Possible to Demonstrate One of the Predicted Specificity Changes in Trnaga Produced by HNO2 Deamination (Fig

A CHANGE IN THE SPECIFICITY OF TRANSFER RNA AFTER PARTIAL DEAM1INATION WITH NITROUS ACID By JOHN CARBON AND JAMES B. CURRY MOLECULAR BIOLOGY DEPARTMENT, ABBOTT LABORATORIES, NORTH CHICAGO, ILLINOIS Communicated by Paid Berg, December 18, 1967 The rapid advances in the structural chemistry of tRNA during the past few years have offered firm support to the concept of a triplet anticodon in tRNA which pairs with a triplet codeword on the RNA message.' This anticodon-codon combination is thought to depend on the usual Watson-Crick base- pairing for specificity, although it is recognized that the specificity patterns are somewhat relaxed in the third or "wobble" position of the triplet,2 and that ribosomal interactions are important in maintaining a high degree of fidelity.3 Changes in the specificity of tRNA observed in strains of E. coli carrying certain suppressor mutations are thought to arise either by mutations at or near the anticodon or amino acid recognition sites of a particular tRNA.4 Recently the amber su +I mutation has been shown to occur in the gene specifying a tRNAtyr, leading to a change in the 5'-base of the anticodon.A If the alteration of a single base of an anticodon is sufficient to change the specificity of tRNA, then the in vitro random deamination of tRNA should lead to a population of molecules with altered specificity. For example, subjection of tRNAGGA with the presumed anticodon UCC to random nitrous acid deamination should yield molecules containing the anticodons, UCU and UUC (Fig. 1). FIG. 1.-Possible anticodon changes in tRNAGly (Glu) (Gly) (Arg) produced by in vitro deamination with nitrous acid. CODONS: - GAA - - GGA - - AGA - Codons are shown with the 5'-end on the left; anticodons, I.: with the 5'-end on the right. It is assumed that the ANTICODONS: CUU HN02 CCU HN0, UCU anticodon of tRNAG% is as shown. Only single base changes are considered. According to current theory, such altered species would insert glycine into polypeptides in response to the arginine or glutamic acid codewords, AGA and GAG. This assumes that such altered molecules would still interact normally with the glycine activating enzyme (see Discussion). Since nitrous acid deamination of tRNA is a random process which inactivates tRNA after only two to three hits per molecule,6 active molecules suffering anticodon hits will be present in minute concentration in HNO2-treated tRNA. The availability of an extremely sensitive in vitro assay7 to measure incorporation of C14-glycine in response to the codewords AGA and GAG has now made it possible to demonstrate one of the predicted specificity changes in tRNAgA produced by HNO2 deamination (Fig. 1). This assay depends upon the use of the alternating polyribonucleotide, poly (AG),* containing only the codewords AGA and GAG and normally specifying the synthesis of only an arginine-glutamic copolypeptide using an in vitro system derived from E. coli.8 This assay was previously used to demonstrate that tRNA from E. coli strains carrying the missense suppressor mutation su+ will introduce C14-glycine into an alternating 467 Downloaded by guest on September 24, 2021 N. A. S. 468 BIOCHEMISTRY: CARBON AND CURRY PROC. copolypeptide with glutamic acid.7'9 We now show that E. coli B tRNA preparations enriched in tRNAcIGA, but free of tRNAargA, will support such poly (AG)-dependent C"4-glycine incorporation after treatment with nitrous acid under controlled conditions. Materials and Methods.