(3H)anisomycin binding to eukaryotic ribosomes

Anisomycin, a well-known inhibitor of eukaryotic ribosomes' peptidyl-transferase activity, specifically binds to the 60 S ribosome subunit. Quantitative studies on [³H]anisomycin binding to yeast and human tonsil ribosomes have shown that a maximum of one molecule of the antibiotic is bound per ribosome in both cases. There is a single type of binding to 60 S subunits but ribosomes themselves are not homogeneous with respect to [³H]anisomycin binding, since the interaction between antibiotic and ribosome occurs with two different affinities. Only ribosomes having the higher type of affinity for [³H]anisomycin are active in catalysing peptide bond formation, as tested in both the puromycin and the fragment reaction assays. Affinity of [³H]anisomycin for ribosomes is higher at 0 °C than at 30 °C. Affinity is decreased in the presence of ethanol.The acetate group in the 3′ position of the pyrrolidine ring of anisomycin is important for the anisomycin—ribosome interaction since deacetylanisomycin appears to have a mode of action similar to anisomycin but has an affinity for the ribosome that is 350 times smaller.The effect of certain peptidyl-transferase inhibitors has been tested on [³H]anisomycin binding to ribosomes. Using either yeast or human tonsil ribosomes a number of sesquiterpene antibiotics of the trichodermin group (trichodermin, trichodennol, fusarenon X and trichothecin) totally block [³H]anisomycin binding whereas puromycin and verrucarin A only partially inhibit the [³H]anisomycin interaction with ribosomes. Gougerotin, blasticidin S and actinobolin have no effect. Tenuazonic acid and sparsomycin inhibit [³H]anisomycin binding to ribosomes but the degree of inhibition differs between yeast and human tonsil ribosomes.     

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.     

Cooperative effects in the peptidyltransferase center of Escherichia coli ribosomes

We have measured the binding isotherms of C--A--C--C--A(3'NH)-[14C]Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes. Analysis of the experimental binding isotherms leads to the following conclusions. A ribosome (or subunit) binds two C--A--C--C--A(3'NH)-Phe molecules. The binding of C--A--C--C--A(3'NH)-Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient tau = 40 +/- 5 or more. The binding of C--A--C--C--A(3'NH)-AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5 X 10(6) M-1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10-25, the association binding constant of puromycin at the acceptor site being (1-2) X 10(4) M-1. The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules absorbed on a ribosome equals 100-200 M-1.     

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling

The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.     

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.     

Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.

A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.     

mRNA-binding site of ribosomes at different stages of translation. II. Affinity modification of Escherichia coli ribosomes bya benzylidene derivative of AUGU6 in pre- and post-translation complexes

Affinity labeling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methyl-amino]benzylidene derivative of AUGU6 (AUGU6-[14C]CHRCl) was studied within the pretranslocational complex ribosome.AUGU6[14C]CHRCl.tRNA(fMet)(P-site).fMetPhe-tR NA(Phe)(A-site) and posttranslocational complex ribosome.AUGU6[14C]CHRCl.fMetPhe-tRNA(Phe)(P-site). Both 30S and 50S subunits were labeled within these complexes, but the extent of 30S subunit modification was 6-8-fold higher than those for 50S subunit. Ribosomal proteins of both subunits were found to be labeled preferentially. Proteins S1, S5, S11, L1 were identified to be crosslinked with AUGU6[14C]CHRCl within the pretranslocational complex and S7--within the posttranslocational complex from the data of two-dimensional electrophoresis in the polyacrylamide gel.     

Characterisation of the binding of virginiamycin S to Escherichia coli ribosomes.

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit.     

fMet-tRNA F Met binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats.

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied in vitro, did not change the factor-dependent binding of fMet-tRNA f Met to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA f Met-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA f Met to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.     

Spatial proximity of the ribosomal mRNA binding center to the 50S subunit]

4-(N-2-chloroethyl-N-methylamino)benzylamide of 5'-heptaadenylic acid was used for affinity labelling of the ribosome in the vicinity of its mRNA-binding centre. This derivative, similar to the free oligonucleotide, stimulates the binding of [14C]-lysyl-tRNA to ribosomes of E. coli and alkylates ribosomes both the 30S and the 50S subunits. The alkylation of ribosomes is inhibited by pre-incubation of ribosomes with polyadenylic acid, which suggests that the chemical modification is a specific one and occurs in the vicinity of mRNA-binding site. The fact, that a short oligonucleotide having an active group on its 5'end attacks the 50S subunit of ribosome may indicate that the mRNA-binding centre is located in the contact region between ribosomal subunits.     

RIBOSOME-MEMBRANE INTERACTION : Nondestructive Disassembly of Rat Liver Rough Microsomes into Ribosomal and Membranous Components

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl₂, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [³H]puromycin into hot acid-insoluble material and from the release of [³H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (~15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.     

Photoaffinity labeling of rat liver ribosomes by N-(2-nitro-4-azidobenzoyl)puromycin

N-(2-Nitro-4-azidobenzoyl)-[3H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes.     

Inhibition of translation in eukaryotic systems by harringtonine.

