Synthesis and Aminoacylation of 3 '-Amino-3 '-Deoxy Transfer

Proc. Nat. Acad. Sci. USA Vol. 70, No. 9, pp. 2671-2675, September 1973

Synthesis and Aminoacylation of 3 '-Amino-3 '-deoxy Transfer RNA and Its Activity in Ribosomal Protein Synthesis (puromycin-tRNA/3'-amino-3'-deoxy ATP/aminoacyl synthetase/ribosomal acceptor site)

THOMAS H. FRASER AND ALEXANDER RICH Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139 Contributed by Alexander Rich, June 12, 1973

ABSTRACT 3'-amino-3'-deoxy adenosine was enzy- tRNA synthetase and phenylalanine. The amino acid is matically converted into 3'-amino-3'-deoxy ATP. This ATP bound to the modified tRNA through the 3'-amino group, as analogue was incorporated into the 3'-terminal adenosine position of Escherichia coli tRNA. The modified tRNA shown by the stability of this aminoacyl-tRNA to base- was aminoacylated with phenylalanine by use of E. coli catalyzed hydrolysis in contrast with the lability of normal phenylalanyl tRNA synthetase. The phenylalanine was aminoacyl-tRNA. Finally, we show that the aminoacyl-tRNA attached to the 3'-amino group of the tRNA, as shown by analogue can be bound to the acceptor site of the ribosome its high resistance to base-catalyzed hydrolysis in contrast with the normal lability of phenylalanyl-tRNA. Amino- where it can accept acetyl-phenylalanine and form a dipep- acyl tRNA synthetases charge the 3'-amino-3'-deoxy tRNA tide; however, this analogue is unable to donate its phenyl- with kinetics that are similar to those of the charging reac- alanine and incorporate it into a growing polypeptide chain. tion in which normal tRNA is the substrate. When phenylalanyl-3'-amino-3'-deoxy tRNA is used in a protein- METHODS synthesizing system directed by poly(U) in vitro, this molecule is capable of receiving an acetyl-phenylalanine 3'-Amino-3'-deoxy A TP Was Prepared by the following from the donor site of the ribosome. However, the ribo- method. 60,umol of 3'-amino-3'-deoxy adenosine (a gift of some is unable to cleave the anmide bond connecting the NIH, Cancer Chemotherapy Cerfter), according phenylalanine to the tRNA molecule; hence, the phenyl- prepared alanyl-3'-amino-3'-deoxy tRNA has acceptor but not to Baker et al. (3), was incubated with 8 ,umol of ATP, 300 donor activity in protein synthesis. Failure ofthe ribosome ,umol of phosphoenol pyruvate, 0.1 mg of pyruvate kinase to cleave the amide bond may be due to its greater sta- (Sigma, 320 units/mg), 10 /g of rabbit-muscle myokinase bility relative to the normal ester bond. However, it may (Boehringer), 220 units of adenosine kinase [prepared from also be due to the fact that the isomerization of the pep- 5 tidyl chain between the 2' and 3' positions of adenosine is rabbit liver by the method of Lindberg et al. (4, 5) ], ,imol of prevented due to the amide bond at the 3' position, and MIgCI2, 500 ,imol of KCl, and 1.25 ml of glycerol in a total cleavage may normally occur with the peptidyl chain on volume of 12.5 ml maintained at pH 5.8 by 7 mMI Tris- the 2' position of adenosine. maleate buffer. After 20 hr at room temperature (25°), the mixture was on a Dowex-1-formate in involves reaction chromatographed The central repetitive reaction protein synthesis column (2 X 15 cm) and eluted with a linear gradient of addition of an aminoacvl-tRNA to the acceptor site on the 0-2.2 \I formate The fractions RNA ammonium (pH 4.5). pooled ribosome specified by messenger (mRNA). Peptide containing 3'-amino-3'-deoxy ATP were made 50% in are formed the ribosomal transferase, bonds by peptidyl ethanol, and the pH was adjusted to 8.5. The modified ATP which transfers the chain from one growing polypeptide was precipitated by addition of 0.1 