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TIBS-461; No of Pages 7 Review TRENDS in Biochemical Sciences Vol.xxx No.x How ribosomes make peptide bonds Marina V. Rodnina1, Malte Beringer1 and Wolfgang Wintermeyer2 1 2 Institute of Physical Biochemistry, University of Witten/Herdecke, Witten, D-58448, Germany Institute of Molecular Biology, University of Witten/Herdecke, Witten, D-58448, Germany Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds – the peptidyl-transferase center – is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate. The ribosome is a ribozyme Most natural catalytic RNAs, or ribozymes, are involved in RNA maturation. They catalyze phosphoryl-transfer reactions that require the activation of either a ribose hydroxyl group (e.g. hammerhead ribozyme, hepatitis delta ribozyme, hairpin ribozyme, self-splicing introns and, perhaps, the spliceosome) or a water molecule (e.g. RNase P) for nucleophilic attack of a phosphodiester bond [1]. Compared with protein enzymes, which are chemically much more diverse, ribozymes possess a limited repertoire of groups that take part in catalysis. Nevertheless, ribozymes use several mechanisms, including general acid-base catalysis, metal ion-assisted catalysis, and substrate-alignment by base-pairing and other interactions. Thus, they act in ways that are similar to protein enzymes [2]. The most abundant natural ribozyme is the ribosome, which is a ribonucleoprotein particle that synthesizes proteins and is the only natural RNA-based polymerase. The ribosome binds two tRNA substrates, one with the growing peptide chain attached by a high-energy ester linkage to its 30 hydroxyl (the peptidyl-tRNA in the P site), and the other with a single amino acid esterified to its 30 hydroxyl (the aminoacyl-tRNA in the A site) (Figure 1). During peptide-bond formation, the a-amino group of the A-site aminoacyl-tRNA attacks the carbonyl carbon of the P-site peptidyl-tRNA to produce a new peptidyl-tRNA that is one-amino-acid longer in the A site and a deacylated tRNA in the P site. The second enzymatic activity associated with the peptidyl-transferase center is the hydrolytic cleavage of the ester bond in peptidyl-tRNA during termination of protein synthesis. In contrast to peptidebond formation, which is an intrinsic activity of the Corresponding author: Rodnina, M.V. ([email protected]). Available online xxxxxx. www.sciencedirect.com ribosome and proceeds without auxiliary factors, peptide release requires specialized release factors that recognize termination codons and promote the hydrolysis of the P-site peptidyl-tRNA. Although studied for decades, it is only in the past few years that enormous progress has been made in understanding ribosome function. Crystal structures have provided much information about the active site for peptide-bond formation. Rigorous kinetic analysis and the development of methods to produce and purify ribosomes with mutations in crucial rRNA residues have revealed the nature of catalysis and the role of ribosome residues. Computational analysis has enabled the reaction trajectories to be modeled, thereby providing direct insight into the mechanism. Structural and mutagenesis studies, enzymology, and computer simulations converge at a consistent picture of the mechanism of the peptidyl-transfer reaction of the prokaryotic ribosome, which is the main focus of this review. Structure of the peptidyl-transferase centre The catalytic center for peptide-bond formation is located on the large ribosomal subunit. The large subunit in bacteria, 50S, is composed of two RNA molecules, 23S rRNA and 5S rRNA, and >30 proteins. The 50S subunit alone can synthesize peptide bonds as rapidly as the 70S ribosome [3]. One approach to studying peptide-bond formation is to crystallize ribosomes with substrates, transition-state analogs and products [4–15]. The high-resolution crystal structures of ribosomes have revealed that the peptidyl-transferase center is composed of RNA only, with no protein within 15 Å of the active site, which supports earlier biochemical evidence of the key role of rRNA, rather than proteins, in the catalysis of peptide-bond formation [4,7,10,11,14]. The only protein that might be involved is L27 because the deletion of as few as three amino acids at the N terminus of L27 leads to impaired activity [16]. The flexible N terminus of L27, which protrudes towards the interface of the bacterial 50S subunit, might contact the 