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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]).
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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,
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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
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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
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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
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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
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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].
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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
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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. The mechanism of
signaling to the peptidyl-transferase center is not known,
but given the progress in ribosome mutagenesis, the availability of efficient translation systems and advances in
structural studies, these questions are now within reach.
Acknowledgements
We thank Niels Fischer for preparing Figure 1b, and Venki Ramakrishnan
and Harry Noller for providing results before publication. Work in our
laboratories is supported by the Deutsche Forschungsgemeinschaft, the
Alfried Krupp von Bohlen und Halbach-Stiftung, and the Fonds der
Chemischen Industrie.
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