-Transfer RNA was purchased from the General Biochemicals Co. or was isolated from E. coli by the method of Zubay,10 except that the preparations were treated with 0.2 M Tris (unneutralized) for 15 min to hydrolyze aminoacyl tRNA. Silicic acid (325 mesh, suitable for chromatography) was obtained from the Fisher Scien- tific Co. This material was prewashed with 1 M HCl and then water until neutral, and the fines were removed by decantation. Labeled amino acids were purchased from the New England Nuclear Corp. E. coli strains HfrRtryB- and HfrR- sut were obtained from P. Berg, Stanford University. Poly U, C, and A were from Miles Chemical Co. Random poly AG (7.4:1) and poly UG (3:1) were prepared using M. lysodeikticus polynucleotide phosphorylase, purified to stage VI by the method of Thanassi and Singer." The in vitro assay for the poly (AG)-dependent incorporation of C'4-glycine into TCA- tungstate insoluble material was carried out as described previously,7 using prewashed ribosomes and a protamine-treated 100,000 X g supernatant prepared from E. coli strain HfrRtryBj,12 The poly (AG)-dependent incorporation C14'arginine alternating copolypeptide with L-glutamic acid was measured in a similar manner,8 except that the 100,000 X g supernatant was rendered virtually tRNA-free by either a preliminary DEAE-cellulose treatment'3 or by protamine treatment to A2W/A2W = 1.5.7 The alternating polydeoxyribonucleotide, d(AG:TC), was prepared by replication of an authentic samplel4 (originally from H. G. Khorana) with DNA polymerase (fraction VII of Richardson et al.'5). RNA polymerase was purified to stage IV by the procedure of Chamberlin and Berg.16 Methylated albumin on silicic acid (MASA) chromatography: Prewashed silicic acid (70 gm) was suspended in 0.05 M Na phosphate buffer (pH 7.0), boiled briefly, cooled, and a solution of 2 gm methylated albuminl7 in 100 ml water added with stirring. Excess albumin was removed by decantation and suction filtration. The filter cake was suspended in a solution of 0.05 M sodium acetate buffer (pH 5.4)-0.3 M NaCl and packed into a 2 X 31-cm column. After equilibration with 2-3 column volumes of the same buffer, 100 mg of E. coli B tRNA in 20 ml buffer was applied to the column. Elution was carried out using a linear gradient to 1.0 M NaCl (2000 ml total volume) in the same buffer, collecting 10-ml fractions. Similar columns (4.4 X 33 cm) were run using 500 mg tRNA and the same total gradient volume with only a slight loss in resolution. Aliquot samples were assayed for C'4-glycine and C14-arginine acceptor ability in the usual manner.'8 The fractions were pooled in groups of 10-20 tubes, and the tRNA precipitated by adding two volumes of ethanol. After isolation by centrifugation, the pellets were washed with ethanol and ether, vacuum-dried, and stored frozen in aqueous solution. Nitrous acid deamination of prefractionated tRNA: The MASA pools were treated with nitrous acid as previously described,6 except that 0.8 M acetate buffer (pH 4.40) was used and the reaction mixture was not maintained at constant pH with an automatic titrator. Polynucleotide-stimulated binding studies: C'4-glycyl tRNA was prepared as previously described.'9 Ribosomal binding was measured by method B of Sl11 et al.,'9 except that the incubation was for 10 min at 37°. Ribosomes were prewashed twice with buffered 3 M KCl by the method of Smith.20 Results.-Any specificity changes induced by nitrous acid deamination of tRNA could be due to (1) a change in or near the anticodon of a tRNA, causing a change in the normal codon-anticodon relationship (Fig. 1), or (2) a change in the aminoacyl tRNA synthetase recognition site, or conformation of the tRNA, such that an incorrect amino acid is esterified onto the terminal nucleotide. In the particular case described here, incorporation of glycine into polypeptides Downloaded by guest on September 24, 2021 VOL. 59, 1968 BIOCHEMISTRY: CARBON AND CURRY 469