30 terminus of the P site tRNA. However, some organisms do not have L27 or any protein groups where the N terminus of L27 is located, which indicates that L27 is not part of an evolutionary conserved mechanism (which is expected to employ identical residues in all organisms). However, it is not excluded that L27 contributes to tRNA positioning at the catalytic site [16]. The acceptor ends of A-site and P-site tRNAs are located in a cleft of the 50S subunit on the side facing the 30S subunit [6,13]. Their universally conserved 30 -terminal CCA residues are oriented and held in place by interactions with 23S rRNA (Figure 2a). The conserved bases A2451, 0968-0004/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2006.11.007 Please cite this article in press as: Rodnina, M.V. et al., How ribosomes make peptide bonds, Trends Biochem. Sci. (2006), doi:10.1016/j.tibs.2006.11.007 TIBS-461; No of Pages 7 Review 2 TRENDS in Biochemical Sciences Vol.xxx No.x Figure 1. Peptide-bond formation on the ribosome. (a) Reaction scheme. The a-amino group of aminoacyl-tRNA in the A site (yellow) attacks the carbonyl carbon of the peptidyl-tRNA in the P site (orange) to produce a new peptidyl-tRNA that is one amino acid longer in the A site and a deacylated tRNA in the P site. The peptidyl-transferase center is on the 50S subunit (green). On the 30S subunit (gray), aminoacyl-tRNA are recognized according to the match between their anticodons and the codon of mRNA in the A site. (b) Structure of the ribosome with bound tRNAs. The model is based on the crystal structure of E. coli ribosomes [12,58]. The tRNA positions in the P site and the A site have been adjusted according to [13]. Ribosomal protein L1 and the L12 stalk [59] are shown for orientation. U2506, U2585, C2452 and A2602 are located at the core of the peptidyl-transferase center [5,9,10] (Figure 2b). The crystal structures of Haloarcula marismortui 50S subunits complexed with different transition-state analogs have revealed that the reaction proceeds through a tetrahedral intermediate with S chirality [9]. The oxyanion of the tetrahedral intermediate is stabilized by a water molecule that is positioned by nucleotides A2602 and U2584 [9]. The only atom within hydrogen-bonding distance of the aamino group mimic is the 20 -OH of A76 of the P-site moiety of the transition-state analog [9]. N3 of A2451, which is within hydrogen-bonding distance of the a-amino group in the pre-reaction state (see later), seems to lose this interaction during the course of the reaction. The rRNA backbone in the peptidyl-transferase center occurs in similar conformations in 50S subunits from H. marismortui with various ligands [6,7,9–11], 50S from Deinococcus radiodurans [17], 70S ribosomes from Thermus thermophilus with a P-site tRNA [14,15] and vacant 70S ribosomes from Escherichia coli [12]. However, elements of the peptidyltransferase center might assume different orientations, for example when a substrate is bound to the A site [10]. Some active-site nucleotides are particularly mobile, such as A2602, which lies between the A and P sites [5,14]. Enzymology of peptidyl-transfer reaction The active sites of enzymes contain residues that participate in the chemical transformation of substrates. The main functions of these residues are to modulate the electrostatic environment and chemical catalysis, including facilitation of proton-transfer reactions and covalent chemistry at the reaction center. General acids, general bases and catalytic nucleophiles represent essential activesite residues because they participate directly in the formation and rupture of covalent bonds. Further contributions to catalysis include electrostatic and structural complementarity to the transition state, reorganization of water, and the use of the binding energy for substrate positioning and lowering the entropy of activation [18,19]. The ribosome does not employ covalent catalysis [20], but all other strategies might be involved. The aim of studying the enzymology of peptidyl transfer is to assess the contribution of different catalytic strategies and to reveal the catalytic role of ribosomal groups at the active site. Before peptide-bond formation, aminoacyl-tRNA must enter the A site of ribosomes that carry a peptidyl-tRNA in the P site. The rate of binding of aminoacyl-tRNA to the A site (accommodation) is in the range of 10 s1 [21], which is significantly slower than the intrinsic rate of peptide-bond