Inhibitor Of Peptide Bond Formation Research Articles

Mononucleotide derivatives as ribosomal P-site substrates reveal an important contribution of the 2'-OH to activity.

The chemical synthesis of various acylaminoacylated mononucleotides is described and their activities as donor substrates for the ribosomal peptide synthesis were investigated using PhetRNA(Phe) as an acceptor. This minimal reaction was characterized in detail and was shown to be stimulated by CMP, cytidine and cytosine. By using several cytidine and cytosine analogs evidence is provided that this enhancement is rather caused by base pairing to rRNA, followed by a structural change, than by a base mediated general acid/base catalysis. Only derivatives of AMP proved active as P-site substrates. Further, a significant contribution of the 2'-OH to activity was indicated by the finding that AcLeu-dAMP was inactive as donor substrate, although it is a good inhibitor of peptide bond formation and thus, is presumably bound to the P-site. However, Di(AcLeu)-2'-OCH(3)-Ade and DiAcLeu-AMP were moderately active in this assay suggesting that the reactivity of the 3'-acylaminoacid ester is stimulated by the presence of the 2'-oxygen group. A model is discussed how further interactions of the 2'-OH in the transition state might influence peptidyl transferase activity.

Structures of Five Antibiotics Bound at the Peptidyl Transferase Center of the Large Ribosomal Subunit

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0 Å resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H. marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

SPARKA Novel Method To Monitor Ribosomal Peptidyl Transferase Activity

The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.

Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site.

A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.

Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes.

The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [(125)I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink.

Mutations in the Peptidyl Transferase Center of 23 S rRNA Reveal the Site of Action of Sparsomycin, a Universal Inhibitor of Translation

Sparsomycin is a universal and powerful inhibitor of peptide bond formation which, in contrast to many other ribosome-targeted antibiotics, does not produce footprints on rRNA. A mutant of an archaeon Halobacterium halobiumhas been isolated that exhibits resistance to sparsomycin. Resistant cells possessed a mutation in the 23 S rRNA, where C2518 (C2499 in Escherichia coli) was substituted by U. Introduction of the C2518U mutation into the chromosomal 23 S rRNA gene of wild-type H. halobiumrendered cells resistant to sparsomycin, demonstrating that a single nucleotide alteration in the rRNA is sufficient to confer resistance. Accordingly, ribosomes containing mutant 23 S rRNA exhibited increased tolerance to sparsomycin in vitro. Mutations of two other nucleotide positions in the peptidyl transferase center, C2471 and U2519 (C2452 and U2500 in E. coli), conferred resistance to low concentrations of sparsomycin. The location of the sparsomycin resistance mutations reveals the possible site of drug binding and/or action. Our findings provide further support for the idea that rRNA may be directly involved in interaction with antibiotics and the catalysis of the peptide bond formation.

A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome.

The chiral synthesis and biochemical properties of electron rich phenolic sulfoxide analogs of sparsomycin

A novel route to activated phenolic sulfoxide analogs of sparsomycin has been developed. These analogs display an enhanced “preincubation effect” as inhibitors of peptide-bond formation. This time-dependent component of inhibition, which is postulated to result from an enzyme-mediated Pummerer rearrangement, is the dominant route to inhibition in these activated analogs.

Sulfoxide configuration in sparsomycin determines time-dependent and competitive inhibition of peptidyl transferase

Sparsomycin, S cR s configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation. In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay. The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin.

A mutation of Saccharomyces cerevisiae leading to resistance to some inhibitors of peptide bond formation

A mutant of the yeast Saccharomyces cerevisiae has been isolated that is resistant to narciclasine, an inhibitor of peptide bond formation on 80S ribsomes. The mutant shows cross-resistance to a number of inhibitors of peptidyl transferase including anthelmycin, a 4-aminohexosyl cytosine antibiotic, which does not compete with narciclasine for its ribosomal binding site. The mutation is within the gene tcm1 or a closely linked gene on chromosome XV; it is expressed in the 60S ribosomal subunit. The parameters of the binding of (3H)narciclasine to ribosomes and ribosomal subunits from both wild-type and mutant strains have been calculated by ultracentrifugation. One molecule of narciclasine is bound per ribosome or per 60S ribosomal subunit, the values of the dissociation constants being 0.054 and 0.13 μm respectively, for 80S and 60S particles from the wild-type cells. Ribosomes of the mutant strain have a lower affinity for narciclasine and trichodermin than ribosomes from wild-type cells. The mutation is semidominant in heterozygous diploid cells.