volume of saturated tRNA to an adjoining aminoacyl tRNA. During this syn- BaBr2; and the precipitate was recovered by cen- the ester low-speed thesis, the peptidyl transferase normally cleaves The barium salt of 3'-amino-3'-deoxy ATP was connecting the amino acid and the 3'-terminal adenosine trifugation. bond solubilized by batch treatment with Dowex-50-Na+ at pH of tRNA and forms a bond between the carbonyl peptide 6.0. The sodium salt was stored frozen in solution at -20°. group of the peptidyl chain and the a-amino group on the adjoining aminoacyl-tRNA. Peptidyl transferase has the ability Preparation of Phenylalanyl-3'-amino-3'-deoxy tRNVA. Snake to synthesize not only the amide bond, but also an ester bond venom phosphodiesterase was used to remove the 3'-terminal (1, 2). Thus a-hydroxyacyl-tRNA is active in protein syn- AMIP from tRNA by a modification of the method of M\Tiller thesis, and ribosomes can catalyze the synthesis of polyesters. and Philipps (6, 7). 25 mg of E. coli B deacylated tRNA We have been interested in the activity of a tRNA analogue (Schwarz-Mann) were incubated at 22° for 2 hr with 0.2 mg in which the 3'-hvdroxvl of the 3'-terminal adenosine is sub- of snake venom phosphodiesterase (Worthington), 0.4,mol of stituted with an amino group. Using 3'-amino-3'-deoxy ATP glycine (pH 9.0), and 10,imol of magnesium acetate in a total as a substrate, we show that tRN\-nucleotidyl transferase volume of 1 ml. The reaction mixture was deproteinized by will incorporate 3'-amino-3'-deoxy A'MP at the 3' terminus of passage over a silicic-acid column (7). tRNA was precipitated tRNA as a substitute for the 3'-terminal AMP. This tRNA in ethanol and redissolved in distilled water. CNIP was re- analogue is capable of aminoacylation using phenylalanyl incorporated in the tRNA with purified tRNA-nucleotidvl transferase to yield tRNA-C-C (7). Abbreviation: Phe-N-tRNA, phenylalanyl-3'-amino-3'-deoxy The reaction mixture for preparation of 3'-amino-3'-deoxy tRNA. tRNA (tRNA-C-C-AN) contained in 2 ml: 8 mg of tRNA- 2671 Downloaded by guest on September 25, 2021 2672 Biochemistry: Fraser and Rich Prot. Nat. Acad. Sci. USA 70 (1978)

3' AMINO-3-DEOXY ADENOSINE NH2 tRNA to accept ['H]ATP was then assayed with tRNA- ADENOSINE KN N nucleotidyl transferase (7).

KINASE HOCH2 N A 100,000 X g supernatant (S-100) was prepared from E. coli D-10, and tRNA-nucleotidyl transferase was removed by 3'-AMINO-3!-DEOXY AMP liquid polymer phase fractionation (8). The preparation was 1 MYOKINASE then passed over a DE-52 column to remove RNA (9). The 3!-AMINO-3' DEOXY ADP S-100 fraction was stored in 20 mM Tris * HCl (pH 7.8)-2 mM 2 PYRUVATE KINASE 2-mercaptoethanol-1 mM magnesium acetate-10% glycerol at -200. 3!-AMINO-3!-DEOXY ATP The tRNA charging reaction contained the following com- tRNA- NH2 ponents in a volume of 1 ml: 0.107 ,umol of [14C]phenylalanine NUCLEOTIDYLTRANSFERASE N N I F tRNA-C-C e (New England Nuclear Corp., specific activity 472 Ci/mol), 3'-AMINO-3'-*DEOXY tRNA OCH2 NN) 1.5 mg of 3'-amino-3'-deoxy tRNA, 0.42 mg of S-100 frac- PHENYLALANYL tion free of tRNA nucleotidyltransferase and RNA, 4 /umol NH OH of dATP, 120 umol of 20 Mmol of magnesium acetate, tRNA SYNTHETASE KCl, IFD CHrCH-CROIHHO and 20 ,umol of 2-mercaptoethanol; it was buffered at pH 7.5 PHENYLALAMNYL-3!