in response to the arginine codeword, AGA, could result from an anticodon deamination in tRNAGGA, or a change in tRNAAGA such that it accepts glycine instead of or in addition to arginine. These possibilities are identical to those described previously as possible changes in tRNA produced by the missense suppressor mutation designated S 36. 9 To determine which of these possibilities is pertinent in the studies described here, it was necessary to prefraction- ate the crude tRNA to separate tRNAGGA from tRNAAGA, and to individually subject tRNA containing these species to deamination. Prefractionation of E. coli B tRNA and localization of tRNA gGGA and tRNA A: Transfer RNA from E. coli B was fractionated over methylated albumin on silicic acid (MASA) columns, resulting in partial separation of the tRNAgly and tRNAarg (Fig. 2). After fractions were combined to form five pools (see Fig. 2),

FIG. 2.-MASA column = 1 | 431 4 | 5 I chromatography of E. coli B Ilk 2A tRNA. See Methods section for x a description of the fractiona- Iva tion conditions. 30* 0-----, optical density at i00 1.6 260 m/L. l A % 7 60 *-4, C'4-glycine acceptor X ItA 2 O-O, C14-arginine acceptor X 400 ability. Fractions were pooled in five . 200 0.4 sections (arrows) for isolation of tRNA. 0 OaA -i0- 90 100 110 120 130 --140 110 120 *7 Tou n"r the tRNA was isolated and assayed for acceptor ability with C14-glycine and C'4-arginine. Note that pool 1 is free of arginine acceptor ability and is threefold enriched in tRNAgly over the unfractionated tRNA (Table 1). Pool 2 tRNA, although over twofold enriched in tRNAgly, is contaminated with a very small quantity of tRNAarg. In order to localize the tRNAglyA, as distinguished from the glycine-specific tRNA's responding to GGU, GGC, and GGG, samples of tRNA from pools 1-5 were charged with C14-glycine of high specific activity, and the binding2' to E. coli ribosomes was measured in the presence of the random polynucleotides, poly AG (7.4:1) and poly UG (3.0: 1). Although random poly AG should stimulate the binding of both tRNAGGA and tRNAGGG, the use of a high A: G ratio in the polynucleotide should minimize response due to any tRNAGG. The results (Table 2) clearly show that binding of C'4-glycyl tRNA from only pool 1 is stimulated by poly AG. Poly UG-stimulated binding is high in pools 1, 2, and 3, however. Note that when unfractionated C'4-glycyl tRNA is used, binding as stimulated by poly AG is considerably lower than is obtained with poly UG. This is in agreement with the results of triplet binding studies on C'4-glycyl tRNA, which have shown that of the four currently accepted trinucleotide codewords for glycine, GGU and GGC promote a high level of Downloaded by guest on September 24, 2021 470 BIOCHEMISTRY: CARBON AND CURRY PROC. N. A. S.

TABLE 1. Amino acid acceptance and poly (AG)-dependent incorporation of C14-glycine and arginine as supported by MASA-fractionated tRNA. Poly (AG)- tRNA, Poly (AG)-Dependent dependent MASA C'4-Amino Acid Acceptance C4L.Glycine Incorp. C 14-arginine pool number (mumoles/pmole nucleotide) (pgomoles/ml/hr) incorp. (see Fig. 1) Glycine Arginine Untreated HNO-treated (,uymoles/ml/hr) 1 2.54 <0.01 1.00 13.6 17 2 2.10 0.08 2.53 5.85 630 3 1.02 0.57 1.40 1.35 992 4 0.30 2.12 0.92 -0.62 411 5 0.098 0.71 0.0 1.04 154 Unfrac- 0.76-0.80 0.55-0.70 0.1-1.2 2.5-6.8 500-570 tionated

Reaction mixtures were as described in the Methods section. tRNA was prefractionated as shown in Fig. 2. Poly (AG)-dependent C14-glycine incorporation is expressed as the g/imoles C1- glycine/ml reaction mixture incorporated per hour above that of control tubes lacking poly (AG) (dAG:TC omitted in stage 1).7 This unstimulated incorporation usually averaged 3-4 gsgmoles/ ml/hr. In the case of poly (AG)-dependent C14-arginine incorporation, the amount obtained in the absence of any added tRNA (about 130 IA~moles/ml/hr) is subtracted in each case. Reaction mixtures contained 5-10 A2so units/ml of tRNA. Nitrous acid treatment was for 3 hr (see Methods). Specific activities were 181 cpm/jumole for C'4-glycine and 54 cpm/pumole for C'4-arginine.

