The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination

The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively. We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis. While mutations at A2451, U2585, and C2063 (E. coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation. This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.     

The G2447A mutation does not affect ionization of a ribosomal group taking part in peptide bond formation

Peptide bond formation on the ribosome is catalyzed by RNA. Kinetic studies using Escherichia coli ribosomes have shown that catalysis (>10(5)-fold overall acceleration) is due to a large part to substrate positioning. However, peptide bond formation is inhibited approximately 100-fold by protonation of a ribosomal group with pKa=7.5, indicating either a contribution of general acid-base catalysis or inhibition by a pH-dependent conformational change within the active site. The function of a general base has been attributed to A2451 of 23S rRNA, and a charge relay system involving G2447 has been postulated to bring about the extensive pKa shift of A2451 implied in the model. Using a rapid kinetic assay, we found that the G2447A mutation, which has essentially no effect on cell growth, lowers the rate of peptide bond formation about 10-fold and does not affect the ionization of the ribosomal group with pKa=7.5 taking part in the reaction. This result does not support the proposed charge relay mechanism involving G2447 and the role of A2451 as general base in the catalysis of peptide bond formation.     

Effect of replacing uridine 33 in yeast tRNAPhe on the reaction with ribosomes

We have determined several kinetic parameters for the reaction of poly(U)-programmed ribosomes with ternary complexes of elongation factor Tu, GTP, and yeast Phe-tRNA analogs with different bases substituted for uridine in position 33. These analogs test whether disruption of the hydrogen bonds normally formed by uridine 33 and steric crowding in the anticodon loop are detrimental to tRNA function on the ribosome. Single-turnover kinetic studies of the reaction of these ternary complexes with ribosomes show that these Phe-tRNA analogs decrease the apparent rate of GTP hydrolysis (kGTP) and the ratio of peptide formed to GTP hydrolyzed. Thus, the substitution of uridine 33 affects not only the selection of a ternary complex by the ribosome but also the selection of an aminoacyl-tRNA in the proofreading reaction. The effects become greater as first one, and then the other, H-bond is disrupted. Steric crowding in the anticodon loop is also important, but does not have as great an effect on the rate constants. An analysis of the elementary rate constants which comprise the rate constant, kGTP, demonstrates that the reduction in kGTP results from a decreased rate of ternary complex association with the ribosome (k1) and that there is little or no effect on the rate of GTP cleavage (k2). An analysis of the rate constants involved in proofreading shows that all the modified (tRNAs have increased rates of aminoacyl-tRNA rejection (k4) but that the rate of peptide bond formation (k3) is unaffected.     

PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.     

What are the roles of substrate-assisted catalysis and proximity effects in peptide bond formation by the ribosome?

The action of the peptidyl transferase center of the large ribosomal unit presents a fundamental step in the evolution from the RNA world to the protein world. Thus, it is important to understand the origin of the catalytic power of this ancient enzyme. Earlier studies suggested that the ribosome catalyzes peptide bond formation by using one of its groups as a general base, while more recent works have proposed that the catalysis is due to proximity effects or to substrate-assisted catalysis. However, the actual nature of the catalytic mechanism remains controversial. This work addresses the origin of the catalytic power of the ribosome by using computer simulation approaches and comparing the energetics of the peptide bond formation in the ribosome and in water. It is found that a significant part of the observed activation entropy of the reference solution reaction is due to solvation entropy, and that the proximity effect is smaller than previously thought. It is also found that the 2'-OH of the A76 ribose, which is associated with a large rate acceleration in the ribosome reaction, does not catalyze peptide bond formation in water. Thus, the catalytic effect cannot be attributed to substrate-assisted catalysis but rather to the effect of the ribosome on the reacting system. Overall, our calculations indicate that the reduction of the activation free energy is mainly due to electrostatic effects. The nature of these effects and their relationship to catalytic factors in modern enzymes is analyzed and discussed.     

Mechanism of peptide bond formation on the ribosome

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.     

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.     

A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.     

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.     

Conformational Sampling of Aminoacyl-tRNA during Selection on the Bacterial Ribosome

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.     

Yeast ribosomal protein deletion mutants possess altered peptidyltransferase activity and different sensitivity to cycloheximide.

The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal peptidyltransferase. Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit. We have developed an in vitro system for the determination of peptidyltransferase activity in yeast ribosomes. Using this system, a kinetic analysis of a model reaction for peptidyltransferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromycin. This reaction (puromycin reaction) followed first-order kinetics. At saturating concentrations of puromycin, the first-order rate constant (k(3)) is identical to the catalytic rate constant (k(cat)) of peptidyltransferase. This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 degrees C. We now present for the first time kinetic evidence that yeast ribosomes lacking a particular protein of the 60S subunit may possess significantly altered peptide bond-forming ability. The k(cat) of peptidyltransferase from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 min(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1). These results suggest that the presence of ribosomal proteins L24 and, to a lesser extent, L39 is required for exhibition of the normal catalytic activity of the ribosome. Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis. However, the absence of L24 caused increased resistance to cycloheximide, a translocation inhibitor. Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 microM cycloheximide for the L24 mutant compared to 0.7 microM for the wild type.     