-AMINO-3'-DEOXY tRNA NH2 with 100 mM Tris HCl. After incubation for 45 min at 370, FIG. 1. Outline of the steps taken to convert 3'-amino-3'- the reaction mixture was deproteinized. deoxy adenosine into phenylalanyl 3'-amino-3'-deoxy tRNA. Phenylalanyl-tRNA (Phe-tRNA) and Acetyl Phenylalanyl- The chemical structures of the starting material and the final tRNA (AcPhe-tRNA). Phe-tRNA was prepared from de- product are shown on the right. Arrows on the left have the en- E. coli B tRNA as described zymes that are used in the reactions next to them. acylated essentially by Bretscher (9), except that unlabeled amino acids were not included and deproteinization was done on a silicic-acid column. AcPhe- C-C, 25 ug of purified E. coli tRNA-nucleotidyl transferase tRNA was prepared according to de Groot et al. (10). Ac-Phe (8), 5 mg of reduced glutathione, 4 umol of 3'-amino-3'-deoxy tRNA was purified as described (11). ATP, 0.75 mg of bovine-serum albumin, 25 umol of magnesium acetate, and 60 Mmol of KCl; it was buffered at pH 9.2 by 50 mM glycine. After 2 hr at 370, the mixture was deproteinized on silicic acid. The ability of 3'-amino-3'-deoxy TABLE 1. Acceptor activity of Phe-N-tRNA Acid-precipitable

100. [14C]AcPhe 030 Incubation % of bound z < Reaction time [14C] AcPhe- > \ \ r tUridine mixture (min) dpm tRNA LUcr 60 Phe-N-Ado (Base)' Complete 0 230 8.2 15 2405 85.9 oJ40- ^ V Complete Phe-N-Ado Complete 30 2671 95.4 20\< * Phe-Ado(Base) Minus poly(U) 15 251 5.4 20-- Minus Phe-N-tRNA 15 81 2.9 |* ^ Phe-Ado 0. 0 3C 60 90 120 Phe Acceptor activity of Phe-N-tRNA was determined in a two- MINUTES _ step assay. In the first step, [14C]AcPhe-tRNA was bound to the FIG. 2 (left). Stability of various aminoacylated tRNAs is ribosome. The incubation mixture contained per ml: 27.6 A260 units shown as a function of time at 370 and pH 9.5. ["4C] Phenylalanine of puromycin-treated ribosomes (13) washed in 1M NH4Cl three was used for AcPhe-tRNA (A) and Phe-tRNA (0), while ['H]- times, 80 usg of poly(U), 0.2 umol of GTP, 0.4 umol of phosphoenol phenylalanine was used for Phe-N-tRNA (-). tRNA was precip- pyruvate, 40 jg of pyruvate kinase (320 units/mg), 750 jug of itated with 10% trichloroacetic acid at different times, and the crude initiation factors (12), 8 umol of magnesium acetate, 80 precipitable counts per min are expressed as a percentage of those Mmol of NH4Cl, 6 umol of 2-mercaptoethanol, and 140 pmol of precipitable at 0 time. purified [14C]AcPhe-tRNA. The mixture was buffered at pH 7.8 FIG. 3 (right). ['4C]Phe-tRNA and ['4C]Phe-N-tRNA were with 50 mM Tris * HCl. After incubation for 10 min at 30°, bind- charged. They were incubated separately at 370 in pH 5.5 am- ing of [14C]AcPhe-tRNA was essentially complete. About 2 pmol monium acetate buffer with 500 Mg/ml of pancreatic RNase for 15 of [14C]AcPhe-tRNA was bound per A260 unit of ribosomes. Bind- min. Reaction mixtures were precipitated in ethanol and centri- ing depended on the presence of poly(U). The mixture was cooled fuged at 15,000 X g for 15 min. The supernatants were analyzed to 00, and 0.5 mg of ['H]Phe-N-tRNA, 1% charged with [3H]- by thin-layer electrophoresis. Where indicated (Base), the superna- Phe-N-tRNA, and 450 pmol of purified elongation factor Tu were tants were incubated with 2 M triethylamine for 30 min at 37°. added per ml. [3H]Phe-N-tRNA had been previously incubated Thin-layer electrophoresis was done in 20% acetic acid-ammo- for 1 hr at 37° in pH 9.5 Tris-HCl to insure that no normal nium buffer at pH 2.7 for 1 hr at 34 V/cm. Origin of the electropho- [3H] Phe-tRNA was present. Incubation at 30° was continued for resis corrected for endosmosis is shown by the position of un- the indicated times. SO,-M Samples were removed and 150 ul of 0.5 charged ['4C]uridine. Phe is ["4C]phenylalanine, Phe-Ado is M Tris - HCl (pH 9.5) was added; the mixture was incubated at [14C]phenylalanyl-adenosine, Phe-N-Ado is [14C]phenylalanyl 370 for 2 hr. 10% Trichloroacetic acid was added, and the pre- 3'-amino-3'-deoxy adenosine. cipitable counts were measured. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) 3'-Amino-3'-deoxy Transfer RNA 2673