TABLE 2. Polynucleotide-stimulated binding to E. coli ribosomes of MASA-fractionated C"4-glycyl tRNA. Amount Bound to Ribosomes (jsrmoles) C14-glycyl tRNA Minus Plus Plus added (,umoles) polynucleotide poly AG (7.4: 1) poly UG (3.0: 1) Pool 1 (33.2 ,slmoles) 0.74 1.47 (+0.73) 3.88 (+3.14) Pool 2 (36.3 MAImoles) 0.89 0.82 (-0.07) 3.89 (+3.00) Pool 3 (32.8 IA;smoles) 0.84 0.41 (-0.43) 1.97 (+1.13) Pool 4 (10.5 A4Lmoles) 0.42 0.23 (-0.19) 0.75 (+0.33) Unfractionated tRNA (31.7 &pmoles) 0.70 0.87 (+0.17) 1.61 (+0.91) Conditions for the binding assay are described elsewhere"s and in the Methods section. Reac- tion mixtures (50 A) contained 0.10 A260 units of poly AG (7.4:1) or 0.22 A260 units of poly UG (3.0: 1). Values in parentheses are obtained by subtracting the binding observed in the absence of polynucleotide. The specific activity of the C14-glycine was 176 cpm/,umole.

binding, while GGA and GGG induce relatively less.22 It is probable then that tRNAGGA is a relatively minor subspecies of tRNAgly, and is present predomi- nantly in tRNA from pool 1. Attempts to localize tRNAAGA in fractionated tRNA by binding studies have been unsuccessful in this and other laboratories, probably because this particular tRNAarg is an extremely minor subspecies in E. coli.23 However, the poly (AG)-dependent incorporation of C14-arginine,5 as measured in a system partially dependent on the addition of exogenous tRNA, is capable of detecting the

presence of extremely small quantities of tRNAarg . Preliminary experiments indicated that when a protamine-treated 100,000 X g supernatant (A280/A260 = 1.5) was used in the absence of exogenous tRNA, poly (AG)-dependent C'4- arginine incorporation was 15-20 per cent of that obtained in the presence of either unfractionated tRNA or tRNA containing only acceptor activity for C14-arginine (prepared by periodate oxidation24 of arginine-protected tRNA). It is apparent that the protamine-treated 100,000 X g supernatant contains Downloaded by guest on September 24, 2021 VOL. 59, 1968 BIOCHEMISTRY: CARBON AND CURRY 471

sufficient tRNAGAG to support glutamic acid incorporation, but is deficient in tRNA A. This assay then may be used as a means to establish the presence of tRNAAGA in fractionated samples of tRNA. When applied to the MASA pools (Fig. 2), maximal stimulation of poly (AG)-dependent C14-arginine incorporation was obtained with pool 3 tRNA, while pool 1 tRNA was completely inactive (Table 1). We are thus reasonably certain that pool 1 tRNA contains tRNAGGA, but lacks tRNA A. Nitrous acid deamination of MASA-fractionated tRNA: Our previous studies have determined that inactivation of tRNA by nitrous acid deamination follows random single-hit kinetics with an apparent target size of approximately 35-60 nucleotides. Reaction is sufficiently slow at pH 4.3 so that an average of only one nucleotide per tRNA molecule is deaminated per hour.6 When unfractionated tRNA was treated with nitrous acid under similar conditions for various lengths of time, samples were obtained which would support an extremely small and variable incorporation of C14-glycine into polypeptides specified by poly (AG). However, the treatment of MASA pools 1-5 (Fig. 2) with nitrous acid at pH 4.4 for three hours resulted in a 14-fold increase in the ability of pool 1 tRNA to support poly (AG)-dependent C'4-glycine incorporation, with only a twofold increase using pool 2 tRNA, and no observable change using pools 3-5 (Table 1). Since pool 1 contains the highest proportion of tRNAGGA (Table 2), we are probably observing the result of deaminations of the 3'-C in the anticodon of tRNAG1GA (see Fig. 1). The poly (AG)-dependent incorporation of C'4-glycine as supported by pool 1 tRNA exposed to nitrous acid for various lengths of time is shown in a Figure 3. This incorporation A I

