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A new beginning of the end of translation
© 2002 Nature Publishing Group http://structbio.nature.com
Måns Ehrenberg and Tanel Tenson
The structures of peptide release factors raise new questions about their mechanism of action in protein
synthesis.
When a ribosome has completed the synthesis of a protein, the polypeptide must be
released from the synthesis machinery to
do its job in the cell. Termination of protein
synthesis is normally signaled by one of
three base triplets in mRNA — UAG, UAA
or UGA. When one of these ‘stop codons’ is
translocated into the aminoacyl-tRNA site
(A-site) of the ribosome, it is recognized by
a protein called a ‘class 1 release factor’. The
release factor (RF) then induces hydrolytic
cleavage of the ester bond connecting the
growing peptide chain to the tRNA on the
ribosome1. Following ester bond hydrolysis, the release factor is rapidly removed
from the ribosome by the G-protein RF32.
In eukaryotes (and in archaea) there is a
single release factor protein, eRF1 (or aRF1
in archaea), that is capable of recognizing
all three stop codons and terminating protein synthesis. In bacteria, however, there
are two release factor proteins, RF1 and
RF2, which recognize UAA or UAG and
UAA or UGA, respectively.
How do release factors specifically recognize stop codons? How do they effect
polypeptide release from the tRNA? Part of
the answer to these questions may come
from the molecular architecture of the
release factors. The crystal structure of RF2
from Escherichia coli, reported in a recent
issue of Molecular Cell3, and that of human
eRF14 provide a view of these molecules at
high resolution. The structures reveal that
both eRF1 and RF2 contain three structural
domains (Fig. 1) that may be directly
involved in their dual functions (stop codon
recognition and hydrolysis of the ester bond
in peptidyl-tRNA). However, it is striking
that little similarity exists between the
structures of eRF1 and RF2, considering
that they carry out very similar tasks, albeit
in different organisms. These observations
raise new issues about the molecular mechanisms of the action of release factors.
Recognition of stop codons
In principle, the function of class 1 release
factors appears to be very simple. If a ribosome is programmed with a stop codon,
the release factor should bind to the ribosomal A-site and allow for a water molecule
BOX 1 Macromolecular mimicry in translation
The crystal structure of the ternary complex aminoacyl-tRNA–EF-Tu–GTP resembles that of
EF-G. The shape and charge distribution of its domain IV are very similar to those of the tRNA
anticodon stem-loop. This suggests that structural mimicry may exist among tRNA and several
protein translation factors14,25, and that these factors function by binding the aminoacyl-tRNA
(A-) site on the ribosome.
There is certainly a functional mimicry between aminoacyl-tRNAs and class 1 RFs: both categories of molecules need to recognize specific codons while simultaneously interacting with
the peptidyl transferase. For RFs, it is likely that the anticodon-recognition region binds the
decoding center while a different part of the structure binds near the peptidyl transferase to
trigger hydrolysis of the peptidyl-tRNA at the P-site. Common A-site binding is supported by
the observations that peptidyl transfer and termination are similarly affected by certain antibiotics26,27. This led to the early suggestion that these reactions are catalyzed by basically the
same mechanism27.
However, it is presently not clear to what extent the concept of macromolecular mimicry is useful. There can be mimicry between translation factors and tRNAs to varying degrees in different cases, and the concept may be totally irrelevant in others. For instance, the structures of
eRF1, RF2 and RRF are sufficiently different from that of tRNA to make the existence of a structural mimicry less than obvious (Fig. 1). Therefore, we need to inspect the putative existence of
mimicry critically in each case.
to hydrolyze the ester bond in the peptidyltRNA at the P-site. In practice, however,
termination at the right moment in protein
synthesis requires careful molecular
design. If a release factor mistakes a base
triplet encoding an amino acid (sense
codon) for a stop codon and prematurely
terminates protein synthesis, the time and
energy invested in making the polypeptide
chain are wasted. Class 1 release factors
must therefore be very good at discriminating between sense and stop codons.
How this task is carried out at the structural level is not known. We do know, however, that discrimination between stop and
sense codons is remarkably good and that
the precision is achieved without repeated
selections by proofreading5.
Recently, Ito et al.6 swapped domains
between RF1 and RF2 and demonstrated
that two of the amino acids in a tripeptide
motif, SPF (Fig. 1), determine whether the
release factor terminates at UAA or UAG as
does RF1, or at UAA or UGA as does RF2.
This result suggests that (i) a linear peptide
sequence in release factors determines their
identity as either RF1 or RF2; (ii) codon
recognition by release factors and tRNA
may have an important common principle,
although in RFs, an amino acid in SPF
determines which base is recognized,
whereas in tRNA, a base in the anticodon
nature structural biology • volume 9 number 2 • february 2002
does the job; and (iii) there could be a
direct contact between the SPF triplet and
the stop codon on the messenger RNA.
