Studies on the Action of Tetracycline and Puromycin*

SUMMARY By means of a convenient method for separating phenylalanyl puromycin from diphenylalanyl puromycin, it was found that, upon reaction with puromycin, phenylalanyl transfer RNA bound to ribosomes at5 to 6 mu Mg++ yields phenylalanyl puromycin exclusively while phenylalanyl-tRNA bound at 13 mu Mg++ yields diphenylalanyl puromycin as well as phenylalanyl puromycin. Tetracycline inhibited mostly the binding of phenylalanyl-tRNA to the acceptor site, but the binding to the donor site may be inhibited at 13 mu Mg++. Puromycin reaction of phenylalanyl-tRNA bound to the donor site was inhibited by the presence of N-ace-tylphenylalanyl-tRNA at the acceptor site.

Puromycin reaction of phenylalanyl-tRNA bound to the donor site was inhibited by the presence of N-acetylphenylalanyl-tRNA at the acceptor site.
It has been suggested that there are two ribosomal sites for the binding of aminoacyl transfer RNA (l-8).
One of the two sites is sensitive to puromycin and has been called the donor site (site 2) (5, 7). The other site has been called the acceptor site (site 1) (5, 7). In the presence of low concentrations of Mg++ (5 to 6 mM), the bound phenylalanyl-tR.NA reacted with puromycin in the absence of G factor; in the presence of relatively high Mg++ (13 mM), two ribosomal sites were presumably occupied by phenylalanyl-tRNA (1). These considerations provided a convenient method of assaying the action of antibiotics such as erythromycin, lincomycin, and streptomycin (9). In this communication, we extended our studies on the ribosomal sites with the method which separates phenylalanyl puromycin from diphenylalanyl puromycin to elucidate the action of tetracycline and puromycin.
Concerning the mode of action of tetracycline, it is believed that tetracycline inhibits the binding of aminoacyl-tRNA to the acceptor site of the ribosomes (10-12). However, results of the NH?-terminal analysis of polyphenylalanine formed from the complex which was prepared in the presence of tetracycline suggested that the binding of aminoacyl-tRNA to the donor site may also be influenced (7). The availability of the method to determine phenylalanyl puromycin as well as diphenylalanyl puromycin quantitatively made it possible to measure the percentage of inhibition of the binding of phenylalanyl-tRNA to the acceptor and the donor site, respectively.
* This research was supported by United States Public Health Service Grant GM-12,053, National Science Foundation Grant GB-18355, and Damon Runyon Memorial Fund for Cancer Research, Grant 799-DT.
It was found that tetracycline inhibits not only the acceptor site binding but also the donor site binding of phenylalanyl-tRNA in the presence of high Mg* (13 ma/r). The reaction of puromycin with aminoacyl-tRNA on the ribosome has been used extensively for studying peptide bond formation. However, it has not been clear whether puromycin approaches the donor site from the side of the acceptor site or not. By use of the complex having N-acetylphenylalanyl-tRNA both on the acceptor and the donor site, we concluded that puromycin approaches the donor site from the side of the acceptor site.

MATERIALS AND METHODS
Escherickia co& Extract and Other hlaterials-Preparation of ribosomes, tRNA from E. coli B, and aminoacyl-tRNA have been described in the previous communications (1). The ribosomes were washed three times with a buffer containing 0.1 M Tris-HCl (pH 7.8), 0.01 M magnesium acetate, 0.06 M potassium chloride, 0.006 M P-mercaptoethanol, and 0.5 M ammonium chloride, and were free from aminoacyl-tRNA transfer factor (T factor) and initiation factors.
Preparation of T factor and G factor was as described previously (13).
Binding of ['4~Phenylalanyl-tRNA to Ribosomes and Isolation of Complex of Ribosmes, poZy(U), and ['4C]Phenylalanyl-tRNA-A typical binding reaction mixture (0.5 ml) for the isolation of the complex, unless otherwise specified, contained 50 mM Tris-HCl (pH 7.2), 6 mM magnesium acetate, 40 mM ammonium chloride, 400 pg of poly(U), 5.2 mg of tRNA containing 8.5 X lo5 cpm of [14C]phenylalanyl-tRNA, and 5.8 mg of ribosomes. This binding condition was called condition A. In some cases, the binding reaction was performed under identical conditions except that the Mg+f concentration was 13 mM. This was called condition B. When the enzymatic binding of aminoacyl-tRNA was carried out, 85 pg of T factor, 0.2 mM GTP, and 2 ml4 dithiothreitol were also added to the above binding mixture.
