Tuep JouRNAL oF Biobocican CHEMISTRY Vol. 236, No. 6, June 1961 Printed in U.S.A The Enzymic Synthesis of Amino Acyl Derivatives of Ribonucleic Acid Il. ISOLATION OF AMINO ACID-ACCEPTOR RIBONUCLEIC ACIDS FROM ESCHERICHIA COLI* E, J. OFENGAND,t M. Dreckmann, AND Paun Bere From the Department of Microbiology, Washington University School of Medicine, St. Louis 10, Missouri and the Department of Biochemistry, Stanford University, Stanford, California (Received for publication, November 8, 1960) The existence of a ribonucleic acid fraction with the unique property of binding amino acids is now well documented (1-6). The mechanism of formation and the structure of these amino acyl ribonucleic acid derivatives have been investigated in a number of laboratories with the following conclusions. Each amino acyl RNA compound is formed by a specific enzyme that catalyzes both the synthesis of the intermediate amino acyl aden- ylate complex and the corresponding amino acyl RNA deriva- tive (2, 3, 7); each amino acid is linked through its carboxyl group to a ribose-hydroxyl group of the terminal adenylic acid residue in a specific RNA chain (8-10); and each amino acid- acceptor RNA chain is terminated at the acceptor end by the identical trinucleotide sequence, RNA... pCpCpA (10-12) and at the other end by pGp...RNA (13, 14). Regarding the structural features, which differentiate RNA chains with respect to the amino acids they accept, essentially nothing is known. In connection with our studies of the mechanism of amino acyl RNA formation and with the objective of determining the chemical and structural features necessary for the formation of a specific amino acyl RNA derivative, a procedure for the isola- tion of the amino acid-acceptor RNA from Escherichia colt was developed (2). The present report describes the details of this method and some physical and chemical properties of the puri- fied RNA. Since this procedure was developed, there have been a number of accounts of the isolation and properties of amino acid-accep- tor RNA from animal tissues (15, 16), yeast (17-19), and bac- teria (14, 20). It is of considerable interest and very likely of fundamental significance that the amino acid-acceptor RNA from these diverse cells shows striking similaritics in molecular weight, secondary structure, and in nucleotide end groups and composition. How closely this similarity prevails at the more detailed level of nucleotide sequence and over-all molecular conformation remains to be seen. EXPERIMENTAL PROCEDURE Materials £, coli was used as the starting material for the isolation of the amino acid acceptor RNA and for the preparation of enzyme * This investigation was supported by grant funds from the National Institutes of Health of the United Public Health Service. { Predoctoral Research Fellow of the National Science Founda- tion; present address, Medical Research Council Unit, Cavendish Laboratory, Cambridge University, Cambridge, England. extracts used in measuring C4-amino acid incorporation into the RNA. The organisms were grown as previously described (21), and extracts were prepared in a Waring Blendor with glass beads (21). The specific amino acyl RNA synthetases were isolated and used as already described (21). DNase was a twice recrystallized preparation purchased from Worthington Bio- chemical Company. The C*amino acid mixture used in the assays was derived from an acid hydrolysate of protein from Chromatium, strain D, grown with C“Q» as sole carbon source. For use in the assay, it was diluted with an unlabeled hydrolysate of Chromatium protein to a specific activity of 3 to 5 x 10° c.p.m. per umole of carbon. pu-Leucine-1-C and pi-valine-1-C™ were purchased from Isotope Specialties, and unlabeled amino acids were ob- tained from the California Corporation for Biochemical Research. Ecteola-cellulose, type 20, was a reagent grade material ob- tained from the Brown Company. Sodium lauryl sulfate, U.S.P. grade, was used without further purification. Methods RNA was routinely assayed for amino acid acceptor activity with the C “amino acid mixture in the presence of a dialyzed extract of Z. coli. The standard conditions were as follows: 50 umoles of sodium cacodylate buffer, pH 7.0, 1 umole of MgCl, 0.2 pmole of ATP, approximately 6 x 10° ¢.p.m. of the Cl amino acid mixture, 0.2 to 8 units of the acceptor RNA and dialyzed EF. colt extract containing 75 to 150 wg of protein. The mixture, in a total volume of 0.5 ml, was incubated for 20 to 60 minutes at 30°. With these amounts of enzyme and RNA, the reaction is complete in 20 minutes; with aged and less active extracts somewhat longer incubation periods were necessary. The incubation was terminated and the amount of C'-amino acyl RNA formed was determined as previously described (3). Fig. 1 shows that the amount of C'-amino acid linked to the RNA was a linear function of the amount of RNA added. a 4 S + £ NY Vv Na y| 7! 8 eo 4 2 18) 1 {18S a 14 S15 415 eh 4"°3 B12 4128 t ° of 1 Ber 19S @ | % 36 69 = ob 4 @ Gok 4+ 35 2 3b -| 3 &°E 1 0 eet Ao KOOMAMPIK—Linear gradient 0.6M 13M AmF-— 9-29 Ame, 30 609030 9 150 230 270 330 390 0 306090 Volume in mi. Fig. 2. Chromatography of amino acid-acceptor RNA on Ee- teola-cellulose. June 1961 TasLe I Isolation of amino acid-acceptor RNA from E. coli Yield of amino +. . . acid acceptor Nucleic acid RNA fraction Specific activity activity content . umoles/RNA is/amol units/g cells muclentide/ RNA nittlentide Crude...... 00, 892 93 4,2 DNase treated ....... 396 73 5.4 Ecteola.........0..0... 266 24 11.0 ing amino acids. For example, fractions recovered from the leading portion of the peak have 50 to 75% higher specific activity than those fractions from the later part of the peak when assayed either with the amino acid mixture or with leucine, valine, isoleucine, or methionine. Rechromatography of the once-chromatographed RNA yields a single peak emerging at an ammonium formate concentration of 0.88 m with an increase in specific activity of about 20%. We have also used this procedure on 20 g batches of dried cells with slight modifications but essentially similar results. The various components were increased proportionately, and the sodium lauryl sulfate extraction was performed in a stainless steel beaker, immersed in a boiling water bath, with a stirrer driven by an overhead motor to ensure adequate mixing during the heating. Five such batches of the crude acceptor RNA fraction (representing about 10 mmoles of RNA nucleotide) were combined and subjected to the DNase step and then chromatog- raphy ona 37- x 4.5-cm column. The buffer gradient for elu- tion was between 0.6 M and 2.1 mM ammonium formate (1200 ml). The peak of acceptor RNA activity was eluted at an ammonium formate concentration of about 0.9 m. In some runs as much as one-third of the optical density applied to the column was removed with the 0.6 mM ammonium formate wash but this material was always inactive as an amino acid acceptor. Analysis of the total RNA content of the dried ccll prepara- tion gave values which averaged about 525 umoles of nucleotide per g. Basing our calculations on the recovery of 24 umoles of RNA nucleotide in the amino acid acceptor fraction (Table I) and correcting for the yicld (68%), we estimate that the amino acid acceptor RNA comprises between 5 to 10% (6.7% in this instance) of the total cellular RNA. Using a value of 3 umoles of RNA nucleotide as being equal to 1 mg of RNA, we estimate that the acceptor RNA constitutes about 1% of the dry weight of F. colt. This value is somewhat higher than the value of 300 mg of acceptor RNA per 300 g wet weight of cells reported by Tissiéres (20). Purity of Acceptor RNA Preparation—Estimations of protein in the acceptor RNA by the method of Lowry et al. (27) gave values which were equivalent to between 7 to 9 ug of serum al- bumin per umole of nucleotide (approximately 2 to 3% on a weight basis). However, this estimate is undoubtedly high, since guanine reacts in the Lowry procedure to yield a color with the same spectrum as that found with proteins. Guanine, | ug, yields a color equivalent to about 7 to 8 ug of serum albumin? Similar values for the protein content of RNA, as determined by this method, were noted by Tissiéres (20). In contrast to the acceptor RNA preparations obtained by phenol extraction of mammalian tissue (1, 7), the present RNA 2 A. D. Kaiser, private