www.sciencedirect.com Please cite this article in press as: Rodnina, M.V. et al., How ribosomes make peptide bonds, Trends Biochem. Sci. (2006), doi:10.1016/j.tibs.2006.11.007 TIBS-461; No of Pages 7 Review TRENDS in Biochemical Sciences Figure 2. Active-site residues of 50S subunits from H. marismortui with either bound substrate (a) or a transition-state analog (b). (a) Base-pairs formed between cytosine residues of the tRNA analogs in the A site (yellow) and P site (orange) with 23S rRNA bases (green) are indicated (PDB code: 1VQN) [10]. The a-amino group of the A-site substrate (blue) is positioned for the attack on the carbonyl carbon of the ester that links the peptide moiety of the P-site substrate (green). (b) Transitionstate analog (TSA) bound to the peptidyl-transferase center (PDB code: 1VQP) [9]. The hydrogen bond between the nucleophilic nitrogen (blue) and the 20 -OH of A76 at the P site is indicated. Structures in (a) and (b) are shown in different orientations. formation, which was estimated to be 300 s1 [22]. Therefore, the mechanism of peptide-bond formation cannot be studied with the native aa-tRNA substrate under current experimental capabilities. This can be circumvented partially by using A-site analogs of aminoacyltRNA, which are either short fragments that mimic the 30 end, or have a weaker attacking nucleophile, or both (Figure 3). Short 30 -end analogs of tRNA, such as puromycin and C-puromycin, which contains an additional cytidine residue that is analogous to C75 of tRNA, bind to the peptidyl-transferase center rapidly and are incorporated at rates of up to 50 s1 [23,24], which enables the chemistry step to be studied without being limited by accommodation. The reactions with short A-site-substrate analogs and the natural aminoacyl-tRNA are similar because they use the same reaction chemistry and are susceptible to the same inhibitors. However, the analogs lack the tRNA body, so the details of substrate positioning might differ between small analogs and full-size tRNAs. The ribosome brings about a 107-fold enhancement in the rate of the peptidyl-transfer reaction compared with the second-order reaction between model substrates in solution [25]. This acceleration is achieved by lowering the entropy of activation, whereas the enthalpy of Vol.xxx No.x 3 Figure 3. A-site substrates and substrate analogs. Reactive nucleophilic groups are circled. A76 is the 30 -terminal residue of tRNA to which the amino acid/growing polypeptide is attached; the rest of the tRNA molecule is not shown for simplicity. Puromycin is O-methyl tyrosine that is linked to N6-dimethyl adenosine via an amide bond; C-puromycin is puromycin with an additional cytidine residue that is analogous to C75 of tRNA. activation is the same for the reaction on the ribosome and in solution [26] (Figure 4). By contrast, enzymes that employ general acid–base or covalent catalysis act by lowering the activation enthalpy of the catalyzed reaction. Thus, the ribosome seems to use mechanisms of catalysis that are largely entropic in origin, such as substrate positioning in the active site, desolvation and electrostatic shielding [25]. If the ribosome used chemical catalysis with ribosomal residues acting as either general acids or bases, the reaction rate should depend on pH. In addition, the pKa of the a-amino group of aminoacyl-tRNA is 8 [27] and, because only the deprotonated form of the nucleophile is active, the reaction rate should increase as pH increases, even if other ionizing groups are not involved. In fact, measurements in the 1960s and 1970s indicated that the reaction rate increases with pH, and the increase per pH unit indicated that a single ionizing group is involved [28,29]. At the time (before the discovery of RNA catalysis) a histidine residue of a ribosomal protein was proposed to act as a catalyst, which prompted a search for catalytic histidines in ribosomal proteins [30]. However, the conditions of these early experiments did not enable the chemistry step to be monitored rigorously, and the observed pH dependence was probably caused by ionization of the a-amino group in many of the experiments. The ambiguities caused by ionization of the a-amino group have been circumvented using an aminoacyl-tRNA www.sciencedirect.com Please cite this article in press as: Rodnina, M.V. et al., How ribosomes make peptide bonds, Trends Biochem. Sci. (2006), doi:10.1016/j.tibs.2006.11.007 TIBS-461; No of Pages 7 