The ribosomal peptidyl transferase

Peptide bond formation on the ribosome takes place in an active site composed of RNA. Recent progress of structural, biochemical, and computational approaches has provided a fairly detailed picture of the catalytic mechanism of the reaction. The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.     

Role of ribosomal protein L27 in peptidyl transfer

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.     

Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues

The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA [Wagner, T., Cramer, F., & Sprinzl, M. (1982) Biochemistry 21, 1521-1529]. This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site. In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited. The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA. If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.     

Analysis of predictions for the catalytic mechanism of ribosomal peptidyl transfer

The reaction mechanism of peptide bond formation on the ribosome is now becoming established by results from both experiments and computer simulations. Here, we analyze predictions from molecular dynamics simulations, as well as from new crystal structures, and examine their implications for the mechanisms of peptidyl transfer and peptidyl-tRNA hydrolysis. A number of computational predictions for the peptidyl transfer reaction, including quantitative energetics, stereochemistry, hydrogen bonding network, and role of solvent molecules, are found to be supported and confirmed by kinetic and structural data. The results show that this type of reaction calculations can provide important links between structure and function that cannot be obtained by experimental means.     

Affinity labelling of yeast ribosomal peptidyl transferase

Summary Using p-nitrophenylcarbamyl-phenylananyl-tRNA (PNPC-Phe-tRNA) and N-Iodoacetyl-phenylalanyl-tRNA as affinity labels we have attempted to identify the components of the aminoacyl-tRNA binding sites located in the vicinity of the peptidyl transferase centre of the yeast ribosome. Both Phe-tRNA derivatives bind to the ribosomal A-site in the presence of 20 mM Mg⁺⁺ ion concentration and can be translocated to the ribosomal P-site in the presence of elongation factor. After the labels have been allowed to react covalently with ribosomes they were found associated with the large ribosomal subunit. Proteins L36, L43, L42, L29, L2, L17/18, L19/20 and proteins L26, L38, L22/23, L7/9, L4/6, L36, L11, L43, L39 were labelled in samples treated with PNPC-Phe-tRNA and N-Iodoacetyl-Phe-tRNA respectively. In contrast, when only the components of the ribosomal P-site were analysed by reacting the treated particles with puromycin fewer spots were labelled, corresponding to proteins L36 and L19/20 using PNPC-Phe-tRNA and proteins L4/6, L36, and L43 using N-Iodoacetyl-Phe-tRNA.     

The involvement of protein L16 on ribosomal peptidyl transferase activity.

Radioactive ribosomes from Escherichia coli were treated with increasing concentrations of NH4Cl in the presence of 50% ethanol. The resulting particles were tested for peptidyl transferase activity as well as for the binding of (U)C-A-C-C-A-Leu-Ac, (U)C-A-C-C-A-Leu, chloramphenicol, lincomycin and erythromycin. At the same time the proteins present in the particles were quantitatively estimated and the amount of each related to the residual activity displayed by the treated ribosomes. It was found that the loss of protein L16 closely paralleled the inactivation of the particles implying an important role for this protein in the structure of the peptidyl transferase center.     

Interaction of arginine with the ribosomal peptidyl transferase centre.

Arginine inhibits the formation of acetylleucyl-puromycin from C(U)-A-C-C-A-LeuAc and puromycin ('fragment reaction'), catalized by Escherichia coli and yeast ribosomes. From 18 different L-amino acids assayed, arginine was the most effective in producing inhibition (50% inhibition at 20 mM, with 1 mM puromycin). L-Argininamide and D-arginine gave about the same inhibition as L-arginine. The inhibition by L-arginine is competitive with respect to puromycin. The plot of the slopes obtained in a Lineweaver and Burk representation versus [Arg]2, and the plot of 1/v versus [Arg]2 at a fixed concentration of puromycin, are linear, which seems to indicate that two arginine molecules must interact at the puromycin binding site to produce inhibition. In addition to the 'fragment reaction', arginine inhibits the non-enzymatic binding of AcPhe-tRNA, C(U)-A-C-C-A-Leu and C(U)-A-C-C-A-LeuAc to ribosomes. However, it does not inhibit poly(U)-directed polyphenylalanine synthesis or the reaction of puromycin with AcPhe-tRNA previously bound to the peptidyl site. The results agree with arginine binding to the acceptor site, and with a sequential mechanism for the 'fragment reaction', puromycin binding first.     