communication. EL. J, Ofengand, M. Dieckmann, and P. Berg 1743 preparation did not contain significant amounts of bound amino acids. Although no direct estimate was made of the amino acid content of the acceptor RNA preparations described here, the fact is that at least 98% of the ability to accept a number of amino acids was lost after exposure of the RNA to periodate under conditions in which bound amino acids protect against periodate inactivation (9). Moreover, there was no significant increase (<5%) in the amount of amino acid which could be incorporated after dilute alkaline treatment of the RNA, a procedure which removes bound amino acids (9). If, under the conditions in which the cells were grown, the RNA were satu- rated with amino acids, then it seems most likely that these bound amino acids were removed during the initial extraction step, since all other procedures have routinely been used for the purification of amino acyl RNA derivatives. The pentose to phosphate ratios in the purified RNA prepara- tions have ranged from 0.96 to 1.08. Based on the phosphate content, the extinction coefficient, #(P) at 260 my in the pres- ence of 0.2 mM NaCl is 7.7 x 10° at pH 7.0 and 8.8 x 10° at pH 12. In most preparations, the deoxypentose content was less than 4%. In certain instances however the RNA preparations obtained from the Vcteola-cellulose column during the large scale procedures were contaminated with deoxypentose-contain- ing oligonucleotides produced during the DNase treatment. Such oligodeoxynucleotides were removed by treatment with the Lehman phosphodiesterase (28). This enzyme converts oligo- deoxynucleotides and single-stranded polydeoxynucleotides to mono- and dinucleotides which are readily removed by dialysis against 0.2 m NaCl. The spectrum of the purified acceptor RNA preparation is characteristic of the nucleic acids (Fig. 3). At pH 7.0 in 0.2m 1.20 T T T T T T Lio wf | ZS ot om Ma MON * $ ° 7 ot Optical Density eB 8 — LL iw le. | — +300 \ -200 \ ‘ 2100 | l f ! | l 220 230 240 250 260 270 Wave length in my Fig. 3. Absorption spectra of amino acid-acceptor RNA. All curves were normalized to a single concentration of the RNA. @--—@, 0.2 wm NaCl-0.001 m sodium cacodylate buffer, pH 7.0; O--—O, 0.2 w NaCl-0.01 n KOH; O——9O, alkaline hydrolysate in 0.2 mM NaCl at pH 7.0. The RNA was hydrolyzed in 0.5 m NaOH for 18 hours at 37°, neutralized with HCl buffered with sodium cacodylate, pH 7.0, and then diluted to the salt concen- tration indicated. 1744 NaCl, it has a peak at 259 my and a minimum at 230 mu. The ratio of the optical density at 260 to 280 my in various prepara- tions was 2.0 to 2.3 and the ratio at 260 to 230 my was 2.2 to 2.3. The optical density of the RNA is about 15% higher at pH 12 (0.01 n KOH) than at pH 7.0, although the optical density is restored to the original value when the pH is returned to neu- trality. After hydrolysis of the RNA with 0.5 N NaOH for 18 hours at 37°, there is an irreversible increase of 44% in the opti- cal density at 260 mu. Nucleotide Composition and End-Group Analysis—The results of chromatographic analysis of an alkaline hydrolysate of the amino acid-acceptor RNA are shown in Table II. With each of the two RNA preparations examined, more than 95% of the nucleoside recovered was adenosine. The adenosine to nucleo- tide residue ratio was 1:89 in one case and 1:93 in the other. If we accept that nucleosides arise only from the terminal nucleo- Tasuie II Nucleotide analysis of purified amino acid-acceptor RNA Digests were prepared by incubating the RNA with 0.3 n NaOH at 37° for 18 hours after which the mixture was adjusted to be- tween pH 6 to 7 with dry Dowex 50-H*. The hydrolysate was then adjusted to about pH 9 to 10 with NH.OH and adsorbed onto a Dowex 1-formate column (10 X 2 em*). The nucleosides were eluted with 0.01 m ammonium formate buffer, pH 3.7, and the nucleotides were eluted with a parabolic gradient established with 500 ml of 0.035 m ammonium formate, pH 3.7, in each of two mix- ing chambers and 500 ml of 0.6 M ammonium formate, pH 3.7, in the reservoir chamber. The elution was carried out at 4° with a flow rate of 0.2 ml per minute. Fractions of about 6 ml were col- lected and the optical density at 250, 260, and 280 mz was deter- mined. The components listed in the table were eluted in the following order: adenosine, cytidvlic, the unidentified com- pound(s),* uridylic, adenylic, and guanylic acids. Concentra- tions were determined with extinction coefficients published by Beaven et al. (29). Recoveries from the column ranged between 93 and 98% of the optical density applied to the column. Amino acid- acceptor RNA Nucleoside or nucleotide “Ribosomal RNA”’ Prepara-| Prepara- tion I tion II moles/100 moles recovered nucleotide Adenosine....................20.. 