reaches a maximum in samples gS treated for three hours and then 0 begins to decrease. Loss of glycine 1.00 a _ acceptor ability by deamination hits is, 1 I 15A ,I. in sensitive regions of the molecule PI~tRNA . - is clearly responsible for this de- 0.50 US crease (Fig. 3). Thus we are prob- a Peel 2 tRNA a ably observing the result of two re- a1 at the anti- 0 o o actions, deamination S b z I I I I codon leading to misreading, as well 1 2 3 4 s as deaminations in other parts of Tim d HN02 ruftN , k the molecule leading to inactivation FIG. 3.-Poly (AG)-dependent C'4-glycine of acceptor ability. For example, incorporation and C 4-glycine acceptor ability after three hours in nitrite solution of E. coli B tRNA as a function of time of ex- at pH 4.4 approximately 50 per cent posure to nitrite solutions at pH 4.4. For reaction conditions see the Methods section. Pool 1 of the tRNAgiy has been inacti- tRNA, poly (AG)-dependent incorporation = vated; however, the remaining 50 0; pool 2 tRNA, poly (AG)-dependent in- per cent shows a high level of incorporation = 0; pool 1 tRNA, C14-glycine acceptance = A. Each incorporation assay corporation in the poly (AG)-depen- contained 10-13 A260 units/ml of the appro- dent assay. priate tRNA. Downloaded by guest on September 24, 2021 472 BIOCHEMISTRY: CARBON AND CURRY PROC. N. A. S.

Properties of the HNOrinduced C"4-glycine incorporation: It might be argued that nitrous acid deaminations are changing the essential conformation of tRNAgiy and in some way inducing a general misreading of triplet codewords. A sample of pool 1 tRNA that had been treated with nitrous acid for three hours was tested for its ability to support in vitro C14-glycine incorporation in the presence of various polynucleotide messages (Table 3). Although incorporation in TABLE 3. Incorporation of C"4-glycine into polypeptides supported by HNO-treated tRNA (pool 1) and various polynucleotides. C4-Glycine Incorporation (Osjmoles/ml/hr) Polynucleotide Expt. 1 Expt. 2 Poly (AG), alternating 22.8 22.0 Poly (AC), alternating 5.9 4.9 Poly U 4.4 6.3 Poly A 5.4 5.1 Poly C 4.4 5.9 None 3.9 5.5 C"4-Glycine incorporation in response to poly (AG) was measured as described previously.7 Poly (AC)-stimulated incorporation was measured by substituting dAC:TG, ATP and CTP in stage 1.7, 25 Poly U, poly A, and poly C were used at a level of 1.5 Amso units/ml reaction mixture. All tubes contained HNO-treated E. coli B tRNA (pool 1), at 12.8 A260 units/ml. the presence of alternating poly (AG) was relatively high (four- to fivefold greater than controls lacking polynucleotide), no stimulation was seen in the presence of poly U, poly A, poly C, or alternating poly (AC).25 Although the arginine codeword, AGA, is read as glycine by the deaminated sample (see below), the threonine codeword, ACA, is not recognized. This is exactly what would be predicted on the basis of deamination of the 3'-C in the anticodon of tRNAGGA. The change we are observing is thus a highly specific one, and not a case of general infidelity of translation. The failure of poly A to stimulate C"4-glycine incorporation indicates that deamination of both cytosines in the anticodon is an extremely rare event, as would be expected. If deamination is occurring randomly at the anticodon of tRNAgA, it follows that tRNA's would be produced which read the codewords AGA and GAA, as shown in Fig. 1. It is possible then that both triplet codewords in alternating poly (AG), AGA and GAG, would be read as glycine, assuming that "wobble" is permitted in the third position so that U could pair with G.2 Previous work has shown that when tRNA from strains of E. coli carrying the su+ mutation is used in the poly (AG)-dependent assay, only the AGA triplet is read as glycine and the system shows a dependence on glutamic acid for full activity (see Fig. 4A).7 9 Similar studies using HNOrtreated tRNA (pool 1) have shown a glutamic acid dependence, but a complete lack of dependence on the presence of unlabeled arginine (Fig. 4A and B). This would indicate that if deamination of the middle C in the anticodon of tRNAGGA does occur, the resulting molecule is either in- activated, or is incapable of pairing with the GAG triplet (see Discussion). The experiments shown in Figure 4 also demonstrate the relative effectiveness of su36 tRNA and HNO2-treated pool 1 tRNA in supporting the incorporation of C14- glycine in response to the arginine triplet, AGA. Although various preparations of these tRNA's have differed somewhat in their specific activities, the suppressor tRNA preparations have generally been somewhat more active. In particular, Downloaded by guest on September 24, 2021 VOL. ,;9, 1968 BIOCHEMISTRY: CARBON AND CURRY 473

