA responds well to AUG; relatively small responses to GUG and UUG were observed compared to those found with E. coZi Met-tRNA. Ser-tRNA preparations from Xenopus neurula, adult liver, and E. COG respond to UCU, UCC, UCA, UCG, AGU, and AGC. However, AGU and AGC were relatively more effective templates for Xenopus Ser-tRNA than for E. coli Ser-tRNA. Responses of amphibian Ser-tRNA to UCG were low compared to those found with E. coli Ser-tRNA. ACU, ACC, ACA, and ACG stimulate bacterial and amphibian Thr-tRNA binding to ribosomes; however, ACG is somewhat less active a template with Xenopus Thr-tRNA than with E. coli Thr-tRNA. ValtRNA preparations from each source respond similarly to GUU, GUC, GUA, and GUG. Responses of AA-tRNA from Xenopus neurula, liver and E. coli to the following codons were qualitatively similar: GAU and GAC, GAA and GAG, for glutamic acid; corresponding to aspartic acid; GGU, GGC, and GGA for glycine (Gly-codon GGG not tested); UUU and UUC, phenylalanine; and UAU and UAC, tyrosine. Recent results with purified AA-tRNA fractions show that mammalian and E. coli AA-tRNA preparations differ in the number and relative abundance of AA-tRNA species and also in codon-anticodon relationships corresponding to certain amino acids (Caskey et al., 1967). It should be emphasized that results obtained with fractionated species of AA-tRNA show that the amount of binding of unfractionated AA-tRNA to ribosomes usually is a function of the concentration of the corresponding species of AA-tRNA. Although no qualitative difference was detected between embryo and adult AA-tRNA for the twelve amino acids studied, quantitative
EMBRYONIC
AND
ADULT
AMPHIBIAN
TRANSFER
RNA
9
differences in response to certain codons were found. For example, differences were found between the responses of embryo and adult Ile-tRNA to AUA, and Glu-tRNA to GAG. However, further study is required to determine whether such differences are biologically significant. It should also be emphasized that the apparent absence of gross qualitative differences between embryonic and adult AA-tRNA by no means precludes the possibility that embryonic differentiation may be dependent upon changes in the codon recognition apparatus which may alter the rate of translation of certain codons. SUMMARY
Nucleotide sequences of thirty-seven RNA codons recognized by aminoacyl-transfer RNA from amphibian embryos were determined and were compared with codons recognized by transfer RNA from adult Xenopus liver and from Escherichia coli. The correspondence between RNA codons and amino acids, determined with aminoacyltRNA from embryos and adults, does not differ grossly for the twelve amino acids examined. However, both Xenopus embryo and adult aminoacyl-tRNA differ from E. coli aminoacyl-tRNA in relative response to certain synonym trinucleotides. It is a pleasure to thank Dr. Donald Brown for sharing his knowledge of Xenopus development with us throughout the course of this study. We are indebted to Theresa Caryk and Norma Zabriskie for excellent technical assistance. REFERENCES ADAMS, J., and CAPECCHI, M. ( 1966). N-Formylmethionyl-sRNA as the initiator of protein synthesis. PTOC. Natl. Acad. Sci. U.S. 55, 147-155. BERNFIELD, M. ( 1965 ). Ribonuclease and oligoribonucleotide synthesis. Synthetic activity of bovine pancreatic ribonuclease derivatives. J. Biol. Chem. 240, 4753-4762. Ribonuclease and oligoribonucleotide synthesis. II. BERNFIELD, 11. ( 1966). Synthesis of oligonucleotides of specific sequence. J. Biol. Chem. 241, 2014-2023. BERNFIELD, M., and NIRENBERG, M. (1965). RNA codewords and protein synthesis: The nucleotide sequences of multiple codewords for phenylalanine, serine, leucine, and proline. Science 147, 479-484. BmMAcohmE, R., TRUPIN, J., NIRENBERG, M., LEDER, P., BERNFIELD, ht., and JAOUNI, T. (1965). RNA codewords and protein synthesis. VIII. Nucleotide sequences of synonym codons for arginine, valine, cysteine, and alanine. Proc. Natl. Acad. Sci. U.S. 54, 954-960.