1.12 | 1.08 | not detected Adenylie acid. ..................,. 18.4 18.6 25.6 Uridylic acid. ..... 2.00.22 16.4 18.1 20.9 Unidentified component*......... 4.10 | 2.94 | not detected Guanylic acid... 0.002... ee. 31.5 | 30.4 31.2 Cytidylic acid........0...00....... 28.5 28.4 22.3 Purines/pyrimidines.............. 1.04 | 1.00 1.31 Adenine + uracil + unidentified component*/guanine + cyto- SINC... eee 0.67 | 0.70 0.87 * This material was eluted from the column immediately pre- ceding the peak containing 2’- and 3’/-UMP. The ratio of the optical density at 280 my to 260 mp was 0.64 and 0.78, respectively. It had an absorption maximum at 268 my in 0.1 N HCl and in 0.1 n KOH. At the alkaline pH, there was a decrease in the absorp- tion at 268 mp of about 15% and the appearance of a distinet shoulder in the region of 290 mu. Insufficient material was avail- able to identify this compound(s) or to determine whether it was a derivative of the pseudouridylic acid type of compound (30). To estimate its concentration, the extinction coefficient of uridylic acid was used. , Linzymic Synthesis of Amino Acyl RNA Derivatives. Ill Vol. 236, No. 6 109 80 60 40 oO H20. @ 02 MNocl N Units of amino acid acceptor activity S n, haoM 8 Oo oo © Leucine @ Valine Nh Oo % of initial amine acid acceptor activity remaini {0 20 30 40 50 00 Minutes at 100° Fig. 4. Effect of heating on amino acid-acceptor activity of RNA. (a) Acceptor RNA, which had been dialyzed for 48 hours against 3 changes of distilled water, was diluted in water or 0.2 m NaCl to a concentration of 6.8 nmoles of RNA nucleotide per ml. One milliliter was heated in a boiling water bath for the times indicated, cooled by immersion in an ice bath and stored at —10° for about 24 hours. The samples were then thawed and assayed with a dialyzed #. colt extract and a C4-amino acid mix- ture as described in ‘‘Methods.”’ (b) RNA nucleotide in water, 1.8 ymoles, was heated as above for the times indicated and then cooled in ice and assayed within 60 to 90 minutes for acceptor activity with leucine and valine, as described previously (8). «370 -360 ogre .350 4“ & ® oO i eo oO af ao iS) oO ! Se. _ Optical Density at 260 mw a 3 >, e, & Oo ° 7 ~‘ a - o@' @e--- nN .o o 20 40 60 80 Temperature Fie. 5. Effect of temperature on the ultraviolet absorption of amino acid-acceptor RNA. RNA was dissolved in 0.1 m sodium cacodylate buffer, pH 7.0. Readings were made as described by Doty et al. (83). Approximately 10 minutes were allowed for the sample to come to thermal equilibrium at each temperature. tide with free 2’- and 3’-hydroxyl groups on the ribose and that there is only one such residue per polynucleotide chain, then it is possible to estimate the minimal molecular weight. This esti- mate, based on an average chain length of 91 nucleotides and on the nucleotide composition, yields a value of 31,000 + 1,500. In this calculation, it is assumed that all the RNA molecules contain a terminal adenylic acid residue unesterified at the 3’- position. Any chains with the 3’-hydroxyl group esterified June 1961 would appear as nucleotides after alkaline hydrolysis and there- fore make our estimate of the chain length and molecular weight too high. Nevertheless, the molecular weight is only about 20% higher than that estimated by Tissiéres (20) from sedimentation, viscosity, and diffusion parameters and also is somewhat higher, or of the same order as, the estimates for acceptor RNA from mammalian liver (15) and yeast acceptor RNA preparations (17, 18). With regard to the over-all nucleotide composition of amino acid-acceptor