RESULTS alanine attached to tRNA-C-C-AN. The presence of an amino Fig. 1 outlines the synthesis of phenylalanyl 3'-amino-3'- group on the 3' position of tRNA-C-C-AN and the high resis- deoxy tRNA (Phe-N-tRNA). 3'-Amino-3'-deoxy adenosine tance to basic hydrolysis of phenylalanine attached to this was incubated with a mixture containing liver adenosine tRNA indicates that the amino acid is attached to the kinase, muscle myokinase, muscle pyruvate kinase, ATP, and terminal adenosine through a base-stable amide linkage phosphoenolpyruvate. 3'-Amino-3'-deoxy ATP was iso- rather than through the base-labile ester linkage found in lated from the reaction mixture by passage through a Dowex normal phenylalanyl-tRNA. 1 column. The analogue elutes from the column at a position Incorporation of 3'-amino-3'-deoxy ATP into the 3' where ADP is normally found because the 3'-amino group terminus of tRNA-C-C was tested in another manner by sub- on the analogue is protonated at this pH and effectively jecting Phe-N-tRNA to extensive digestion with ribonuclease. neutralizes one of the negative charges on the 3'-amino-3'- In normal aminoacyl-tRNA, this digestion liberates the deoxy ATP. The final yield for the synthesis of the ATP terminal adenosine with its attached amino acid as an intact analogue was 60%, based on the amount of 3'-amino-3'-deoxy molecule. Both ['4C]Phe-tRNA and ['4C]Phe-N-tRNA were adenosine in the initial reaction mixture. subjected to ribonuclease digestion and analyzed by electro- E. coli tRNA was incubated with snake venom phospho- phoresis at pH 2.7. An autoradiogram of the thin-layer plate diesterase to remove the terminal AMP. Because some of the is shown in Fig. 3. Phe-adenosine and Phe-N-adenosine exposed CMPs at the 3' end of the molecule were also re- have about the same mobility, although Phe-N-adenosine moved, CTP was added to the digested tRNA mixture to- migrates slightly more rapidly. This is probably due to slightly gether with tRNA-nucleotidyl transferase before the analogue more protonization of the amide carbonyl oxygen than the was incorporated. Subsequently, tRNA-C-C was incubated ester carbonyl oxygen at this pH. After incubation for 30 min with a 100-fold molar excess of 3'-amino-3'-deoxy ATP in the 601 ,I . presence of tRNA-nucleotidyl transferase. After 2 hr at 370 606 _ the product was isolated by deproteinization and ethanol pre- 7 cipitation of the reaction mixture. The amount of analogue accepted by tRNA-C-C was estimated by incubating the 40- 00-_5- product with [3H]ATP in the presence of tRNA-nucleotidyl transferase. In different preparations 25-50% of the tRNA accepted [3H]ATP. This finding suggested that 50-75% of the C-, z U tRNA-C-C had accepted the 3'-amino-3'-deoxy ATP on the 20,- 3' terminus to make tRNA-C-C-AN. 0J At this stage of the investigation we did not attempt to

demonstrate directly the presence of 3'-amino-3'-deoxy 0 7, 0- adenosine on the 3' terminus of tRNA. Instead, we chose to 0 20 40 60 0 5 10 15 20 incubate the analogue tRNA with aminoacyl tRNA syn- MINUTES MINUTES thetases in the presence of ['4C]phenylalanine to see whether FIG. 4 (left). Charging of tRNA and 3'-amino-3'-deoxy tRNA tRNA-C-C-AN could be charged and, if so, whether it had with 15 ['4C]aminoacids. Three different tRNA preparations were incubated with [14C]aminoacids and S-100 fraction free of RNA properties that were different from those of unmodified and tRNA-nucleotidyl transferase. Intact tRNA (tRNA-C-C-A) tRNA. dATP was used as the energy source in the amino- (0), 3'-amino-3'-deoxy tRNA (tRNA-C-C-AN) (0) and tRNA acylation reaction rather than ATP, because ATP would be from