Fat body protein associated with the larval diapause of the southwestern corn borer, Diatraea grandiosella: Synthesis and characteristics

1. 1. The proteins of the larval fat body of the southwestern corn borer, Diatraea grandiosella, were subjected to isoelectric focusing and disc electrophoresis in polyacrylamide gels to examine the diapause-associated protein (DAP). 2. 2. The fat bodies of last instar pre-diapausing larvae, as well as those of non-diapausing larvae which had been treated with juvenile hormone or a juvenile hormone mimic, were shown to store DAP. 3. 3. The protein fraction has an isoelectric point of 5.9, and appears to be a single polypeptide unit with a molecular weight of about 35,000. 4. 4. The protein was resistant to hydrolysis at 37°C by midgut enzymes and trypsin. 5. 5. De novo synthesis of DAP was demonstrated both in vitro and in vivo in the fat body of pre-diapausing larvae using radioactive leucine. 6. 6. Isolated fat bodies of 33-day old last instar pre-diapausing larvae incubated in macromolecule-free Grace's medium incorporated [ 3H]leucine selectively into DAP. 7. 7. Experiments employing [ 14C] and [ 3H]leucine and cycloheximide, an inhibitor of peptide bond formation, confirmed the synthesis of DAP in vivo. 8. 8. DAP was released unchanged from the fat body of mid-diapausing larvae in vitro. Since the protein does not accumulate in the haemolymph in vivo it may be structurally modified before or after release in vivo. 9. 9. The possible diapause-related functions of DAP are appraised.

Binding of [ 3H]narciclasine to eukaryotic ribosomes A study on a structure-activity relationship

[ 3H]Narciclasine is a specific inhibitor of peptide bond formation on eukaryotic ribosomes and binds to 60-S ribosomal subunits. Binding of [ 3H]narciclasine to yeast ribosomes is inhibited by many other inhibitors of peptide bond formation including anisomycin, several sesquiterpene antibiotics (trichodermin, trichothecin, fusarenon X and verrucarin A) several Cephalotaxus alkaloids (harringtonine, homoharringtonine and isoharringtonine), several Amaryllidaceae alkaloids (pretazettine, haemanthamine, lycorine, pseudolycorine and dihydrolycorine) and the narciclasine derivatives trans-dihydronarciclasine, trans-dihydronarciclasine acetonide and isonarciclasine. Binding is also inhibited, although to a very small extent, by methylnarciclasine and cisdihydronarciclasine. In contrast, no inhibition of [ 3H]narciclasine binding was observed in the presence of certain other inhibitors of peptide bond formation including blasticidin S, gougerotin, sparsomycin and puromycin.

Bruceantin, a novel inhibitor of peptide bond formation

The effects of bruceantin on a number of steps of the protein synthesis process have been studied using resolved model systems from both yeast and reticulocytes. Bruceantin is a potent inhibitor of polyphenylalanine synthesis as directed by poly(U). However, inhibition is less pronounced on protein synthesis as directed by endogenous mRNA and the compound inhibits the poly(U) system only poorly if added after polyphenylalanine synthesis has been initiated. Peptide bond formation as assayed in both the fragment reaction and in the puromycin reaction with a preformed initiation complex containing ribosomes and [ 35S]Met-tRNA F is totally blocked by bruceantin. Neither the enzymic binding of Phe-tRNA to reticulocyte ribosomes nor the formation of the 35S-labeled tRNA · ribosome initiation complex is inhibited by bruceantin. The binding of [ 14C]trichodermin to yeast ribosomes is strongly inhibited by bruceantin. A Klotz plot shows that both these drugs bind to ribosomes in mutually exclusive fashion and it can be calculated that bruceantin binds to the peptidyltransferase center with K d = 0.34 μM. This high affinity is considerably lower for polyribosomes ( K d = 557 μM), which may explain the earlier finding that bruceantin only stabilizes polyribosomes at high drug concentrations.

Initiation of translation with native 40-S ribosomal subunits.

We have studied the steps taking place in initiation of protein synthesis in rabbit reticulocyte cell-free systems by using native 40-S subunits. Binding of the initiator Met-tRNAf to the native 40-S subunits took place without additions of initiation factors, in the presence or absence of poly(A,G,U) but GTP is required. There is no requirement of mRNA for the binding reaction, suggesting the presence of eIF-2 and eIF-3 in native 40-S subunits. Polydextran sulphate and N-ethylmaleimide block the binding of Met-tRNAf to native 40-S subunits, whereas pactamycin and edeine A1 enhance the binding. Aurintricarboxylic acid inhibits the poly(A,G,U)-dependent stimulation of the reaction and releases some initiation factor(s) from native 40-S subunits. Addition of 60-S subunits to the native 40-S · Met-tRNAf complex in the absence of eIF-5 leads to a 80-S · Met-tRNAf complex, reactive with puromycin, suggesting that this initiation factor was present in the native 40-S subunits. Formation of methionyl-puromycin with the 80-S · Met-tRNAf complex is blocked by inhibitors of peptide bond formation, such as anisomycin, trichodermin, trichothecin, bruceantin and harringtonine. Pactamycin, edeine A1, aurintricarboxylic acid, pyrocatechol violet and adrenochrome do not inhibit peptide bond formation but lead to the formation of an unusual 40-S · Met-tRNAf complex which, after the joining of the 60-S subunit, is not reactive in the puromycin reaction.