Review 4 TRENDS in Biochemical Sciences Vol.xxx No.x Figure 4. Entropic catalysis by the ribosome. Activation parameters are shown for the second-order uncatalyzed (knon) and ribosome-catalyzed peptide-bond formation at substrate limitation (kcat/KM) or saturation (kcat) [25,26]. Abbreviations: S1, P-site substrate; S2, A-site substrate; P, reaction products. Reproduced, with permission, from Ref. [25]. derivative in which a hydroxyl group replaces the amino group as reactive nucleophile [22,24,31]. The ribosome readily accepts hydroxy-Phe-tRNA as a substrate and catalyzes the formation of an ester bond instead of a peptide bond. The accommodation of hydroxy-Phe-tRNA is not rate-limiting because the formation of an ester bond is slow and, therefore, hydroxy-Phe-tRNA can be used to monitor the pH-dependence of the reaction with the fulllength substrate. Measuring the reaction rate at pH 6–9 reveals that changes in pH do not affect the reaction rate [22]. This indicates that catalysis by the peptidyl-transferase center is independent of pH, which argues against the involvement of ionizing groups of the ribosome in chemical catalysis and indicates that general acid–base catalysis is not used to a great extent. Peptide-bond formation between full-length peptidyl-tRNA and aa-tRNAs with a native amino group is also independent of pH [22]. Although the accommodation step, which is rate-limiting, might mask part of a potential pH effect, these results are consistent with protonation and deprotonation events having either small or no influence on the reaction. In contrast to full-length substrates, a pronounced pH-dependence has been observed with the minimal A-site substrate, puromycin. Protonation of a ribosomal group (pKa 7.5) reduces the rate of reaction by 150-fold [23,24], which is much less than expected for an essential base. This effect reflects a conformational rearrangement of active-site residues that impairs catalysis but does not take place with full-length aa-tRNA. This conclusion is corroborated by the observation that the reaction between A-site C-puromycin and P-site peptidyl-tRNA is not influenced by the ionization of ribosomal groups, which indicates that the presence of the cytidine residue (which mimics C75 of the A-site tRNA and, presumably, its interaction with G2553) (Figure 2a), is sufficient to induce and stabilize the active conformation of the peptidyl-transferase center [23]. Are bases of 23S rRNA involved in catalysis? Identification of the ribosomal residues that form the catalytic site has raised the question of the possible roles of these rRNA residues in catalysis. The effects of mutating several 23S rRNA bases that are either in the, so-called, inner shell of the active site (A2541, U2506, U2585 and A2602) (Figure 2b) [24,32–36] or adjacent to it (G2447) [34,35,37], and the non-canonical pair (A2450-C2063) [32,38] have been examined. Strikingly, none of these mutant ribosomes (except those with the A2450GC2063U mutation, see later) have defects in the rate of peptide-bond formation with either full-length, intact aatRNA or C-puromycin [33,36,37]. However, eight of the nine mutants exhibited a strong reduction in the rate of reaction with puromycin (30–9400-fold reduction compared with wild-type ribosomes) [24,33,36,37]. The largest decrease in reaction rate with the native aminoacyl-tRNA (200-fold) occurred with the A2450G-C2063U double mutation [32]. It is likely that replacing the ionizing A+-C pair with a G-U pair is less isosteric than expected and that this disturbs the structure of the active site [32]. The role of A2451 deserves particular comment because the notion that A2451 acts as a catalytic residue in peptidyl transfer has entered biochemistry textbooks. Kinetic analysis of A2451U and G2447A mutants (the latter residue forms an essential part of the charge-relay system that is postulated to bring about the required pKa shift of A2451) in two organisms, E. coli and Mycobacterium smegmatis, argues strongly against an essential role of A2451 in peptide-bond formation [33,36,37]. Rather, 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 23S rRNA [36]. A2451 seems to function as a pivot point in stabilizing the ordered structure of the active site, rather than by taking part in chemical catalysis [36]. Which other groups might be involved? A group that is within hydrogen-bonding distance of the nucleophilic group of transition-state analogs is the 20 -OH of A76 of