Use of 50 S-binding Antibiotics to Characterize the Ribosomal Site to Which Peptidyl-tRNA Is Bound*

Five antibiotics (puromycin, erythromycin, linco- mycin, sparsomycin, and virginiamycin MI) that bind specifically to the 60 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation of fluorescence from probes at- tached to the a-amino group of Phe-tRNA. Derivatives of three probes with differing chemical characteristics and environmental sensitivities were used: a coumarin, an aminonaphthalenesulfonate, and a pyrene. The effects of the antibiotics on the fluorescence of labeled AcylPhe-tRNAs in the two states, while generally qualitatively similar, are nonetheless quantitatively distinct, as are the calculated binding constants for the antibiotics. Puromycin, as reported earlier, binds to both the puromycin-reactive and -unreactive states, but its dissociation constant is higher for the latter state. Erythromycin binds tightly to ribosomes bearing labeled AcylPhe-tRNA in either the puromycin-reac-tive or -unreactive state. Its effect on the fluorescence of the labeled tRNA is very similar in the two states, except with the pyrene probe, where it has a larger effect in the puromycin-reactive state. Lincomycin and sparsomycin bind to both ribosomal states, but both bind more tightly to the puromycin-reactive

Use of 50 S-binding Antibiotics to Characterize the Ribosomal Site to Which Peptidyl-tRNA Is Bound* (Received for publication, April 13, 1992) Obed W. Odom and Boyd HardestyS From the Department of Chemistry and Biochemistry, Clayton Foundation Biochemical Institute, University of Texas, Austin, Five antibiotics (puromycin, erythromycin, lincomycin, sparsomycin, and virginiamycin MI) that bind specifically to the 60 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation of fluorescence from probes attached to the a-amino group of Phe-tRNA. Derivatives of three probes with differing chemical characteristics and environmental sensitivities were used: a coumarin, an aminonaphthalenesulfonate, and a pyrene. The effects of the antibiotics on the fluorescence of labeled AcylPhe-tRNAs in the two states, while generally qualitatively similar, are nonetheless quantitatively distinct, as are the calculated binding constants for the antibiotics. Puromycin, as reported earlier, binds to both the puromycin-reactive and -unreactive states, but its dissociation constant is higher for the latter state. Erythromycin binds tightly to ribosomes bearing labeled AcylPhe-tRNA in either the puromycin-reactive or -unreactive state. Its effect on the fluorescence of the labeled tRNA is very similar in the two states, except with the pyrene probe, where it has a larger effect in the puromycin-reactive state. Lincomycin and sparsomycin bind to both ribosomal states, but both bind more tightly to the puromycin-reactive state, the extent of the difference varying with the identity of the fluorescent probe. Virginiamycin M1 binds to ribosomes with AcylPhe-tRNA in the puromycin-reactive site, but its binding could not be detected to ribosomes with AcylPhe-tRNA in the puromycin-unreactive site.
' The abbreviations used are: AcylPhe-tRNA, Phe-tRNA acylated with any moiety at its a-amino group; AcPhe-tRNA, Phe-tRNA acetylated a t its a-amino group; Acp3Ud7, 3-(3-amino-3-carboxypro-py1)uracil at position 47 of E. coli tRNAPh'; AEDANS-SAcPhe-tRNA, Phe-tRNA mercaptoacetylated at its a-amino group and then reacted with IAEDANS; CITC, 3-(4-isothiocyanatophenyl)-7-diethylamino-4-methylcoumarin; CPM, 3-(4-maleimidophenyl)-7-diethylamino-4methylcoumarin; CPM-SAcPhe-tRNA, Phe-tRNA mercaptoacetylated at its a-amino group and then reacted with CPM; DCIA, 3-(4-iodoacetylaminophenyl)-7-diethylamino-4-methylcoumarin; IAEDANS, 5-[(2-iodoacetylaminoethyl)amino]naphthalene-l-sul-puromycin-reactive state, the A and P sites, respectively, of the classical two-site model of ribosome function (Watson, 1964). This result was not expected from the latter model, in which peptidyl-tRNA is in the P site immediately before peptidyl transfer and in the A site immediately after peptidyl transfer. The result also demonstrates that deacylated tRNA, peptidyl-tRNA, and puromycin can be bound simultaneously to the same ribosome. The finding is consistent with other data, indicating movement of the newly deacylated tRNA, but not the nascent peptide, that is associated with the peptidyl transferase reaction (Hardesty et al., 1986;Odom et al., 1990). Moazed and Noller (1989) reported that nearly all of the same bases on 23 S RNA are protected by AcPhe-tRNA from chemical modification whether it is in the puromycin-reactive or -unreactive