The gradients were centrifuged for 65 min at 48,000 rpm in a Beckman-Spinco rotor (SW 50.1), and the 70 S ribosome portions were collected as the ribosomal complex.
Reaction of Bound [14C]Phenylalanyl-tRNA with Puromycin-The reaction mixture (0.5 ml) for the puromycin reaction contained 20 mM Tris-HCl (pH 7.2), 13 mM magnesium acetate, 80 mM NH&l, 0.2 mM GTP, 24 pg of G factor, 1 mM puromycin, and 46 Action of Tetracycline and Puromycin Vol. 247, No. 1 0.3 ml of the fraction containing the ribosomal complex isolated as described above. The reaction mixture was incubated for 60 min at 22", mixed with 2 ml of 0.01 M Tris-HCl (pH 7.8), and shaken well with 6 ml of ethyl acetate. The ethyl acetate extract (0.5 ml) was mixed with 5 ml of Bray's solution (14), and the radioactivity was measured by liquid scintillation counter. The ethyl acetate extract (5 ml) wa,s evaporated in a vacuum, redissolved in 0.6 ml of 0.5 M acetic acid, and applied on a Sephadex G-15 column as described below.
Approximately 70 to 75% of the bound [14C]phenylalanyl-tRNA reacted with puromycin under these conditions. Separation of Phenylalanyl Puromycin and Diphenylalanyl Puromycin by Xephadex G-15 Column Chrmatoglaphy-The puromycin derivatives of phenylalanine formed as described in the preceding section were analyzed by Sephadex G-15 column chromatography (15). The puromycin derivative in 0.5 ml of 0.5 M acetic acid was applied to a Sephadex G-15 column (0.8 x 150 cm) which had been equilibrated with 0.5 M acetic acid. The elution was carried out by 0.5 M acetic acid with a flow rate of 1 ml per 12 to 15 min. Each fraction (1 ml) was collected and mixed with 5 ml of Bray's solution and the radioactivity of the [14C]phenylalanyl puromycin derivatives was measured.
The elution profile is shown in Fig. 1.
The dinitrophenyl phenylalanine is soluble in ether. Thus, under these procedures, lOO'% of radioactivity should become ether soluble.
On the other hand, under identical conditions [14C]diphenylalanyl puromycin would yield only 50% of its radioactivity as ether-soluble material because of insolubility of phenylalanine into ether. The materials of the first peak (Fractions 38 to 42 in Fig. 1, A and B) and the second peak (Fractions 48 to 58 in Fig. 1B) were separately pooled, dried in a vacuum, and dissolved in 0.2 ml of 1 $Zo triethylamine. Dinitrophenylation was started by addition of 0.2 ml of 5% solution of dinitrofluorobenzene in alcohol and carried out for 4 hours at room temperature.
After the dinitrophenylation was completed, 6 N HCl (0.2 ml) was added to the reaction mixture, and free dinitrophenol was removed by ether extraction. Under these acidic conditions, most of the dinitrophenyl [14C]phenylal-any1 puromycin derivatives remained in the aqueous layer. The concentration of HCl of these aqueous layers was brought to 6 N by the addition of concentrated HCl, and acid hydrolysis was performed at 110' for 6 hours. The resulting hydrolysate was diluted to 0.6 N HCl with water, and ether-soluble materials extracted three times with 5-ml portions of ether. The radioactivities in the combined ether extracts and aqueous layer were compared.
With material of the first peak, a major portion (about 90%) of its radioactivity was converted to ether-soluble material indicating that the first peak represents phenylalanyl puromycin.
When the second peak was subjected to the identical NH*-terminal analysis, about 53yo of the radioactivity was converted to the ether-soluble material showing that the second peak represented diphenylalanyl puromycin. JJateriaZs-Preparation of N-acetylphenylalanyl-tRNA was performed as described previously (16) Our previous studies, on the mode of action of antibiotics (9), were dependent on this assumption. It was therefore desirable to obtain the evidence which would indicate that this is indeed the case. From this assumption it follows that we should have mostly phenylalanyl puromytin when the binding was carried out at low Mgf+ (condition A) while diphenylalanyl puromycin as well as phenylalanyl puromytin would be formed when the binding was carried out at high Mg* (condition B). Fig. 1A shows that the puromycin derivative formed under condition A gives one peak corresponding to a phenylalanyl puromycin upon passage through the Sephadex G-15 column.