The Cephalotaxus alkaloids harringtonine, homoharringtonine and isoharringtonine inhibit protein synthesis in eukaryotic cells. The alkaloids do not inhibit, in model systems, any of the steps of the initiation process but block poly(U)-directed polyphenylalanine synthesis as well as peptide bond formation in the fragment reaction assay, the sparsomycin-induced binding of (C)U-A-C-C-A-[3H]Leu-Ac, and the enzymic and the non-enzymic binding of Phe-tRNA to ribosomes. These results suggest that the Cephalotaxus alkaloids inhibit the elongation phase of translation by preventing substrate binding to the acceptor site on the 60-S ribosome subunit and therefore block aminoacyl-tRNA binding and peptide bond formation. However, the Cephalotaxus alkaloids do not inhibit polypeptide synthesis and peptidyl[3H]puromycin formation in polysomes. Furthermore, these alkaloids strongly inhibit [14C]trichlodermin binding to free ribosomes but hardly affect the interaction of the antibiotic with yeast polysomot interact with polysomes and therefore only inhibit cycles of elongation. This explains the polysome run off that has been observed by some workers in the presence of harringtonine.     

Interaction of substrates and substrate-like inhibitors with the peptidyltransferase center from Escherichia coli ribosomes

The binding isotherms of CACCA(3'NHPhe----Ac) and CACCA(3'NHPhe) to E. coli ribosomes and 50S subunits were measured. A theoretical model of adsorption for the case of cooperative interaction between two ligands adsorbed on a ribosome was designated. The analysis of the experimental binding isoterms leads to the following conclusions. A ribosome (or subunit) binds one CACCA (3'NHPhe----Ac) molecule to donor site of the peptidyl transferase center, but two CACCA (3'NHPhe) molecules to both donor and acceptor sites. The binding of CACCA (3'NHPhe) to ribosomes (or subunits) is a cooperative process, characterized by the cooperativity coefficient tau = 40 +/- 5 or more. When model substrates CACCA-Phe, CACCA-Leu and CACCA-Val were taken instead of CACCA (3'NHPhe) in the incubation mixture with ribosomes, dipeptides were obtained even in the case, when ratio [model substrate]: [ribosome] (in moles) was much lower than 1. Puromycin binding to acceptor site with constant (1-2) X 10(4) M-1 also stimulates CACCA(3'NHPhe----Ac) adsorption to the donor site of ribosomes with cooperativity coefficient being equal to 1.5-2.5. It is also shown that cytidine 5'-phosphate binding to the donor site increases kappa cat of the reaction of minimal donors with CACCA-Phe by 1.5 orders of magnitude but has no effect on Km of this reaction. These facts point out that cytidine 5'-phosphate being adsorbed on the corresponding area of the donor site leads to the conversion of low-productive complex [ribosome + minimal donor substrate + acceptor substrate] into high-productive complex [ribosome + minimal donor substrate + acceptor substrate + cytidine 5'-phosphate].     

The proteins of the messenger RNA binding site of Escherichia coli ribosomes.

Oligo(U) derivatives with [14C]-4-(N-2-chloroethyl-N-methylamino)benzaldehyde attached to 3'-end cis-diol group via acetal bond, p(Up)n-1UCHRCl as well as with [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to 5'-phosphate via amide bond, ClRCH2NHpU(pU)6 were used to modify 70S E. coli ribosomes near mRNA binding centre. Within ternary complex with ribosome and tRNAPhe all reagents covalently bind to ribosome the extent of modification being 0.1-0.4 mole/mole 70S. p(Up)n-1UCHRCl alkylates either 30S (n=5,7) or both subunits (n=6,8). rRNA is preferentially modified within 30S subunit. ClRCH2NHpU(pU)6 alkylates both subunits the proteins being mainly modified. The distribution of the label among proteins differ for various reagents. S4, S5, S7, S9, S11, S13, S15, S18 and S21 are found to be alkylated within 30S subunit, proteins L1, L2, L6, L7/L12, L19, L31 and L32 are modified in the 50S subunit. Most proteins modified within 30S subunit are located at the "head" of this subunit and proteins modified within 50S subunit are located at the surface of the contact between this subunit and the "head" of 30S subunit at the model of Stoffler.     

70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.     

Binding of ricin A chain to rat liver ribosomes: Relationship to ribosome inactivation

Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [³H] NEM was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [³H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [³H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg²⁺. At optimal Mg²⁺ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg²⁺, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 X 10^?8 M. [³H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 X 10^?7 M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.     

Comparison by photoaffinity labeling of the proteins involved in GTP hydrolysis from 70S ribosomes and a 5S RNA-protein complex from Bacillus stearothermophilus.

Photoaffinity labeling of 70S ribosomes from B. stearothermophilus by [³H]-1-(4-azidophenyl)-2-(5′-guanyl) pyrophosphate (APh-GDP) in the presence of fusidate and elongation factor G (EF-G) results in incorporation of tritium in the 50S proteins BL2, BL10 and BL22. Irradiation of the corresponding 5S RNA-protein complex in the presence of the GDP derivative gives only incorporation of tritium in BL10 and BL22. The proteins BL10 and BL22 comigrate in two dimensional gel electrophoresis with the 50S ribosomal proteins EL11 and EL18 from E. coli. The result suggests that the region at or near the guanine nucleotide binding site of the ribosome and the complex are the same. Since previous work has shown that the latter two are labeled upon irradiation of the ribosome with [³H]-APh-GDP, it is concluded that ribosomes from E. coli and B. stearothermophilus have structurally related GTPase sites.     

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the structural specificity of puromycin binding to Escherichia coli ribosomes

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.