site. Considered together these observations appear to indicate that peptidyl-tRNA is in a similar site or position on the 50 S subunit before and after the peptidyl transfer reaction has taken place, i.e. with or without deacylated tRNA also bound to the ribosome. They prompt the question of why peptidyl transfer to puromycin is blocked when deacylated tRNA is prebound to the ribosome. To explore this problem further we have covalently attached fluorescent probes to the a-amino group of Phe-tRNA by way of a mercaptoacetyl bridge, creating fluorescent peptidyl-tRNA analogues (Odom et al., 1990). These AcylPhe-tRNA derivatives function as analogues of peptidyl-tRNA. Binding of certain 50 S subunit-specific antibiotics causes significant perturbations in fluorescence from the ribosome-bound AcylPhe-tRNAs. The system provides a sensitive way to monitor the environment of the peptidyl-tRNA on the 50 S subunit in the puromycin-reactive and -unreactive sites. Here we report the effects of five antibiotics on fluorescence from three ribosome-bound fluorescent AcylPhe-tRNAs as a method of comparing the puromycin-reactive and -unreactive states. The effects of lincomycin, sparsomycin, and virginiamycin MI, in addition to puromycin and erythromycin, were tested with pyrene, CPM, and AEDANS as fluorescent acyl derivatives. Each of the former three antibiotics is known to be an inhibitor of peptidyl transfer and to bind to the 50 S subunit (for reviews see Ottenheijm et Di-Giambattista et al., 1989). Binding of lincomycin and erythromycin is mutually exclusive (Fernandez-Muiioz et al., 1971).
They are thought to bind at or near loop V of 23 S RNA, since dimethylation of adenine at position 2058 confers resistance to both antibiotics (Skinner et al., 1983). However, in contrast to lincomycin, erythromycin usually causes no inhibition of peptidyl transfer at least with short peptidyl chains (Cundliffe, 1986). Sparsomycin has been reported to fonic acid; poly(A), poly(adeny1ic acid); poly(U), poly(uridy1ic acid); pyrene-SAcPhe-tRNA, Phe-tRNA mercaptoacetylated at its a-amino group and then reacted with N-( 1-pyrene)maleimide; RP-HPLC, reversed-phase high-performance liquid chromatography; s4U8, thiouracil at position 8 of E. coli tRNAPhe; EF, elongation factor. Values for K d a t 0 "C, the temperature at which the puromycin reaction is usually carried out, are somewhat lower (Table I).
It is difficult to measure binding of puromycin in the puromycin-reactive site. After binding, peptidyl transfer to puromycin occurs rapidly with subsequent release of peptidylpuromycin from the ribosome. By measuring fluorescence at low temperature and immediately after the addition of puromycin, however, it is possible to separate binding from the covalent reaction, with CPM-SAcPhe-tRNA and pyrene-SAcPhe-tRNA. In the case of AEDANS-SAcPhe-tRNA the peptidyl transferase reaction is so rapid even a t 0 "C that it has not been possible to separate these events. With CPM-SAcPhe-tRNA, puromycin binding affects fluorescence in a similar manner when the tRNA is in either the puromycinreactive or -unreactive site, giving a 7-nm blue shift and slight increase in intensity as reported previously (Odom et al., 1990). Utilizing this shift, we estimate the K d for puromycin binding to ribosomes carrying CPM-SAcPhe-tRNA in the puromycin-reactive site to be about 60 p~ at 0 "C, about 8fold lower than for ribosomes carrying CPM-SAcPhe-tRNA in the puromycin-unreactive site. With pyrene-SAcPhe-tRNA in the puromycin-reactive site puromycin binding gives little direct effect on the fluorescence so that a K d value cannot be determined fluorescently. It is also possible to estimate dissociation constants for puromycin by measuring the effect of puromycin concentration on the rate of peptidyl transfer to puromycin (see, for example, Pestka, 1972). By performing the puromycin reaction at 0 "C for 10 min, satisfactory saturation curves for puromycin are obtained using AcPhe-tRNA, CPM-SAcPhe-tRNA, or pyrene-SAcPhe-tRNA. Under these conditions less than 30% of the peptidyl-tRNA analogues have reacted at saturating puromycin concentrations. Values for K d calculated by this method are shown in Table I. The value obtained with CPM-SAcPhe-tRNA (48 p~) is similar to the 60 p~ obtained by fluorescence. With AEDANS-SAcPhe-tRNA the rate of the peptidyl transferase reaction with puromycin is too high for binding to be measured reliably, as indicated above. There is considerable variation in Kd depending on the type of AcylPhe-tRNA used, nonfluorescent AcPhe-tRNA giving the highest value (109 p~) and pyrene-SAcPhe-tRNA giving the lowest value (21 p~) .