As expected from the function of the G factor, the addition of the factor did not influence the nature or the quantity of the product as shown in this figure.
On the other hand, when the complex was made under condition B, the addition of the G factor and GTP caused a large increase of formation of phenylalanyl puromycin and diphenylalanyl puromycin as shown in Fig. 1B. Effect of Tetracycline on Binding of Phenylalanyl-tRNA--It has been proposed that tetracycline has an exclusive inhibitory effect on the binding of aminoacyl-tRNA to the acceptor site (l&12). On the other hand, during the studies on the relationship between the acceptor site and the donor site, it was observed that, under certain conditions, tetracycline may also inhibit the binding of phenylalanyl-tRNA to the donor site (7). It was therefore desirable to determine exactly the percentage of inhibition of the binding of aminoacyl-tRNA to each site. In the experiment shown in Table I the ribosomal complex was prepared under various conditions in the presence or absence of tetracycline.
The puromycin derivatives of [lJC]phenylaline were formed from these complexes and analyzed with Sephadex G-15 column.
It should be pointed out that the inhibitory effect of tetracycline on the binding reaction was much more pronounced when the binding reaction was carried out in the presence of high Mg++ or T factor.
In order to determine which site was more susceptible to tetracycline, the formation of puromycin derivatives of the bound phenylalanyl-tRNA was studied with or without G factor. AS shown in Table I the addition of G factor stimulated the formation of phenylalanyl puromycin derivatives when the ribosomal complex prepared at high Mgf+ (13 mM) or in the presence of T factor was used. Table I also shows the results of the analysis of the puromycin derivatives formed from these complexes. The relative amounts of phenylalanyl puromycin and diphenylalanyl puromycin were calculated.
With these values, it was possible to estimate the distribution of phenylalanyl-tRNA reactive with puromycin between the donor and the acceptor site. The principles used in calculating the distribution of phenylalanyl-tRNA between the two sites are (a) phenylalanyl puromycin formed in the absence of G factor was assumed to be located at the donor site; (b) diphenylalanyl puromycin was derived from phenylalanyl-tRNA bound to two sites; thus the  The reaction mixture for the preparation of the ribosomal com-mation of puromycin derivatives of [%]phenylalanine, and the plex was described in the text except that it contained 4 mg of ribo-column chromatography of the puromycin derivatives were carsomes, 32Opg of poly(U), and 1.8 X 106 cpm of [14C]phenylalanyl-ried out as described in the text. Where indicated 15 ag of G tRNA (4 mg of tRNA); in some cases, 85rg of T factor, 2 mM dithi-factor was added to the reaction mixture for the formation of pureothreitol, and 0.2 mu GTP were added. The isolation of the mycin derivatives.  The calculations of the amount of the puromycin-reactive ['*Clphenylalanyl-tRNA at the acceptor and the donor site were carried out as follows.
For example, from the ribosomal complex formed in the presence of 6 mM Mg*, 6190 cpm of the puromycin derivatives of [lQZ]phenylalanine were formed in the absence of the G factor (line 1 in Table I). Of this, 92yo (5695 cpm) were phenylalanyl puromycin and 8% (495 cpm) were diphenylalanyl puromycin.
Since diphenylalanyl puromycin must come from the phenylalanyl-tRNA at the donor and the acceptor site, 248 cpm (495 X $. = 248) were assigned to each of these sites. The phenylalanyl puromycin (5695 cpm) must come entirely from the donor site. In the presence of the G factor, 6640 cpm of puromycin derivatives of [r%]phenylalanine were formed (line 2 in Table I). Of this, 89% (5910 cpm) and 11% (730 cpm) were ['4C]phenylalanyl puromycin and diphenylalanyl puromycin, respectively.
Thus, 215 cpm (5910 -5695 = 215) of phenylalanyl puromycin were formed due to the addition of G factor. We regard that this was bound at the acceptor site and translocated to the donor site by the G factor. The diphenylalanyl puromycin formed due to the addition of G factor was 235 cpm (730 -495 = 235). We assign 117 cpm (235 X + = 117) to eachsite.