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.     

Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction

The ribosome is the macromolecular machine responsible for protein synthesis in all cells. Here, we establish a kinetic framework for the 50S modified fragment reaction that makes it possible to measure the kinetic effects that result from isotopic substitution in either the A or P site of the ribosome. This simplified peptidyl transferase assay follows a rapid equilibrium random mechanism in which the reverse reaction is nonexistent and the forward commitment is negligible. A normal effect (1.009) is observed for (15)N substitution of the incoming nucleophile at both low and high pH. This suggests that the first irreversible step is the formation of the tetrahedral intermediate. The observation of a normal isotope effect that does not change as a function of pH suggests that the ribosome promotes peptide bond formation by a mechanism that differs in its details from an uncatalyzed aminolysis reaction in solution. This implies that the ribosome contributes chemically to catalysis of peptide bond formation.     

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.     

pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.     

Investigation of the ribosomal peptidyl transferase center using a photoaffinity label

The synthesis of a peptidyl-tRNA photoaffinity analog, 2-nitro-4-azidophenoxy-4′-phenylacetyl-phenylalanyl-tRNA^Phe is described. Covalent attachment of this analog to Escherichia coli 70 S ribosomes requires poly(U)-stimulated binding prior to photolysis. Peptidyl site binding is indicated by the ability of puromycin to release the peptidyl moiety from non-photolyzed samples. Covalently attached 2-nitro-4-azidophenoxy-4-phenylacetyl-Phe-tRNA^Phe can subsequently participate in peptidyl transfer with [³H]Phe-tRNA^Phe bound at the aminoacyl site. This means that the covalent reaction does not produce sufficient distortion of the peptidyl site and its bound tRNA to inactivate the peptidyl transference. If peptidyl transfer with [³H]Phe-tRNA^Phe is allowed to proceed before photolysis, covalent reaction can still occur. In all cases, the main reaction products are two 50 S ribosomal proteins, L11 and L18. The results strongly indicate that these two proteins either form part of the peptidyl transferase center or are located adjacent to it. Presumably, α-halocarbonyl affinity reagents used previously failed to identify these two proteins because they lack easily accessible, reactive nucleophilic groups.     

Pre-steady-state kinetics of ribosomal translocation

The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique. The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP. The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe. Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM). The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol). The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site. The reactions take place simultaneously to form an intermediate post-translocation complex. The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states. In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process. The slow process has not been assigned.     

The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA.

The peptidyl transfer site has been localized at the centre of domain V of 23S-like ribosomal RNA (rRNA) primarily on the basis of a chloramphenicol binding site. The implicated region constitutes an unstructured circle in the current secondary structural model which contains several universally conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed in vivo on a plasmid-encoded rRNA (rrnB) operon and each one yielded dramatically altered phenotypes. Cells exhibiting A2060----C or A2450----C transversions were inviable and it was shown by inserting the mutated genes after a temperature-inducible promoter that the mutant RNAs were directly responsible. In addition, a G2502----A transition caused a decreased growth rate, probably due to a partial selection against mutant ribosome incorporation into polysomes, while an A2503----C transversion produced a decreased growth rate and conferred resistance to chloramphenicol. All of the mutant RNAs were incorporated into 50S subunits, but while the two lethal mutant RNAs were strongly selected against in 70S ribosomes, the plasmid-encoded A2503----C RNA was preferred over the chromosome-encoded RNA, contrary to current regulatory theories. The results establish the critical structural and functional importance of highly conserved nucleotides in the chloramphenicol binding region. A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface.