The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.     

The modification of the peptidyl transferase activity of 50-S ribosomal subunits, LiC1-split proteins and L16 ribosomal protein by ethoxyformic anhydride.

Ethoxyformic anhydride abolishes the peptidyl transferase activity of 50-S ribosomal subunits, LiC1 split proteins and L16. Hydroxylamine treatment results in reactivation. Erythromycin exhibits significant protection with 50-S ribosomal subunits. With LiC1 split proteins and L16 significant protection was exhibited only after reconstitution. The results indicate that the ethoxyformic anhydride is reacting with approximately six histidines in LiC1 split proteins and one in L16. Since L16 has been reported to contain a single histidine, the results presented indicate the involvement of this histidine in peptidyl transferase activity.     

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.     

SPARK--a 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.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Mapping the path of the nascent peptide chain through the 23S RNA in the 50S ribosomal subunit.

Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes. The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome. Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures. The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids). Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781. Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I. These cross-linking results are correlated with other types of topographical data relating to the 50S subunit.     

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.     

Inhibition of the peptidyl transferase A-site function by 2'-O-aminoacyloligonucleotides

New “non-isomerizable” analogs of the 3′-terminus of AA-tRNA, C-A(2′Phe)H, C-A(2′Phe)Me, C-A(2′H)Phe and C-A(2′Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3′-O-AA-derivatives were active acceptors of Ac-Phe in the peptidyl transferase reaction, while the 2′-O-AA-derivatives were completely inactive. Both 2′- and 3′-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3′-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3′-terminus of 2′-O-AA-tRNA.     

Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center

One of the oldest questions in RNA science is the role of nucleotide modification. Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications. Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding. Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations. Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others. The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed.     

The photochemical inactivation of peptidyl transferase activity.

The photochemical oxidation of the 50-S ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first-order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivation exhibits a maximum at pH 7.5. Erythromycin at a low concentration (0.04 mumol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect. The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.     

Structure of a protein L23-RNA complex located at the A-site domain of the ribosomal peptidyl transferase centre

Protein L23 from the ribosome of Escherichia coli is the primary ribosomal product cross-linked to affinity-labelled puromycin; it lies, therefore, within the A-site domain of the peptidyl transferase centre on the 50 S subunit. We have characterized this functional domain by isolating and sequencing the RNA binding site of protein L23; it consists of two main fragments of 25 and 105 nucleotides that strongly interact and are separated by 172 nucleotides in the primary sequence. The higher-order structure of the RNA moiety was probed by chemical reagents, and by single-strand and double-strand-specific ribonucleases; a secondary structural model and a tertiary structural interaction are proposed on the basis of these data that are compatible with phylogenetic sequence comparisons.Several nucleotides exhibited altered chemical reactivity, both lower and higher, in the presence of protein L23, thereby implicating a large proportion of the RNA structure in the protein binding. The sites were located mainly at the extremities of the helices and at nucleotides that were putatively bulged out from the helices.The RNA moiety and an adjacent excised fragment contain several highly conserved sequences and a modified adenosine. Such sequences constitute important functional domains of the RNA and may contribute to the putative role of this RNA region in the peptidyl transferase centre.     

Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

Photo-reactive 3-(4'-benzoylphenyl)propionyl-Phe-tRNA bound to the A- or the P-site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U-2584 and U-2585 at the A-site and A-2451 and C-2452 at the P-site. Minor crosslinks from both sites were observed at nucleotides A-2503 to U-2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo-crosslinking. In addition, tetracycline was also shown to photo-crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A- and P-site.     

Uncovering the enzymatic pKa of the ribosomal peptidyl transferase reaction utilizing a fluorinated puromycin derivative

The ribosome-catalyzed peptidyl transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site substrate and a second unidentified group thought to reside in the rRNA peptidyl transferase center. Here we report the synthesis and activity of the beta,beta-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pK(a) of the nucleophilic alpha-amino group (<5.0) such that it is deprotonated at all pHs amenable to ribosomal analysis (pH 5.2-9.5). In the 50S modified fragment assay, this substrate reacts substantially faster than puromycin at neutral or acidic pH. The reaction follows a simplified pH profile that is dependent only upon deprotonation of a titratable group within the ribosomal active site. This feature will simplify characterization of the peptidyl transferase reaction mechanism. On the basis of the reaction efficiency of the doubly fluorinated substrate compared to the unfluorinated derivative, the Bronsted coefficient for the nucleophile is estimated to be substantially smaller than that reported for uncatalyzed aminolysis reactions, which has important mechanistic implications for the peptidyl transferase reaction.     

Effect of cytidine-5'-monophosphate on peptidyl transferase activity.

The transfer reaction with pA-fMet as a donor substrate is strongly stimulated by CMP, whereas the transfer reaction with CpApCpCpA-acLeu as a donor substrate is inhibited by CMP. These results indicate that the donor site of peptidyl transferase contains specific binding sites for the terminal adenosine and for the cytidylic acid residue in the terminal sequence CpCpA of tRNA and that an attachment of proper nucleotides to the donor site induces a conformational change in peptidyl transferase.     

Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS.     

Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.     

The ribosomal peptidyl transferase center: structure, function, evolution, inhibition

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring ribozymes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome

Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3' adenosine of yeast tRNA(Phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-O-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA(Phe) and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center.