The range of K d values obtained suggests that the affinity for puromycin is dependent on the nature of the peptidyl moiety. This is consistent with the finding of Pestka (1972) that polysomes show much higher affinity for puromycin than ribosomes carrying only AcPhe-tRNA.
Erythromycin-Erythromycin was shown previously to affect the fluorescence of ribosome-bound CPM-SAcPhe-tRNA and AEDANS-SAcPhe-tRNA (Odom et al., 1991). The effects were similar whether the tRNAs were bound to the puromy-

Dissociation constants for puromycin for ribosomes bearing various
AcylPhe-tRNAs in the puromycin-reactive or -unreactive site Values given for the puromycin-reactive site were determined from measurements of rates of the puromycin reaction, except for the value in parentheses, which was determined by fluorescence titration. All values given for the puromycin-unreactive site were determined by fluorescence titration. All measurements were performed at 0 "C. An error analysis is given under "Experimental Procedures." cin-reactive or -unreactive site. Using pyrene-SAcPhe-tRNA we find a somewhat larger disparity in the magnitude of the fluorescence perturbation produced by erythromycin in the two states, a 180% increase in fluorescence intensity being produced in the puromycin-reactive state (Fig. 1) in contrast to only a 30% increase in the puromycin-unreactive state (Table 11). Without erythromycin the fluorescence quantum yield of puromycin-unreactive pyrene-SAcPhe-tRNA is about 25% higher than that of puromycin-reactive material. The affinity for erythromycin of ribosomes bearing any of the labeled AcylPhe-tRNAs in either the puromycin-reactive or -unreactive site is relatively high. In fact, the antibiotic appears to bind more strongly to such ribosomes than to empty ribosomes, since substoichiometric concentrations of erythromycin give disproportionately large effects on fluorescence. For example, 0.1 p~ erythromycin gave more than half of the maximal erythromycin effect when added to 0.6 p~ ribosomes containing 25 nM CPM-SAcPhe-tRNA (0.25 times the concentration of erythromycin) in either the puromycin-reactive or -unreactive site (data not shown). This result indicates that erythromycin binds preferentially to the ribosomes to which CPM-SAcPhe-tRNA is bound. Thus, although erythromycin cannot bind to ribosomes containing nascent peptides longer than a few amino acid residues (Tai et al., 1974;Odom et al., 1991), the fluorescent peptidyl-tRNA analogues used in this study enhance erythromycin binding suggesting that the size and character of the peptidyl moiety may be critically important. Interestingly, the reaction with puromycin of CPM-SAcPhe-tRNA, but not that of the other .... , ribosome blank.  AI and Ax,,. are the changes in fluorescence intensity and emission maximum, respectively, observed after the addition of 5 mM lincomycin. Measurements were made at 20 "C.