which the 3'-terminal AMP had been removed and CMP added on the 3' position of unreacted tRNA-C-C by any added back (tRNA-C-C) (A) were all tested. Trichloroacetic acid- tRNA-nucleotidyl transferase present in the S-100 prepara- precipitable radioactivity is plotted as a function of incubation tion. S-100 fraction free of tRNA-nucelotidyl transferase time at 37°. tRNA-C-C-A and tRNA-C-C-AN curves were nor- catalyzed aminoacylation with 15 [14C]aminoacids to the malized to account for the percentage of molecules that had a 3'- same extent as unfractionated S-100 preparation, indicating terminal AMP or 3'-amino-3'-deoxy AMP, respectively. 90% of that the aminoacyl tRNA synthetases were active in the the tRNA-C-C-A molecules had a 3'-terminal AMP, while 50% preparation. After aminoacylation with radioactive phenyl- of the tRNA-C-C-AN molecules had a 3'-terminal 3'-amino-3'- the reaction deoxy AMP, the remainder being tRNA-C-C. alanine, mixture was deproteinized. Phe-N- FIG. 5 (right). In vitro protein synthesis directed by Poly(U) tRNA was then characterized by demonstrating its resistance with either ['4]CPhe-tRNA (0) or Phe-N-tRNA (0). The incuba- to base-catalyzed hydrolysis. Fig. 2 shows the hydrolysis of tion mixture contained per ml: 0.8 mg of poly(U) (Sigma, molecular aminoacyl-tRNA for both Phe-N-tRNA and unmodified weight > 100,000), 4.7 A260 units of ribosomes washed three times Phe-tRNA. On incubation at 370 (pH 9.5), Phe-tRNA is with 1 M NH4Cl, 0.2,mol of GTP, 2 ,umol of phosphoenolpyru- rapidly hydrolyzed with a half-life of 7 min. In contrast to vate, 35 uAg of pyruvate kinase (Sigma, 405 units/mg), 0.34 mg of this, the phenylalanine attached to the tRNA-C-C-AN is tRNA-nucleotidyl transferase and RNA-free S-100 fraction, 15 largely resistant to hydrolysis. During the first 15 min, about Mmol of magnesium acetate, 80 ,umol of NH4Cl, 6 Mmol of 2-mer- 10% of the phenylalanine was no longer precipitable by 10% captoethanol, and 7.4 mg of ['4C]Phe-tRNA (2.2% charged with trichloroacetic acid, but after that the amount of base-resis- [I4C]phenylalanine) or 6.7 mg of [14C]Phe-N-tRNA (1.6% tant phenylalanine did not change appreciably. This initial charged with ['4]C phenylalanine). The mixture was buffered at decrease may be due to contaminating Phe-tRNA. Acetyla- pH 7.8 with 50 mM Tris * HCl. After the indicated times of incubation at 300, 50-,ul samples were removed, and 50 Mul of 0.1 M tion of the a-amino group of phenylalanyl-tRNA stabilizes EDTA and 5 ,l of pancreatic RNase (10 mg/ml) were added. it somewhat to base-catalyzed hydrolysis (14), and Fig. 2 also Incubation was continued for 15 min at 300. The samples were shows its hydrolysis. Although AcPhe-tRNA has a half-life precipitated at 00 with 10% trichloroacetic acid and filtered on of about 25 min, it is distinctly more labile than the phenyl- glass-fiber filter paper. 0, no poly(U). Downloaded by guest on September 25, 2021 2674 Biochemistry: Fraser and Rich Proc. Nat. Acad. Sci. USA 70 (1973)

in 2 M triethylamine, the radioactivity in Phe-adenosine this binding depends upon the presence of poly(U) (Table 1). migrates with the phenylalanine marker while Phe-N- AcPhe-tRNA is believed to bind to the peptidyl or P-site adenosine is unaffected by this incubation. These results are of ribosomes. Phe-N-tRNA was added to the ribosomes and interpreted as confirming the existence of 3'-amino-3'-deoxy incubated at 300. The reaction mixture was then treated at adenosine at the 3' terminus of tRNA where it is amino- pH 9.5 under conditions that hydrolyze the ester linkage be- acylated by phenylalanine through a base-stable amide link- tween AcPhe and tRNA, but are unable to break the amide age. linkage