peptidyl-tRNA in the P site [9,10]. This has a crucial role in the reaction on both isolated 50S subunits [20] and 70S ribosomes [39] but not in the uncatalyzed www.sciencedirect.com Please cite this article in press as: Rodnina, M.V. et al., How ribosomes make peptide bonds, Trends Biochem. Sci. (2006), doi:10.1016/j.tibs.2006.11.007 TIBS-461; No of Pages 7 Review TRENDS in Biochemical Sciences reaction [25,40]. Substitution of 20 -OH of A76 by either hydrogen (20 -deoxy) or fluor (20 -fluoro) reduce the activity 106-fold [39]. Notably, there are no catalytic Mg2+ ions or monovalent metal ions in the vicinity of the 20 -OH of the Psite tRNA [9,14] that might either promote catalysis directly or shift the intrinsically high pKa of the 20 -OH towards neutrality. The essential role of the 20 -OH of A76 of the P-site substrate indicates that the ribosome uses substrate-assisted catalysis (i.e. a mechanism in which a functional group of the substrate contributes to catalysis). Several protein enzymes use substrate-assisted catalysis, including GTPases, serine proteases, type II restriction endonucleases, lysozyme and hexose-1-phosphate uridylyltransferase [41]. Evidence for the involvement of additional groups in catalysis comes from recent crystal structures [9] and is supported by molecular dynamics calculations [42,43]. The 20 -OH of the ribose moiety of A2451 seems to be part of the intricate hydrogen-bond network in the active site and to interact directly with the crucial 20 -OH group of the P-site tRNA. Consistently, substitution of the 20 -OH of A2451 by hydrogen impairs peptidyl-transferase activity [44]. Computational analysis One role of the 20 -OH of A76 of the P-site tRNA has been suggested following computational analysis [42,43]. Molecular-dynamics simulations and free energy-perturbation simulations, in combination with an empirical valence-bond description of the reaction energy surface have been used to examine possible catalytic mechanisms. Simulations of the reactant and tetrahedral intermediate states of the peptidyl-transferase center reveal a stable, pre-organized, hydrogen-bond network that is poised for catalysis (Figure 5). The peptidyl-transferase center might, thus, be viewed as a rigid environment of pre-organized dipoles that do not need to rearrange during the reaction. Figure 5. Concerted proton-shuttle mechanism. The P-site and A-site tRNA substrates are blue and red, respectively, ribosome residues are green, and ordered water molecules that stabilize the developing charges are gray. The attack of the a-NH2 group on the ester carbon results in a six-membered transition state, in which the 20 -OH group of the A-site A76 ribose moiety donates its proton to the adjacent 30 oxygen while simultaneously receiving one of the amino protons [9,42]. Alternatively, the water molecule (*) might be used for a proton shuttle. Modified, with permission, from Ref. [60]. Vol.xxx No.x 5 According to molecular-dynamics simulations, the most favorable mechanism does not involve general acid–base catalysis by ribosomal groups [42]. Rather, the catalytic effect is of entirely entropic origin, which is in accordance with experimental results [25], and is associated with the reduction of solvent reorganization energy rather than with either alignment or proximity of the substrate [42]. The 20 -OH of A76 of the P-site tRNA might take part in a proton shuttle that bridges the attacking a-amino group and the leaving 30 oxygen, and several shuttle pathways can be envisaged [9,42,43,45,46]. The attack of the a-amino group on the ester carbon might result in a six-membered transition state, in which the 20 -OH group donates its proton to the adjacent 30 oxygen while simultaneously receiving one of the amino protons. Such a scenario does not require a pKa shift of the 20 -OH group because of the concerted nature of the bond-forming and bond-breaking events, and is in line with earlier suggestions that are based on biochemical evidence with model substrates [47] and quantum-dynamic simulations [45]. The mechanism of peptide-bond formation The combined evidence supports strongly the idea that entropic catalysis provides the major catalytic mechanism of peptide-bond formation on the ribosome [25,42]. The main supporting observations from structural analysis are the precise alignment of the A-site and P-site substrates by interactions of their CCA sequences, and of the nucleophilic a-amino group of the A-site