i30 15 e +Glu & Arg A : 0E ~~~~~~~~~Glu

X - 10 S 10+G lu

FIG. 4.-PoNo additions

10= g m a 5 -+ Arg 0~~~~~~ 0~~~~~~~~~~~~~

Z%0 ~~~~~~5C~~~la~~~~1005 10 10 15 20 su036 tRNA, A260 units/mi HNO2 tRNA, A2Wunits/ml

FIG. 4.-Poly (AG)-dependent incorporation of C'4-glycinle into polypeptides as a function of added (A) Su' tRNA and (B) HNO2-tRNA (pool 1). M\inus amino acids =0; plus i-glutamic acid = 0; plis L-arginine = A; plus L-glutamic acid and L- arginine = El. Unlabeled amino acids were added at 133 mjumoles/ml reaction mixture. dose-response curves exhibited by the HN02-treated tRNA consistently show a sharp attenuation in response at higher tRNA levels (Fig. 4B). Although the reason for this nonlinear response is not clear at present, it may be caused in part by the presence of inactive but inhibitory deaminated tRNA in these preparations. Discussion.-Although there is no clear-cut chemical evidence that we have induced an anticodon change in tRNAglA, our results can best be interpreted in favor of this possibility. The ability to support poly (AG)-dependent C14- glycine incorporation after HNO2-treatment appears only in tRNA containing tRNAGGA, but not in samples containing tRNA A. This would seem to elimi- nate the possibility that we are in some way changing an enzyme recognition site on tRNAAGA So that it accepts glycine by mistake. Furthermore, the observed change appears specific for the codeword AGA and is exactly in line with what the current codon-anticodon theory would predict. The failure to see the other predicted single-base change in the anticodon of tRNAGGA (Fig. 1) is somewhat puzzling. Perhaps "wobble" does not apply in the case of this particular tRNA, and tRNAGA, if formed, will not read the GAG triplet in our assay. Alternative possibilities would be that the glycine-activating enzyme will not recognize a tRNAgly with an altered second base in the anticodon, or that competition with the normal tRNAgAG is sufficient to prohibit any C14-glycine incorporation in response to GAG. It is interesting in this regard that a missense suppressor which would insert glycine in place of glutamic acid (GAA) in the tryptophan synthetase A protein mutant A46 has never been discovered, despite repeated efforts to do so.4 Perhaps a change of the second base in the anticodon of tRNAGGA can only lead to an inactive molecule. It has been suggested that the activating enzymes could use the anticodon region as a means of recognizing the correct tRNA in the amino acid loading reaction.26 The work reported here can be taken as direct evidence against this theory, at Downloaded by guest on September 24, 2021 474 BIOCHEMISTRY: CARBON AND CURRY PROC. N. A. S.