between phenylalanine and tRNA-C-C-AN (see Fig. Experiments were next done to see whether various differ- 2). If Phe-N-tRNA is bound to ribosomes in the acceptor or ent tRNAs had been modified by 3'-amino-3'-deoxy AMP A-site, then [14C]AcPhe may be transferred to Phe-N-tRNA and were able to be aminoacylated. This was tested by in- with formation of AcPhe-Phe-N-tRNA. This material would cubating 3'-amino-3'-deoxy tRNA with a mixture of 15 be stable to incubation at pH 9.5, whereas when normal Phe- ['4C]aminoacids in the presence of a crude preparation con- tRNA is added to the ribosome, the ester bond in AcPhe- taining all of the E. coli tRNA synthetases. The kinetics of Phe-tRNA would be hydrolyzed by the incubation and the charging of the mixture of [14C ]aminoacids with three different radioactivity would no longer be precipitable by trichloro- substrates are illustrated in Fig. 4. There is no detectable acetic acid. As shown in Table 1, after 30 min of incubation charging of tRNA-C-C, and the overall charging of tRNA- over 95% of the ['4C]AcPhe is acid-precipitable after incuba- C-C-A and tRNA-C-C-AN are rather similar. tRNA-C-C-AN tion at pH 9.5. This precipitability depends upon the presence is somewhat slower in reaching its maximum; it charges of both poly (U) and Phe-N-tRNA. These experiments suggest rapidly for about 20 min and then slowly increases. Normal that Phe-N-tRNA was able to accept [14C]AcPhe from the tRNA reaches a maximum near 20 min and thereafter there P-site of the ribosomes even though the experiments de- is some decrease in the amount of trichloroacetic acid-pre- scribed in Fig. 5 suggested that it was unable to promote syn- cipitable ["4C]aminoacids. This decline may be associated thesis of polyphenylalanine. with a slow digestion of the 3' end of the tRNA due to con- Another experiment was done with an incubation similar taminating exonucleolytic activity. In contrast to this, no to that of Table 1 except that after 15 min of incubation with decline is noted for tRNA-C-C-AN, possibly because the Phe-N-tRNA, a large amount of unlabeled normal Phe-tRNA amino acid is not so readily removed once tRNA is amino- was added. If AcPhe-Phe-N-tRNA were able to donate to this acylated and, therefore, it may be protected from exonu- unlabeled normal Phe-tRNA, the amount of acid-precipitable cleolytic attack. It has been reported that amino acids ester- radioactivity resistant to incubation at pH 9.5 would decrease. ified to the 3' end of tRNA protect it from exonucleolytic di- However, the results were unaffected by addition of normal gestion (6). However, this protection is partial rather than Phe-tRNA. This experiment supports our interpretation that complete, suggesting that in the presence of aminoacyl tRNA ribosomes are unable to break the amide linkage in Phe-N- synthetases, amino acids may be continually removed and tRNA to allow it to act as a donor in polypeptide synthesis. added back to the tRNA. The fact that the tRNA-C-C-AN curve continues to rise suggests that the rate of discharging DISCUSSION is reduced when an amide bond is involved in aminoacylation. The similarity in the total chargability of the mixture of Several experiments are reported here in which 3'-amino-3'- 3'-amino-3'-deoxy tRNAs as compared with normal tRNA deoxy adenosine derivatives are used by several different en- suggests that all the amino acids were being incorporated and zymes with an efficiency similar to that of the normal sub- were therefore active with tRNA-C-C-AN as substrates for strates. In reactions involved in synthesis of Phe-N-tRNA, the different aminoacyl tRNA synthetases. five different enzymes are able to accommodate the analogue