RNA, two features are noteworthy. The first is that a compound representing approximately 3 to 4% of the total nucleotide was eluted from the column just preceding uridylic acid, whereas this material was not detected in the ribosomal RNA hydrolysate. Although authentic pseudouridylic acid was eluted in this region of the chromatogram, this material had somewhat different spectral properties than those reported for pseudouridylic acid (80) (Legend to Table II). Because of the limited amounts of this material, no further characterization was carricd out. More recent analyses of the amino acid-ac- ceptor RNA of E. colt have shown that pseudouridylic acid and thymine ribonucleotide comprise about 3% of the total nucleo- tides (31). This unusually high content of pseudouridylic acid has also been reported for the acceptor RNA from mammalian tissue and from yeast (17, 19). The second feature of the amino acid-acceptor RNA is the relatively close correspondence between the purine and pyrimidine content. Moreover, the total adenylic acid is almost equal to the total uridylic acids (assuming the unknown component to be a derivative of uridylic acid) and the guanylic acid is roughly equal to the cytidylic acid. This type of nucleotide equivalence, which resembles that found in DNA, has also been reported by other investigators (14, 15, 31, 32) but it has not been observed with the RNA isolated from ribosomes (26). Effect of Heating on Purified Amino Acid-Acceptor RNA— At 100°, the amino acid-acceptor RNA is slowly inactivated in 15 7 T T T T T T T a. iw 2 So Cc cpm/ml. x2-10% [oI 2 Ny el ITN NH4OH ZL So on 62M AmF y Fy RNA emoles/ml bo S 0 120 240 480 Eluafe volume, ml. Fic. 6A. 360 E. J. Ofengand, M. Dieckmann, and P. Berg 1745 |) T T T T T T T b. n° . f 7 211 Nl x he 4 E ‘G09 a uO sf 27 : 7 Zi Or E05 1 e ; { (7N NH4OH e - o2 Mame - 303 0 i Y has 0 120 240 360 480 Eluate volume mi. Fie. 6B Fic. 6. Chromatography of C'-valyl and C*-leucyl RNA on Ecteola-cellulose. (a) C'-valyl RNA containing 0.75 mymole of valine per pmole of RNA nucleotide was prepared as already de- scribed (3). RNA nucleotide, 75 umoles, was adsorbed to an Ecteola-cellulose column, (formate form, 10.3 X 1.1 em*). A linear buffer gradient was established with 200 ml of 1.47 m am- monium formate buffer, pH 4.7, in the reservoir and 200 ml of 0.65 m buffer in the mixing chamber. Fractions of about 5 ml were collected at 0.3 ml per minute and both the optical density at 260 mu and the C'* content were determined. We recovered 88% of the C4 and 70% of the optical density. The fractions shown by the dotted portion of the main peak, which represented about 20% of the total radioactivity and had a specific activity of 3.7 mumoles of valine per zmole of RNA nucleotide, were pooled, concentrated, and treated as described in Table III. (6) C'*-leucy] RNA nucleo- tide, 85 wmoles, containing 1.0 mumoles of leucine per umole of RNA nucleotide, was adsorbed to the same type of column men- tioned above (10.6 em X 1.1 cm?). The gradient, rate of elution, fraction size, and method of analysis were as described above. We recovered 87% of the C' and 68% of the optical density. The material taken for further study (dotted portion of the main peak) contained 19% of the total radioactivity and 3.8 mumoles of leucine per umole of RNA nucleotide. its ability to accept amino acids (Fig. 4a and b). This occurs at about the same rate whether the heating is carried out in distilled water or in 0.2 m NaCl at pH 7.0. Inactivation of the specific leucine- and valine-specific RNA chains was slower than the total population of amino acid-acceptor RNA chains. Because the amino acid acceptor activity of the RNA was only slowly inactivated by heating at 100°, another criterion for examining the effect of heating on the structure of the RNA was investigated. Prompted by the studies of Doty ef al. (33), we examined the effect of heating on the optical density of the RNA (Fig. 5).2 Commencing at about 40°, there is a hy- perchromic shift at 260 mp which attains a maximum at about 3 We express our gratitude to Dr. Charles Dekker, Department of Biochemistry, University of California, Berkeley, for allowing us to use his equipment. 