Cooperative and Antagonistic Interactions of Peptidyl‐tRNA and Antibiotics with Bacterial Ribosomes

There is a single-site interaction of [methylene-14C]thiamphenicol and [methylene-14C]chloramphenicol with run-off ribosomes with dissociation constants Kd = 6.8 micronM and Kd = 4.6 micronM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A-site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P-site and other in the A-site. The lower-affinity bindings (dissociation constants Kd = 6.4 micronM and Kd = 1.5 micronM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A-site. The higher-affinity bindings (dissociation constants Kd = 2.3 micronM and Kd = 1.5 micronM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P-site. Therefore binding of nascent peptides to the A-site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P-site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptide bond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl-tRNA is placed in the P-site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics. There is a single-site interaction per ribosome for [35S]thiostrepton, which does not appear to be affected by the attachment to the ribosomes of mRNA, tRNA and nascent peptides either to the A or the P-site. [N-methyl-14C]Lincomycin, [N-methyl-14C]erythromycin, [G-3H]streptogramin B and [G-3H]-streptogramin A bind to run-off ribosomes and polysomes totally free from nascent peptides. However, these antibiotics do not interact with ribosomes bearing nascent peptides either in the A or the P-site and therefore are not active on preformed polysomes. Thus lincomycin and streptogramin A only interact with free ribosomes and 50-S subunits and block the early rounds of peptide bond formation prior to polysome formation. Erythromycin and streptogramin B do not inhibit either initiation or the first round of peptide bond formation. However, erythromycin and streptogramin B, prebound to the ribosome, block peptide elongation probably by steric hindrance with the growing oligopeptide chain when this reaches a certain critical length.

Antibiotics and compounds affecting tanslation by eukaryotic ribosomes. Specific enhancement of aminoacyl-tRNA binding by methylaxnthines.

The mode and site of action of inhibitors of translation (initiation, elongation and termination of protein synthesis) in eukaryotic systems is reviewed. The isolation and characterization of a factor is described that binds Ac-Phe-tRNA to form a complex made up of binding factors, Ac-Phe-tRNA, and ribosome. The binding of Ac-Phe-tRNA probably occurs at the ribosomal site involved in the binding of the initiator substrate Met-tRNAF. The effect of inhibitors of the intitiation phase of protein synthesis on the nonenzymic Ac-Phe-tRNA binding to ribosomes is investigated. The two sites translocation model for translation in eukaryotic cells is presented and the effects of inhibitors on the various steps of protein synthesis are determined empirically. The site of action of inhibitors of peptide bond formation at the ribosomal peptidyl transferase center is elucidated. The action of inhibitors of translocation is sutdied in model cell-free systems from human cells. In addition, a number of methylxanthines are shown to enhance the elongation phase in polypeptide synthesis by stimulating the enzymic binding of aminoacyl-tRNA. The effect of caffeine, theophylline and its derivatives are shown to be fairly specific and dependent on the ribosome concentration. Aminophylline is shown to have a similar effect but also enhances aminoacyl-tRNA synthetase activity at low Mg++ concentrations, probably displacing the optimal concentration of Mg++ in the reaction. This second effect of aminophylline appears to be due to the ethylenediamine moiety of aminophylline since it is also observed in the presence of different polyamines but not in the presence of caffeine or theophylline.

The involvement of sulphydryl groups in the peptidyl transferase centre of eukaryotic ribosomes.

Treatment of mammalian ribosomes with N-ethylmaleimide enhances up to 100% the ribosome efficiency in the "fragment reaction assay" for peptide bond formation by increasing the affinity of the substrate C-A-C-C-A-Leu-Ac for the donor site. This stimulation in peptidyl transferase activity was not observed when yeast ribosomes were treated in a similar manner. Stimulation of the peptidyl transferase activity of mammalian ribosomes was also observed by treatment with either p-chloromercuribenzoic acid or 5,5'-dithiobis-(2-nitrobenzoic acid) or 5,5'-dithiobis-(2-nitropyridine) or the maleimide-derived antibiotic showdomycin. N-Ethylmaleimide treatment also enhances C-A-C-C-A-Leu binding to the acceptor site of the peptidyl transferase centre. However, neither binding of N-Ac-Phe-tRNA in the presence of ethanol, nor binding of Phe-tRNA to the ribosomes is stimulated by N-ethylmaleimide. The antibiotic tenuazionic acid (a selective inhibitor of peptide bond formation by mammalian ribosomes) appears to require for its inhibitory effect the ribosome sulphydryl residues, since its inhibitory action on the fragment reaction is greatly decreased in ribosomes treated with N-ethylmaleimide.