substrate with residues of 23S rRNA in the active site [9,10,48–50]. The most favorable mechanism of catalysis involves intra-reactant proton shuttling via the 20 -OH of A76 of the P-site tRNA, which follows the attack of the A-site a-amino group on the P-site ester bond (Figure 5) [9,42]. The reaction does not involve chemical catalysis by ribosomal groups but might be modulated by conformational changes at the active site [22,23,33–37]. In addition to bringing the reactive groups into close proximity and precise orientation relative to each other, the ribosome might work by providing a pre-organized electrostatic environment that reduces the free energy needed to form the highly polar transition state, shielding the reaction against bulk water, helping the proton shuttle forming the leaving group, or a combination of these effects. The ribosome is an ancient RNA catalyst that accelerates the peptidyl-transfer reaction by a factor of 107 [25]. It is much less efficient than many protein enzymes, which use chemical catalysis and accelerate reactions by up to 1023fold [51]. Apparently, evolutionary pressure has had a much larger influence on increasing the speed and fidelity of the rate-limiting steps of protein synthesis, which do not involve chemistry, such as substrate binding [52], than on the chemistry step of peptide-bond formation. This has enabled the ribosome to retain its catalytic strategy during the evolution of a pre-biotic translational ribozyme into a modern ribosome. Thus, the catalytic mechanism employed by the ribosome seems to be a fossil from the RNA world. Future perspectives It is presumed that the catalytic mechanism of peptide-bond formation on the ribosome is highly conserved in all www.sciencedirect.com Please cite this article in press as: Rodnina, M.V. et al., How ribosomes make peptide bonds, Trends Biochem. Sci. (2006), doi:10.1016/j.tibs.2006.11.007 TIBS-461; No of Pages 7 Review 6 TRENDS in Biochemical Sciences organisms. Given the high degree of sequence conservation of rRNA, in particular at the peptidyl-transferase center [4,53,54], the active site for the reaction is likely to consist of rRNA in all organisms. However, the details of the positioning of groups in the peptidyl-transferase active site might differ between species [4,17]. Most of the biochemical data available have been obtained with E. coli ribosomes and, recently, with ribosomes from the Gram-positive bacterium M. smegmatis [36], and information from other organisms is scarce. One of the future challenges is to obtain structural and mechanistic information for eukaryotic ribosomes. The fundamental aspects of the mechanism of peptide-bond formation have been revealed, so a major challenge is to probe the mechanism of the second important function of the peptidyl-transferase center, the hydrolytic cleavage of the ester bond in peptidyl-tRNA during the termination of protein synthesis. Crystal structures indicate that binding of the tRNA CCA end to the A site, which mimics the action of termination factors by inducing peptide release, promotes a conformational rearrangement at the active site to move the ester group of peptidyl-tRNA into a position that enables the attack of the water molecule [10]. In the context of this model, release factors presumably promote the conformational rearrangement of at least a subset of the rRNA nucleotides that are responsible for activation. The characterization of these structural changes is another challenge. Finally, the hydrolytic activity of the peptidyl-transferase center can be influenced from inside the peptide exit tunnel, presumably by inducing an inactive conformation of the catalytic center via allosteric effects. There are several examples of regulatory peptides that traverse the exit tunnel and inhibit release factor-dependent peptidyl-tRNA hydrolysis in the peptidyl-transferase center, including the 22residue peptide product of an ORF upstream of the gp48 gene of human cytomegalovirus [55] and the TnaC leader peptide that, together with tryptophan, regulates the transcription of the tryptophanase operon in E. coli [56,57]. In both cases, termination at a stop codon is blocked. This yields ribosomes that carry unhydrolyzed peptidyl-tRNA in the P site and are stalled at the end of the coding sequence of the leader peptide, which inhibits transcription of the downstream gene by an attenuation mechanism. 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