1746 Tasir III Partial separation of leucine- and valine-specific acceptor RNA chains The C!4-valyl and C'4-leucyl RNA were recovered from the pooled fractions indicated in Fig. 6, a and b, respectively, by alco- hol precipitation. The amino acids were removed in the presence of the appropriate amino acyl RNA synthetase, PP, and AMP (3). By this procedure, 98% of the valine and 94% of the leucine were removed. This method produced no inactivation of leucine or valine acceptor sites when the original RNA was similarly treated. The RNA preparations were then assayed for their abil- ity to accept the amino acids shown in the table with the stand- ard assay (3). RNA purified as: Amino acid tested Original RNA 1 Leucyl {| Valyl RNA | RNA mpmoles amino acid incorporated / umole RNA nucleotide Leucine........ 2... 0.95 3.43 1.33 Valine... 0... cee eee eee 0.56 0.85 3.50 Tsoleucine................0..0000. 0.41 1.72 1.30 Methionine. ..................... 0.26 1.58 1.50 Ratio of incorporation of leucine to valine... ee. 1.70 4.00 0.38 80°. The total increase in the optical density is about 23% and the midpoint of the transition is at 59°. On cooling, the original optical density is restored. According to Doty ef al. (33), such temperature-induced hyperchromic shifts indicate the existence of a secondary structure resulting from hydrogen-bonded bases arranged in helical regions. The fact that the transition is reversible (in our experiments with respect to the ultraviolet absorption properties) makes it difficult to determine whether a specific structure is or is not required for biological activity. In an attempt to study this problem, we have determined the ability of the acceptor RNA to bind leucine and valine at ele- vated temperatures. With 20- to 50-fold excess of the leucyl- and valyl RNA synthetases (so that heat inactivation of the enzymes during the incubation does not effect the final yield of amino acyl RNA) and the conditions already described (3), the yield of leucyl- and valyl-_ RNA formed at 25, 30, 37, 47, and 55° was determined. Essentially no difference in the yzeld of leucyl- and valyl RNA (less than 10%) was observed up to and includ- ing 55°. These experiments show that at a temperature at which one-half the maximal hyperchromic shift occurs there is little or no difference in the amount of leucyl- or valyl-RNA formation. Although these results suggest that a specific secondary structure of the RNA is unessential for the enzymatic formation of a specific amino acyl RNA derivative, we can not eliminate the possibility that the enzymes react with that por- tion of the molecules with the native configuration and that the amino acy! RNA has a somewhat higher temperature transition range. Sedimentation Coefficient of Amino Acid-Acceptor RNA—The sedimentation coefficient of the amino acid-acceptor RNA in 0.1 M sodium cacodylate buffer, pH 7.0, at concentrations of 0.068 and 10 zmoles of RNA nucleotide per ml (with ultraviolet and schlieren optics, respectively) was 3.9 and 3.8. Since at the time these experiments were performed the only published value available was 1.8 § for rat liver amino acid-acceptor RNA (1), Enzymic Synthesis of Amino Acyl RNA Derwatives. It Vol. 236, No. 6 it seemed possible that our values reflected the properties of the bulk of the RNA rather than the specific amino acid-acceptor component. Sedimentation of C%-valyl RNA was therefore carried out with a separation cell (34) and the procedure de- scribed by Schachman (35). C'-valyl RNA at a concentration of 2.2 umoles of nucleotide per ml, in 0.01 m potassium acetate buffer at pH 4.4, containing 0.2 m NaCl, was centrifuged so that in one run approximately 50% and in another run approximately 68% of the RNA remained in the upper compartment of the separation cell. The average szo,.. of the radioactivity was 4.5 + 0.2, whereas that of the ultraviolet and schlieren boundary was 4.2 + 0.6 and 3.7 + 0.1, respectively. It is therefore clear that the component to which the valine is bound sediments with the bulk of the ultraviolet-absorbing component. The somewhat lower 820,» values obtained from the schlieren boundaries may be due to the difficulty in