labeled AcylPhe-tRNAs, is greatly inhibited by erythromycin.' Lincomycin-Changes in fluorescence caused by binding of lincomycin to ribosomes carrying each of the three fluorescently labeled AcylPhe-tRNAs in either the puromycin-reactive or -unreactive ribosomal site are shown in Table 111. The largest change is observed using pyrene-SAcPhe-tRNA, which in the puromycin-reactive site undergoes a 195% increase in fluorescence upon addition of lincomycin (Fig. 1). In the puromycin-unreactive site the increase in fluorescence is only 30%. These changes are similar to those given by erythromycin, as considered above. They are consistent with the conclusion that the two antibiotics have overlapping binding sites. Lincomycin, like erythromycin, quenches the fluorescence of AEDANS-SAcPhe-tRNA to about the same extent in either ribosomal site. However, lincomycin, in contrast to erythromycin, shifts the emission maximum of CPM-SAcPhe-tRNA in opposite directions in the two ribosomal sites, causing a red shift in the puromycin-reactive site and a blue shift in the puromycin-unreactive site. The perturbation of fluorescence obtained upon binding of lincomycin was used to obtain saturation curves from which dissociation constants were calculated (Table 111). Large differences in the dissociation constant are observed depending on which of the fluorescently labeled AcylPhe-tRNAs is bound and whether it is in the puromycin-reactive or -unreactive site. That lincomycin binding, like that of erythromycin, is influenced by the nature of the peptidyl moiety is indicated from the previous observation that it does not bind to native polyribosomes unless their nascent peptides are first removed (Contreras and Vazquez, 1977). In the puromycin-reactive site the lowest K d (approximately 2 p~) for lincomycin is obtained with AE-DANS-SAcPhe-tRNA. By contrast, with ribosomes carrying CPM-SAcPhe-tRNA or pyrene-SAcPhe-tRNA in the puromycin-reactive site the Kd values are 20 and 300 pM, respectively. In the puromycin-unreactive site K d values for lincomycin are near 1 mM with all of the labeled AcylPhe-tRNAs tested. Thus in all cases the affinity of lincomycin for the puromycin-reactive state is greater than that for the puromycin-unreactive state, but the magnitude of the difference varies from about %fold for pyrene-labeled tRNA to 600-fold for AEDANS-labeled tRNA.
Sparsomycin-Sparsomycin causes relatively small changes in the fluorescence of all of the labeled AcylPhe-tRNAs in the ribosomal puromycin-reactive site (Table IV and Fig. 1). Kd values were estimated from these changes. However, in general the fluorescence perturbations induced by sparsomycin are less than those produced by the other antibiotics tested. Previous observations (Pestka, 1974) indicated that sparsomycin binding is enhanced rather than inhibited by the 0. W. Odom and B. Hardesty, manuscript in preparation. tRNAPhe An effect of sparsomycin on fluorescence from pyrene-and CPM-AcylPhe-tRNA in the puromycin-unreactive site could not be detected, as considered in the text.
* A I and Axmax are the changes on fluorescence intensity and emission maximum, respectively, after the addition of 20 p~ sparsomycin. Measurements were at 20 "C.
presence of nascent peptides, suggesting that the antibiotic does not compete with the nascent peptide for a common binding site. This is also suggested by the fact that the Kd for sparsomycin binding to ribosomes bearing labeled deacylated tRNA is higher than that for ribosomes bearing AcylPhe-tRNA (Table IV). The fluorescence titrations for the puromycin-reactive state indicate very tight binding of sparsomycin with AEDANS-SAcPhe-tRNA, but somewhat weaker binding with pyrene-SAcPhe-tRNA and CPM-SAcPhe-tRNA, the Kd with the latter being about 0.1 p~ (Table IV).
Sparsomycin had no detectable effect on fluorescence from any of the three AcylPhe-tRNAs bound in the puromycinunreactive site but did cause quenching and a small blue shift in the fluorescence of AcPhe-tRNA fluorescently labeled on thiouracil in position 8 with a coumarin derivative. This effect required relatively high sparsomycin concentrations (20 p M or higher, data not shown). However, surprisingly, it was found that sparsomycin has a large effect on the fluorescence of AEDANS-SAcPhe-tRNA in the puromycin-unreactive site in the presence of puromycin. Previously we had shown (Odom et al., 1991) that puromycin alone causes a large increase in the fluorescence of AEDANS-SAcPhe-tRNA in this site. Sparsomycin causes a decrease and red shift in this fluorescence (Fig. 2). A double-reciprocal plot (not shown) constructed from the sparsomycin saturation curve gave an apparent Kd of 27 p~ for sparsomycin. This is about 3 orders of magnitude higher than the corresponding value with AE-DANS-SAcPhe-tRNA in the puromycin-reactive site. If one assumes that sparsomycin and puromycin are competitive for binding as previously reported (Goldberg and Mitsugi, 1967;Pestka, 1972) then the true Kd for sparsomycin binding in the presence of puromycin would be the apparent Kd divided by