The total [r%]phenylalanyl-tRNA bound to the donor site is therefore 5695 + 248 + 117 = 6060 cpm and that bound to the acceptor site is 215 + 248 + 117 = 580 cpm. The sum of these values (6060 + 580 = 6640) represents total puromycin-reactive [r%]phenplalanyl-tRNA bound in the presence of 6 mM Mg++ (line 2 in Table  I Table I. radioactivity of this fraction was divided by two and equally distributed to the acceptor and donor site; and (c) phenylalanyl puromycin dependent on the presence of G factor was allotted to the acceptor site. The results of these calculations are summarized in Table II. It is clear from Table II that the effect of tetracycline was mainly on the acceptor site when the binding reaction was carried out at 6 mM Mg++ or in the presence of T factor.
On the other hand, when the binding reaction was carried out at 13 mM Mg", the effect of tetracycline on the binding of phenylalanyl-tRNA to the donor site became significant. However, even under these conditions, the inhibition of the binding of phenylalanyl-tRNA to the acceptor site was much larger than that to the donor site. It should be noted that the assumption (a) used in calculating these values may not necessarily be correct in view of the fact that a fair amount of nonenzymatic translocation of diphenylalanyl-tRNA takes place (17). If one assumes that similar nonenzymatic translocation takes place with phenylalanyl-tRNA bound at the acceptor site, the inhibitory effect of tetracycline at the donor site becomes less than a few percentage even in the presence of 13 mM Mg++.
In view of the fact that approximately equal amounts of phenylalanyl-tRNA are bound to donor and acceptor site in the presence of 13 mM Mg++ (2), nonenzymatic translocation of phenylalanyl-tRNA is probably not as much as that with diphenylala,nyl-tRNA. This is because non-enzymatic translocation of phenylalanyl-tRNA would give a far greater amount of phenylalanyl-tRNA at the acceptor site. At any rate, one can conclude that a major action of the inhibitory effect of tetracycline is on the acceptor site. It can also be seen in this table that the stimulation of the binding of [r4C]phenylalanyl-tRNA by T factor was mainly on the acceptor site in confirmation of our previous conclusion with the experiment where NH%terminal analysis of polyphenylalanine was employed to determine the amount of bound phenylalanyl-tRNA at these sites (2).

Studies on Puromycin
Action and Influence of Aminoacyl-tRNA at Acceptor Site on Reactivity of Aminoacyl-tRNA Bound at Donor Site-Since the puromycin reaction with the donor sitebound aminoacyl-tRNA is analogous to the peptide bond formation between aminoacyl-tRNAs bound at the donor and acceptor sites (B-20), it is reasonable to assume that puromycin would attack the aminoacyl-tRNA at the donor site from the side of the acceptor site. If this assumption is correct, one would expect that the presence of nonreactive aminoacyl-tRNA at the acceptor site would hinder the reactivity of the aminoacyl-tRNA at the donor site. The report contrary to this expectation (21) prompted us to examine this possibility with the present system. In the experiment described in Table III phenylalanyl-tRNA than with phenylalanyl-tRNA, the rate of formation of puromycin derivative was studied after a short incubation period.
As can be seen from the figure, at the initial period of the reaction a marked inhibition of the puromycin reaction of N-acetyl["C]phenylalanyl-tRNA at the donor site was observed. It should be pointed out that no G factor was added in this experiment.
Thus, the inhibitory effect of [r'%]phenylalanyl-tRNA at the acceptor site was stronger than that of Nacetyl[12C]phenylalanyl-tRNA because the former inhibited the puromycin reaction for two possible reasons, i.e. steric hindrance, as well as formation of N-acetyldiphenylalanyl-tRNA, while the latter inhibited only because of the possible steric hindrance. These results are consistent with the concept that puromycin attacks the donor site from the direction of the acceptor site.
The experiments described above were based on the assumption that the binding sites for N-acetylphenylalanyl-tRNA were the same as those for phenylalanyl-tRNA, and N-acetylphenylalanyl-tRNA behaved similarly to phenylalanyl-tRNA with respect to the binding to ribosomes.
To ascertain that this assumption was correct, the binding sites for N-acetylphenylalanyl-tRNA were compared with those for phenylalanyl-tRNA. As shown in Table IV