estimating the movement of the small boundary resulting from the low concentration of valyl RNA used. Attempt to Isolate Amino Acid-specific RNA Chains—Evidence presented in the previous paper (3) led to the conclusion that amino acid-acceptor RNA is composed of a population of poly- nucleotide chains each specifie for a particular amino acid. This hypothesis was based on the finding that each amino acid was bound to a specific site and that these sites were the terminal nucleotides of each RNA chain. Further support for this idea was achieved by a partial physical separation of the RNA chains specific for leucine from those specific for valine. In separate experiments, C™-valyl and C-leucyl RNA were chromatographed on Ecteola-cellulose columns as shown in Fig. 6a and b. The leading fractions of each peak (dotted portion of each peak) which possessed the highest specific ac- tivity and accounted for approximately 20% of the radioac- tivity applied to the column were pooled and the RNA was re- covered by alcohol precipitation. The leucine-C™ or valine-C¥ was removed and the RNA was tested for its capacity to accept a number of amino acids (Table IIT). The data show that the RNA preparations selected for their acceptor activities for valine or leucine have about 7 and 3.5 times higher activity for accept- ing these amino acids than does the original RNA fraction. More significantly, however, the marked alteration in the ratio of leucine to valine acceptor activity in the isolated fractions suggests that the chromotography resulted in a separation otf the Icucine- and valine-specific RNA chains. It should be pointed out that these same fractions are also enriched about 3- to 4-fold for isoleucine and methionine acceptor activity. We interpret these results as indicating that although the RNA chains specific for leucine and valine normally chromatograph towards the center or trailing portion of the peak, the addition of the amino acid changes their chromatographic properties so that they are eluted somewhat earlier and thus overlap the fractions specific for isoleucine and methionine. Although these experiments suggest that a partial physical separation of the leucine and valine acceptor RNA activity can be achieved, they also demonstrate the limited usefulness of this approach for isolating an RNA that is specific for a single amino acid. Separation of acceptor activities for different amino acids by chromatography have also been reported by Smith ef al. (36). More recent studies by Holley et al. (37) with coun- tercurrent distributions have demonstrated the separation of certain specific amino acid acceptor activities. Our own and these latter studies (86, 37), however, suffer from the difficulty of June 1961 attempting to separate one specific type of RNA chain from perhaps as many as 20 others with very similar physical proper- ties. A more effective approach would appear to be that ex- ploited by Brown e¢ al. (88) and by Zamecnik et al. (39). In these two cases the separation depends on selective adsorption of a specific amino acy] RNA (88) or on the alteration of all but a single class of amino acid-specific RNA chains (39). SUMMARY Amino acid-acceptor ribonucleic acids have been isolated from Escherichia coli by extraction of dried cells with sodium laury! sulfate, fractionation with salt and ethanol precipitation, and chromatography on Ecteola-cellulose. Alkaline hydrolysis of the acceptor ribonucleic acid yields a single nucleoside, adenosine, and roughly equivalent amounts of adenylic and uridylic acids and of guanylic and cytidylic acids. The minimal molecular weight, based on the nucleotide compo- sition and on the assumption of a single terrninal nucleotide unesterified in the 2’- and 3’-hydroxyl groups, is 31,000 + 5%. The optical density at 260 my of the ribonucleic acid in 0.2 mM NaCl, pH 7.0, increases (about 23%) as the temperature is raised between 40 and 80° and returns to the original value on cooling. When the ribonucleic acid is tested for its ability to accept leucine and valine at temperatures up to 55°, no difference is found in the yield of leucyl- and valyl ribonucleic acid sug- gesting that a specific secondary structure