least in terms of changes in the 3'-end of the anticodon (5'-end of codon). The recent report5 that the amber suj+1 (tyrosine) suppressor is different from a normal tyrosine tRNA only in the base at the 5'-end of the anticodon (3 '-end of codon) is further evidence that anticodon changes may occur without changing activating enzyme recognition. Even though an anticodon change does not change the specificity of the activating enzyme-tRNA recognition process, it may alter the KM of the interaction. This is particularly relevant here, since it is known that the level of A36 suppression is strongly dependent on the amount of glycine- activating enzyme in the cell.9 The work reported here suggests an additional approach to the study of tRNA derived from cells carrying genetic suppressors. For example, a careful chroma- tographic comparison of su + tRNA7 and the HNO2-treated pool 1 tRNA, both of which contain a tRNA capable of reading AGA (arginine) as a glycine codeword, could help answer questions concerning the mechanism of action of this missense suppressor. The authors are grateful to Dr. Paul Berg for many helpful discussions. Excellent technical assistance was provided by Miss Diane Jones. * The term poly (AG) is meant to signify a polyribonucleotide containing adenylic and guanylic acid residues in alternating sequence. The omission of the parentheses, as in "poly AG " signifies a random copolymer. For examples, see Zachau, H. G., D. Dutting, H. Feldman, F. Melchers, and W. Karau, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 31 (1966), p. 417. 2 Crick, F. H. C., J. Mol. Biol., 19, 548 (1966). 3 Gorini, L., G. A. Jacoby, and L. Breckenridge, in Cold Spring Harbor Symposia on Quanti- tative Biology, vol. 31 (1966), P. 657. 4Brody, S., and C. Yanofsky, these PROCEEDINGS, 50, 9 (1963). 6 Goodman, H. M., J. D. Smith, J. N. Abelson, A. Landy, F. Sanger, B. G. Barrell, and S. Brenner, Abstracts, 7th International Congress of Biochemistry, Tokyo, 1967 (International Union of Biochemistry, 1967), p. 673. 6 Carbon, J. A., Biochim. Biophys. Acta, 95, 550 (1965). 7 Carbon, J., P. Berg, and C. Yanofsky, these PROCEEDINGS, 56, 764 (1966). 8 Jones, D., S. Nishimura, and H. G. Khorana, J. Mol. Biol., 16, 454 (1966). 9 Carbon, J., P. Berg, and C. Yanofsky, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 31 (1966), p. 487. 10 Zubay, G., J. Mol. Biol., 4, 347 (1962). l' Thanassi, N. M., and M. F. Singer, J. Biol. Chem., 241, 3639 (1966). 12 Brody, S., and C. Yanofsky, J. Bacteriol., 90, 687 (1965). 13 Yamane, T., and N. Sueoka, these PROCEEDINGS, 50, 1093 (1963). 14Byrd, C., E. Ohtsuka, M. W. Moon, and H. G. Khorana, these PROCEEDINGS, 53, 79 (1965). 15 Richardson, C. C., C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Chem., 239, 222 (1964). 16 Chamberlin, M., and P. Berg, these PROCEEDINGS, 48, 81 (1962). 17 Mandell, J. D., and A. D. Hershey, Anal. Biochem., 1, 66 (1960). 18 Muench, K. H., and P. Berg, Biochemistry, 5, 970 (1966). 19 Sl, D., D. S. Jones, E. Ohtsuka, R. D. Faulkner, R. Lohrmann, H. Hayatsu, H. G. Khorana, J. D. Cherayll, A. Hampel, and R. M. Bock, J. Mol. Biol., 19, 556 (1966). 20 Smith, J. D., J. Mol. Biol., 8, 772 (1964). 21 Nirenberg, M., and P. Leder, Science, 145, 1399 (1964). 22 SOH1 D., E. Ohtsuka, D. S. Jones, R. Lohrmann, H. Hayatsu, S. Nishimura, and H. G. Khorana, these PROCEEDINGS, 54, 1378 (1965). 23 Marshall, R. E., C. T. Caskey, and M. Nirenberg, Science, 155, 820 (1967). 24 Baldwin, A. N., and P. Berg, J. Biol. Chem., 241, 839 (1966). 2 Nishimura, S., D. S. Jones, and H. G. Khorana, J. Mol. Biol.,-13, 302 (1965). For example, see Miura, K., in Progress in Nucleic Acid Research, ed. J. N. Davidson and W. E. Cohn (New York: Academic Press, Inc., 1967) vol. VI, pp. 39-82. Downloaded by guest on September 24, 2021