Minimal transition state charge stabilization of the oxyanion during peptide bond formation by the ribosome

Peptide bond formation during ribosomal protein synthesis involves an aminolysis reaction between the aminoacyl α-amino group and the carbonyl ester of the growing peptide via a transition state with a developing negative charge, the oxyanion. Structural and molecular dynamic studies have suggested that the ribosome may stabilize the oxyanion in the transition state of peptide bond formation via a highly ordered water molecule. To biochemically investigate this mechanistic hypothesis, we estimated the energetic contribution to catalytic charge stabilization of the oxyanion using a series of transition state mimics that contain different charge distributions and hydrogen bond potential on the functional group mimicking the oxyanion. Inhibitors containing an oxyanion mimic that carried a neutral charge and a mimic that preserved the negative charge but could not form hydrogen bonds had less than a 3-fold effect on inhibitor binding affinity. These observations argue that the ribosome provides minimal transition state charge stabilization to the oxyanion during peptide bond formation via the water molecule. This is in contrast to the substantial level of oxyanion stabilization provided by serine proteases. This suggests that the oxyanion may be neutralized via a proton shuttle, resulting in an uncharged transition state.     

Substrate-assisted catalysis of peptide bond formation by the ribosome

The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.     

Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome

The catalytic mechanism of peptide bond formation on the ribosome is not known. The crystal structure of 50S ribosomal subunits shows that the catalytic center consists of RNA only and suggests potential catalytic residues. Here we report rapid kinetics of the peptidyl transferase reaction with puromycin at rates up to 50 s(-1). The rate-pH profile of the reaction reveals that protonation of a single ribosomal residue (pK(a) = 7.5), in addition to protonation of the nucleophilic amino group, strongly inhibits the reaction (>100-fold). The A2451U mutation within the peptidyl transferase center has about the same inhibitory effect. These results suggest a contribution to overall catalysis of general acid-base and/or conformational catalysis involving an ionizing group at the active site.     

Peptide bond formation does not involve acid-base catalysis by ribosomal residues

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).     

Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp > Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.     

Essential mechanisms in the catalysis of peptide bond formation on the ribosome

Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.     

Mechanism of peptide bond formation on the ribosome--controversions

During past five years there have been published many experimental data concerning structure and function of the ribosome. With the presentation of atomic structures we obtained a new data about composition of peptidyl transferase center. It is now obvious that PTC is composed entirely of rRNA. It is also known that the proper substrate alignment is the major factor for ribosome's catalytic activity. However, more detailed mechanism of peptide bond formation on the ribosome still remains unclear. Several issues remain unsolved. For example, are there any chemical components coming from ribosome itself, that enhance the rate of the reaction? Do intact ribosomes perform peptidyltransfer in the same way as the isolated ribosomal subunits that have been the source of most of the data? In this article we present different opinions and controversions around peptide bond formation on the ribosome.     

Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs

The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.     

Kinetic studies on the interaction between a ribosomal complex active in peptide bond formation and the macrolide antibiotics tylosin and erythromycin

The inhibition of peptide bond formation by tylosin, a 16-membered ring macrolide, was studied in a model system derived from Escherichia coli. In this cell-free system, a peptide bond is formed between puromycin (acceptor substrate) and AcPhe-tRNA (donor substrate) bound at the P-site of poly(U)-programmed ribosomes. It is shown that tylosin inhibits puromycin reaction as a slow-binding, slowly reversible inhibitor. Detailed kinetic analysis reveals that tylosin (I) reacts rapidly with complex C, i.e., the AcPhe-tRNA. poly(U).70S ribosome complex, to form the encounter complex CI, which then undergoes a slow isomerization and is converted to a tight complex, CI, inactive toward puromycin. These events are described by the scheme C + I <==> (K(i)) CI <==> (k(4), k(5)) CI. The K(i), k(4), and k(5) values are equal to 3 microM, 1.5 min(-1), and 2.5 x 10(-3) min(-1), respectively. The extremely low value of k(5) implies that the inactivation of complex C by tylosin is almost irreversible. The irreversibility of the tylosin effect on peptide bond formation is significant for the interpretation of this antibiotic's therapeutic properties; it also renders the tylosin reaction a useful tool in the study of other macrolides failing to inhibit the puromycin reaction but competing with tylosin for common binding sites on the ribosome. Thus, the tylosin reaction, in conjunction with the puromycin reaction, was applied to investigate the erythromycin mode of action. It is shown that erythromycin (Er), like tylosin, interacts with complex C according to the kinetic scheme C + Er <==> (K(er)) CEr <==> (k(6), k(7)) C*Er and forms a tight complex, CEr, which remains active toward puromycin. The determination of K(er), k(6), and k(7) enables us to classify erythromycin as a slow-binding ligand of ribosomes.