Experiments were next done to determine the activity of substrates with apparent ease. In the reaction involving Phe-N-tRNA in protein synthesis. The first experiment was myokinase, 3'-amino-3'-deoxy ANIP will act as a phosphate done with a polyphenylalanine synthetic system directed by acceptor to form the corresponding diphosphate analogue poly(U), with either [14C]Phe-tRNA or [14C]Phe-N-tRNA. and, in addition, the triphosphate analogue can act as a donor Phe-N-tRNA was not converted into an RNase-stable, tri- for transferring a phosphate to AMIP. In the synthetase reac- chloroacetic acid-precipitable product (Fig. 5). This is in tion shown in Fig. 4, the kinetic curve is an aggregate involv- marked contrast to the rapid production of an RNase-stable, ing several different synthetases in the reaction mixture. Al- trichloroacetic acid-insoluble product when normal Phe-tRNA though the overall charging kinetics are similar, the detailed was used. The small amount of Phe-N-tRNA converted into charging with purified aminoacyl tRNA synthetases and a trichloroacetic acid-precipitable product may be due to the specific 3'-amino-3'-deoxy tRNAs should be examined. The presence of nascent polypeptide chains on the ribosomes since fact that the kinetics appear similar to those in which normal treatment of these ribosomes with puromycin reduced this in- substrate is used may be explained by the recent finding (15) corporation by more than 50%. This effect, however, sug- that suggests that the synthetase reaction involves the initial gested that although Phe-N-tRNA could not be incorporated aminoacylation of the 2'-hydroxyl group with subsequent into an acid-precipitable product, it nonetheless might be transfer of the amino acid between the 2' and 3' position. If active as an acceptor of nascent polypeptide chains. this reaction mechanism is correct, it is likely that the amino Phe-N-tRNA may be inactive in protein synthesis either acid is transferred to and remains in the 3' position of the because it fails to bind to the ribosome or because the ribosome tRNA-C-C-AN in view of the great stability of Phe-N-tRNA is unable to break the amide linkage between phenylalanine at high pH. If the rate-limiting step in the charging reaction and tRNA. This was tested directly with a preparation in is addition of amino acid to the 2' hydroxyl, the kinetics which [14C]AcPhe-tRNA was bound to the ribosome in the might be very similar when the charging of tRNA-C-C-AN presence of poly(U). This material binds to the ribosomes and is compared with normal tRNA substrate. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) 3'-Amino-3'-deoxy Transfer RNA 2675

When a-hydroxyacyl-tRNAs are used as a substrate, isomerization of the amino acid to the 2' position. If, indeed, ribosomes can catalyze polyester formation (1, 2). The ester the translocation of peptidyl-tRNA from the A-site to the P- linkage between the a-hydroxy acid and the terminal adenosine site is associated with transfer of the peptidyl chain from the is broken by the ribosome, which then forms an ester linkage 3' position to the 2' position, or if this isomerization is needed between the a-hydroxy acids. Repetition of this process re- before donation of the peptidyl chain, then Phe-N-tRNA sults in formation of polyesters. In the present experiment we would be inactive not because of the enhanced stability of the are asking whether it is possible for ribosomes to break an peptide bond to cleavage by peptidyl transferase, but be- amide linkage between the amino acid and the 3'-terminal cause of impairment of transfer from the 3' to the 2' position. adenosine of tRNA in order to form an amide linkage in the It is clear that