(G-3H)gougerotin binding to ribosomes. Heterogeneity of eukaryotic ribosomes.

The quantitative interaction of [G-3H]gougerotin with Escherichia coli and Saccharomyces cerevisiae ribosomes is studied in this contribution. While E. coli ribosomes show an homogeneous single-site interaction with the antibiotic (dissociation constant = 1.5 μM), S. cerevisiae ribosomes are heterogeneous for [G-3H]gougerotin binding: there is a single-site interaction with dissociation constant = 0.9 μM in 28% of the ribosomes and another single binding site with dissociation constant = 12 μM in 58% of the ribosomes. Antibiotic interaction takes place in all cases with the larger ribosomal subunit. Gougerotin binding is affected by the nature of the ribosomal preparation. Thus, gougerotin has a much stronger affinity for E. coli washed ribosomes than for ribosomes reconstituted from their subunits. This difference is reduced when tRNAphe is prebound, in the presence of poly(U), to the reconstituted ribosomes. Gougerotin is also able to detect differences between the poly(U) · ribosome · Ac-Phe-tRNA complexes formed with either washed or reconstituted ribosomes. The effect of cations, pH, ethanol, temperature and inhibitors of peptide bond formation on [G-3H]gougerotin binding to E. coli and S. cerevisiae ribosomes has also been studied. Only those antibiotics that act on the peptidyl transferase centre of both prokaryotic and eukaryotic ribosomes (blasticidin S, sparsomycin and actinobolin) completely inhibit the binding of [G-3H]-gougerotin. In contrast, griseoviridin strongly enhances [G-3H] gougerotin binding to both types of ribosomes.

Preparation of pure [ 3H]anisomycin by tritium exchange labeling

The antibiotic anisomycin is a very useful tool in studying protein synthesis since it is a specific inhibitor of the peptidyl transferase centre of eukaryotic ribosomes (5–7). By tritium exchange labeling followed by chromatographic and electrophoretic purification, we have obtained [ 3H]anisomycin of specific activity 285 mCi/mmole, and the methodology followed is described in this paper. This method is useful in preparing tritium labeled antibiotics other than anisomycin provided that the nonradioactive compound has the following characteristics: (a) a chemical structure resistant to the method required for tritium labeling, (b) ionic groups, and (c) chromophore groups with absorption maxima in the uv or visible part of the spectrum. Since these circumstances concur frequently in a number of chemical structures, a method essentially similar to that described in this work might be widely used. The method was not applicable to amicetin, blasticidin S, and fusidic acid, as these antibiotics were broken down during the tritium labeling. However, gougerotin, a well known inhibitor of peptide bond formation by prokaryotic and eukaryotic ribosomes (2–7), has been tritiated and purified following a method very similar to that described in this contribution to [ 3H]gougerotin (110 mCi/mmole) (16).

The trichodermin group of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes

We have studied the effects of a number of sesquiterpene antibiotics of the trichodermin group on different steps of the elongation phase in human tonsils and yeast cell-free systems. We have observed that these antibiotics are more active in the human tonsils than in the yeast cell-free systems. When a similar system is considered the antibiotic trichodermin is more active than its derived alcohol trichodermol. Sesquiterpene antibiotics of the trichodermin group do not affect non-enzymatic binding of Ac-Phe-tRNA and either enzymatic or non-enzymatic binding of Phe-tRNA to human tonsil ribosomes. These antibiotics are also without effect on translocation to the P site of Ac-Phe-tRNA bound to the ribosomal A site. All the sesquiterpene antibiotics of the trichodermin group tested (trichodermin, trichodermol, verrucarin A, fusarenon X) are very active inhibitors in the peptide bond formation step. This inhibitory effect of antibiotics of the trichodermin group was observed in the “puromycin reaction” and in the “fragment reaction” assays. Binding of the substrates CACCA-Leu-Ac and UACCA-Leu, respectively, to the donor and the acceptor sites of the peptidyltransferase centre was also studied in this work. We have observed in this system an inhibitory effect in binding of substrates to both the donor and the acceptor site of the peptidyltransferase centre by all the antibiotics of the trichodermin group.