10
MARSHALL
AND
NIRFJBERG
D., and LITTNA, E. (1964). RNA synthesis during the development of Xenopus kzeuis, the South African clawed toad. J. Mol. Biol. 8, 669-687. BROWN, D. and LITTNA, E. (1966). Synthesis and accumulation of low molecular weight RNA during embryogenesis of Xenopus Laevis. J. Mol. Biol. 20, 95-112. BRUNNGRABER, E. (1962). A simplified procedure for the preparation of soluble RNA from rat liver. Biochem. Biophys. Res. Commun. 8, l-3. CASKEY, C. T., BEAUDET, A., WILCOX, M., and NIRENBERG, M. (1967). Differences in RNA codon recognition as a function of cellular tRNA content. Federation PTOC. 26, 349. CLARK, B., and MARCKER, K. ( 1966). N-Formyl-methionyl-sribonucleic acid and chain initiation in protein synthesis: Polypeptide synthesis directed by a bacteriophage libonucleic acid in a cell-free system. Nature 211, 378-380. DAWID, I. (1965). Deoxyribonucleic acid in amphibian eggs. J. Mol. Biol. 12, 581-599. GORINI, L., and BECKWITH, J. (1966). Supp ression. Ann. Rev. Microbial. 20, 401422. KELLOGG, D., DOCTOR, B., LOEBEL, J., and NIRENBERG, M. (1966). RNA codons and protein synthesis. IX. Synonym codon recognition by multiple species of valine-, alanine-, and methionine-sRNA. Proc. Natl. Acad. Sci. U.S. 55, 912-919. LEDER, P., SINGER, M., and BRIMACOMBE, R. (1965). Synthesis of trinucleoside diphosphates with polynucleotide phosphorylase. Biochemistry 4, 1561-1567. MARCKER, K. and SANGER, F. (1964). N-Formyl-methionyl-sRNA. J. Mol. Biol. 8, 835940. MARSHALL, R., CASKEY, C. T., and NIRENBERG, M. (1967). Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA. Science 155, 820-826. MOLDAVE, K. ( 1963). The preparation of C4-aminoacyl soluble-RNA. Methods Enzymol. 6, 757-761. NAKAMOTO, T., and KOLAKOFSKY, D. ( 1966). A possible mechanism for initiation of protein synthesis. PTOC. Natl. Acad. Sci. U.S. 55, 606-613. NIE~~KOOP, P. P., and FABER, J. (1956). “Normal Table of Xenopus Laevis ( Daudin) .” North-Holland Publ., Amsterdam. NIRENBERG, M. ( 1963). Cell-free protein synthesis directed by messenger RNA Methods Enzymol. 6, 17-23. NIRENBERG, M. ( 1965). Protein synthesis and the RNA code. Harvey Lectures Ser. 59, 155-185. NIRENBERG, M., and LEDER, P. (1964). RNA codewords and protein synthesis. I. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science 145, 1399-1407. NIRENBERG, M., LEDER, P., BERNFIELD, M., BRIMACOMBE, R., TRUPIN, J., ROTTMAN, F., and O'NEAL, C. (1965). RNA co d ewords and protein synthesis VII. On the general nature of the RNA code. Proc. Natl. Acad. Sci. U.S. 53, 11611168. SBLL, D., OHTSUKO, E., JONES, D., LOHRMANN, R., HAYATSU, H., NISHIMURA, S., and KHORANA, H. (1965). Studies on polynucleotides. XLIX. Stimulation of the binding of aminoacyl-sRNA’s to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids. PTOC. Natl. Acad. Sci. U.S. 54, 13781385. BROWN,
EMBRYONIC
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AMPHIBIAN
TRANSFER
RNA
11
T., and THACH, R. ( 1966). Role of the formylmethionine codon AUG in phasing translation of synthetic messenger RNA. J. Mol. Biol. 19, 74-90. WEBSTER, R., ENGELHARDT, D., and ZINDER, N. ( 1966). In uitro protein synthesis; chain initiation. Proc. Natl. Acad. Sci. U.S. 55, 155-161. SUNDARARAJAN,