further experiments will be necessary in order polypeptide chain. PheN-tRNA is able to enter the acceptor to differentiate between these alternative explanations. site and is active in receiving AcPhe that is transferred to it by the peptidyl transferase enzyme. This acceptor activity We thank Dr. Harry B. Wood and the Cancer Chemotherapy is not surprising in view of the well-known activity of puro- program for supplying us with the 3'-amino-3'-deoxy adenosine. We thank Dr. Georg R. Philipps for the tRNA nucleotidyl trans- mycin, which is structurally similar to the 3' end of Phe-N- ferase and Eliot Jewkowsky for purified elongation factor Tu. We tRNA. For this reason, the analogue could be described as acknowledge the skillful technical assistance of Richard Parker. "puromycin-tRNA." However, the peptidyl transferase ap- This research was supported by grants from the National Insti- parently is unable to cleave the amide bond so that Phe-N- tutes of Health, National Science Foundation, and the National tRNA is unable to donate its amino acid to form an amide Aeronautics and Space Administration. linkage. The reasons for this inability of Phe-N-tRNA to act as a 1. Fahnestock, S. & Rich, A. (1971) Nature New Biol. 229, 8-10. donor in protein synthesis are unknown. There are two pos- 2. Fahnestock, S. & Rich, A. (1971) Science 173, 340-343. sible explanations that should be considered. One is that the 3. Baker, B. R., Schaub, R. E. & Kissman, H. M. (1955) J. peptidyl transferase breaks the amide bond at a markedly re- Amer. Chem. Soc. 77, 5911-5915. duced rate compared to the rate at which the ester bond is 4. Lindberg, B., Klenow, H. & Hanson, K. (1967) J. Biol. broken This is not Chem. 242, 350-356. during polypeptide synthesis. explanation 5. Lindberg, B. (1969) Biochim. Biophys. Acta 185, 245-247. unreasonable in view of the greater stability of the amide link- 6. Miller, J. P., Hirst-Burns, M. E. & Philipps, G. R. (1970) age in contrast to the relative lability of the ester linkage. Biochim. Biophys. Acta 217, 176-188. We cannot rule out a small donor activity of PheN-tRNA to 7. Miller, J. P. & Philipps, G. R. (1971) J. Biol. Chem. 246, form a trichloroacetic acid-insoluble polyphenylalanine pro- 1274-1279. 8. Miller, J. P. & Philipps, G. R. (1970) Biochem. Biophys. Res. duct since, as shown in Fig. 5, Phe-N-tRNA forms a smal! Commun. 38, 1174-1179. amount of RNase-stable, acid-insoluble precipitate compared 9. Bretscher, M. S. (1968) J. Mol. Biol. 34, 131-136. with the control, which does not contain poly(U). However, 10. deGroot, N., Lapidot, Y., Panet, A. & Wolman, Y. (1966) a large part of this appears to be due to nascent polypeptide Biochem. Biophys. Res. Commun. 25, 17-22. chains on the ribosome, as this product is reduced by more than 11. Lucas-Lenard, J. & Haenni, A. L. (1969) Proc. Nat. Acad. Sci. USA 63,93-97. 50% when puromycin-treated ribosomes are used. Another 12. Anderson, J. S., Bretscher, M. S., Clark, B. F. C. & Marcker, possible explanation for the failure of the analogue to act as a K. A. (1967) Nature 215, 490-492. donor may be that Phe-N-tRNA is never translocated into the 13. Harris, R. & Pestka, S. (1973) J. Biol. Chem. 248, 1168-1174. peptidyl site that is necessary for donor activity. Although the 14. Haenni, A. L. & Chapeville, F. (1965) Biochim. Biophys. Acta 114, 135-148. former explanation seems more favorable, we cannot rule out 15. Sprinzl, M., Scheit, K. H., Sternbach, H., von der Haar, F. & the latter. In addition, aminoacylation of the amino group on Cramer, F. (1973) Biochem. Biophys. Res. Commun. 51, 881- the 3' position by way of an amide bond effectively prevents 887. Downloaded by guest on September 25, 2021