may be necessary for the amino acy} ribonucleic acid synthetases. The s2o.. for valylribonucleic acids is 4.5 + 0.2 8 which is essentially the same as the sedimentation coefficient of the major portion of the ultraviolet-absorbing material of the ribonucleic acid. Attempts to isolate ribonucleic acid chains specific for leucine or valine resulted in partial resolution of the two types of ribo- nucleic acid chains, although there was little or no separation of either of these from acceptor ribonucleic acid chains specific for isoleucine or methionine. REFERENCES 1. Hoaaranp, M. B., Stepnenson, M. L., Scorr, J. F., Hecur, L. 1, ann Zamecnix, P. C., J. Biol. Chem., 231, 241 (1958). 2. Bera, P., anp Orenaanp, E. J., Proc. Natl. Acad. Sci. UV. S., 44, 78 (1958). 3. Berc, P., Bercmann, F. H., Orencanp, E. J., anp Digcx- MANN, M., J. Biol. Chem., 236, 1726 (1961). 4. Hotiey, R. W., J. Am. Chem. Soc., 79, 658 (1957). 5. Wetss, 8. B., Acs, G., anp Lipmann, F., Proc. Natl. Acad. Sci. U. S., 44, 189 (1958). 6. Scrwzet, R. 8., Bovarn, F. G., Auten, E. H., anp Guass- MAN, l., Proc. Natl. Acad. Sct. U. S8., 44, 173 (1958). 7. Lipmann, R., Hiusmann, W. C., Harrmann, G., Boman, H. G., anp Acs, G., J. Cell. Comp. Physiol., 54, 75 (1959). 8. Zacnau, H. G., Acs, G., anp Lipmann, F., Proc. Natl. Acad. Scr. U. S., 44, 885 (1958). E. J. Ofengand, M. Dieckmann, and P. Berg 1747 9. Preiss, J.. Berg, P., Orencanp, E. J., Beremann, F. H., AND DirckMaNN, M., Proc. Natl. Acad. Sct. U. S., 45, 319 (1959). 10. Hecut, L. J., Steruenson, M. L., ann Zamecnig, P. C., Proc. Natl. Acad. Sci. U. S., 45, 505 (1959). li. Preiss, J., Dieckmann, M., ano Berg, P., J., Biol. Chem., 236, 1748 (1961). 12. CanaLuakis E.8., anpD Herpert, E., Proc. Natl. Acad. Sci. U.S., 46, 170 1960). 13. Sincer, M. F., ano Canvoni, G. L., Biochim. et Biophys. Acta, 39, 182 (1960). 14, Zinuic, W., ScHacTscHaBEL, D., ano Krone, W., Z. Physiol. Chem., 318, 100 (1960). 15. ALLEN, In. H., Guassman, E., Corpses, E., anp Scuweet, R. S., J. Biol. Chem., 235, 1068 (1960). 16. Gotptuwarir, D., J. Biol. Chem., 234, 3245 (1959). 17. Monier, R., SrerHenson, M. L., anp ZamMeEcwix, P. C., Bio- chim. et Biophys. Acta, 48, 1 (1960). 18. OTaka, E., anD Osawa, S., Nature (London), 185, 921 (1960). 19. Osawa, 8., anp OtTaxa, E., Biochim. et Biophys. Acta, 36, 549 (1959). 20. Tissibres, A., J. Molecular Biol., 1, 365 (1959). 21. Beramann, F. H., Bere, P., anp Digcxmann, M., J. Biol. Chem., 236, 1735 (1961). 22. Aupaum, H. G., ano Umsreit, W. W., J. Biol. Chem., 167, 369 (1947). 23. Discue, Z., in E. Cuarcarr anp J. M. Davipson (Editors), The nucleic acids, Vol. 1, Academic Press, Inc., New York, 1955, p. 285. 24, Fiskn, C. H., ano SussaRow, Y., J. Biol. Chem., 66, 375 (1925). 25. CuEen, P.8., Toripara, T. Y., anD WARNER, H., Anal. Chem., 28, 1756 (1956). 26. Spaur, P. F., anp Tissires, A., J. Molecular Biol., 1, 237 (1959). 27. Lowry, O. H., Roseproucu, N.J., Farr, A. L., Aanp RANDALL, R. J., J. Biol. Chem., 193, 265 (1951). 28. Leaman, I.R., J. Biol. Chem., 285, 1479 (1960). 29. Beaven, G. H., Houipay, E. R., anp Jounson, E. A., in E. Cuarcarr anp J. M. Davipson, (Editors), The nucleic acids, Vol. 1, Academic Press, Inc., New York, 1955, p. 493. 30. Coun, W., J. Biol. Chemr., 235, 1488 (1960). 31. Dunn, D. B., Smaru, J. D., anp Spaur, P. F., J. Molecular Biol., 2, 113 (1960). 32. Dunn, D. B., Biochim. et Biophys. Acta, 34, 286 (1959). 33. Dory, P., Borprker, H., Fresco, J. R., Haseixorn, R., AND Litt, M., Proc. Natt. Acad. Sct. U. S., 45, 482 (1959). 34. TiseLius, A., Peperson, K. O., ano SvepBErG, T., Nature (Lendon), 140, 848 (1937). 35. ScuacuMan, H. K., in §. P. CoLowick anp N. O. Kapian (Editors), Methods in enzymology, Vol. 4, Academic Press, Inc., New York, 1957, p. 32. 36. SmitH, K. C., Corpss, E., anp Scuweert, R. S., Biochim. et Biophys. Acta, 33, 286 (1959). 37. Houiey, R. W., Arcar, J.. anp Doctor, B. P., Ann. N.Y. Acad. Sci., 88, 745 (1960). 38. Brown, G. L., Brown, A. V. W., anp Gorpon, J., in Brook- haven symposium in biology, No. 12, U. 8S. Brookhaven Na- tional Laboratory, Upton, N. Y., 1959, p. 47. 39. Zamecnix, P. C., SrepHenson, M. L., anv Scort, J. F., Proc. Natl. Acad. Sct., U. S., 46, 811 (1960).