(1 + ([P]/K,)) where [PI is the puromycin concentration and
Kp is the dissociation constant for puromycin, previously calculated to be about 0.5 mM at 20 "C (Odom et al., 1990). Since the puromycin concentration was 2 mM, the true Kd for sparsomycin would be 1/5 of the apparent Kd, still much higher than the value obtained for the puromycin-reactive state. However, the same apparent K d value for sparsomycin was obtained whether the sparsomycin titration was performed in the presence of 0.5 or 2 mM puromycin, indicating little or no competition for binding between sparsomycin and puromycin. We also have evidence, contrary to literature reports cited above, that sparsomycin and puromycin can be bound simultaneously to ribosomes bearing AcylPhe-tRNA in the puromycin-reactive site.' Virginiamycin M1-Binding of virginiamycin MI to ribosomes also was measured by its effect on the fluorescence from the AcylPhe-tRNAs. First the effect of preincubation of ribosomes with virginiamycin MI on binding of deacylated tRNAPhe, AcylPhe-tRNA, or AcylPhe-tRNA after deacylated tRNA was determined. The deacylated tRNAPhe was labeled at either the 5'-phosphate, or at the thiouracil at position 8, both with coumarin derivatives. The AcylPhe-tRNAs used were labeled at these positions or at the a-amino group with CPM. Table V indicates that preincubation with virginiamycin M, has little effect on binding of deacylated tRNA or AcylPhe-tRNA alone, but largely prevents the binding of AcylPhe-tRNA after prebinding of deacylated tRNA. This AcylPhe-tRNA would be bound into the puromycin-unreactive site. A previous report (Cocito and Kaji, 1971) has indicated that virginiamycin M1 blocks EF-Tu-dependent binding of aminoacyl-tRNA to the acceptor site of ribosomes. It also    appears to prevent puromycin binding? We (Odom et al., 1990) and others (Moazed and Noller, 1989) have presented evidence that puromycin-unreactive AcylPhe-tRNA is bound in a site distinct from the unblocked aminoacyl-tRNA binding site, presumably the site for puromycin binding. In any event, the present results clearly indicate that virginiamycin M1 prevents binding of AcylPhe-tRNA to ribosomes to which deacylated tRNA is prebound. Other experiments with virginiamycin M, utilized ribosomes bearing prebound fluorescently labeled AcylPhe-tRNA in either the puromycin-reactive or -unreactive site. In the latter situation, however, no effect of virginiamycin M1 could be detected, supporting the conclusion that virginiamycin M, does not bind to such ribosomes. Thus binding of virginiamycin M, to ribosomes and binding of AcylPhe-tRNA into the puromycin-unreactive site appear to be mutually exclusive. Virginiamycin M, does bind to ribosomes carrying labeled AcylPhe-tRNA in the puromycin-reactive site, and to ribosomes bearing 5"phosphate-labeled deacylated tRNA, as indicated by its perturbation of their fluorescence (Table VI), as well as by its inhibition of the reaction of the AcylPhe-tRNAs with puromycin (data not shown). The largest direct effect of virginiamycin M, on fluorescence occurs with AEDANS-SAcPhe-tRNA, with which a 7-nm red shift and more than a 2-fold increase in fluorescence intensity are observed. With pyrene-SAcPhe-tRNA there is little direct effect of virginiamycin M, while with CPM-AcylPhe-tRNA there is only a slight red shift in the fluorescence spectrum. Titration with virginiamycin MI of ribosomes carrying AEDANS-SAcPhe-tRNA indicates a Kd for virginiamycin M, of about 2 p M , much higher than the value of < 10 nM determined for binding of virginiamycin M1 to empty ribosomes.' The binding of virginiamycin M, to ribosomes carrying CPM-SAcPhe-tRNA is even weaker, for which fluorescence titration indicates a Kd near 10 pM. Since there is little direct effect of virginiamycin M1 on fluorescence from bound pyrene-SAcPhe-tRNA, competititon between lincomycin and virginiamycin (to be reported in more detail elsewhere) was used to obtain a Kd of 0.5 p~.

DISCUSSION
Earlier results (Hardesty et al., 1986;Odom et al., 1990) indicated that peptidyl-tRNA was in a similar physical position on 50 S ribosomes before and after the peptidyl transferase reaction and that puromycin as well as deacylated tRNA could be bound to the ribosomes in the latter situation. Peptidyl transfer to puromycin does not take place in the latter situation. These observations raise the question of how deacylated tRNA blocks the peptidyl transferase reaction and about the nature of the peptidyl-tRNA binding site in the two situations. The effect of the antibiotic binding on fluorescence 0. W. Odom and B. Hardesty, unpublished results.