While the study of Ito et al.6 rationalizes
how bacterial RF1 and RF2 can recognize
different stop codons, it does not reveal
how they discriminate between U and
other bases in the first position of a codon.
Discrimination between bases in the first
codon position is essential to avoid premature termination at sense codons, and the
ability of peptide release factors to do so
with high precision must therefore be one
of the major organizing principles behind
their three-dimensional structure.
Hydrolysis of the ester bond
The second major task of the release factors is to induce hydrolysis of the ester
bond in a peptidyl-tRNA. The ribosome
itself can hydrolyze the ester bond in peptidyl-tRNAs even in the absence of release
factors. This activity is stimulated by high
pH, deacylated tRNA at the A-site and
organic solvents7. This observation suggests that the peptidyl transferase center
in the ribosome may be involved in terminating peptide synthesis. Moreover, it
suggests that the action of peptide release
factors may be indirect — that is, when a
release factor interacts with its cognate
stop codon at the A-site, it may induce a
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© 2002 Nature Publishing Group http://structbio.nature.com
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Fig. 1 The similarities and differences between the crystal structure of tRNA and the structures of proteins involved in termination of translation and
subsequent recycling of ribosomes to a new round of initiation. The anticodon of tRNA and the peptides proposed to be involved in codon recognition are shown in green. The conserved CCA end of tRNA contacting the peptidyl transferase center and the GGQ peptide are shown in red. Domain
numbering for eRF1 and RF2 is shown in blue.
conformational change at the peptidyl
transferase center that is primed to
hydrolyze the ester bond.
While it is not yet known how contacts
between a release factor and a ribosome
programmed with a stop codon can turn
on the hydrolytic reaction, one structural
element of this mechanism has been identified. There is very little homology
between the peptide sequences of class 1
release factors from different organisms,
with one notable exception. There exists a
universally conserved tripeptide, Gly-GlyGln (GGQ)8, which is in domain III of RF2
and in domain II of eRF1 (Fig. 1).
Substitutions of amino acids in the GGQ
motif do not impair stop codon recognition of the release factors but do impair or
abolish their ability to release the peptide
from tRNA (ref 9; A. Zavialov, unpublished
result). This suggests that the GGQ motif
may be in direct contact with the peptidyl
transferase center, similar to the amino
acid-carrying CCA end of a tRNA. If the
GGQ motif and the stop codon-recognizing peptide motif engage the ribosome
simultaneously, the distance between these
two motifs should correspond to the distance between the peptidyl transferase center of the large subunit and the decoding
site of the small ribosomal subunit, which
is 75 Å. This also implies that peptide
release factors mimic tRNAs, where the
distance between the anticodon and the
CCA end is ∼75 Å (Fig. 1; Box 1).
Functional domains of eRF1
As there is no SPF motif in eRF1, Song
et al.4 tentatively identified a codon recognizing region in eRF1 based on the hypothesis that eRF1 functionally mimics a tRNA.
Assuming that the GGQ motif in eRF1
mimics the 3′ CCA of a tRNA, conserved
amino acids, including a tetrapeptide
motif (NIKS) on the tip of domain I may
mimic the anticodon of tRNA (Fig. 1). One
obvious problem with this interpretation is
that the maximal distance between the
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GGQ motif in eRF1 and the tip of domain
I is much longer than the maximal distance
of 75 Å. The notion that domain I is
involved in codon recognition is supported
by genetic evidence10, which was used to
suggest that a conserved groove near but
not at the tip of domain I is the anticodon
mimic in eRF11,4. The distance between
this anticodon-like sequence and the GGQ
motif is ∼80 Å (ref. 4), a value very close to
75 Å. However, if one assumes, as do
Vestergaard et al.3, that the NIKS motif
itself is the anticodon mimic, the anticodon-like sequence would be separated
from the GGQ motif by 99.5 Å, a distance
much longer than 75 Å.
Implications of the RF2 structure
With the rich, complex background of the
termination of protein synthesis, what does
the structure of RF2 add to our knowledge
of this important process? The most unexpected finding by Vestergaard et al.3 is that
the distance between the GGQ motif and
the codon-recognizing SPF motif is merely
23 Å, much shorter than the expected 75 Å
if the release factor indeed mimics aminoacyl-tRNA. To save the mimicry paradigm,
Vestergaard et al.3 suggest instead that the
tRNA anticodon loop-stem is mimicked by
the three-helix domain (Fig. 1) of RF2. In
this way, they can dock the structure of RF2
to the ribosome; however, there is so far no
independent evidence that this region confers codon specificity. Furthermore, the SPF
motif identified genetically by Ito et al.6 is
not anywhere near the mRNA in this
model. It therefore seems that the crystal
structure of RF2 is incompatible with the
hypothesis that it mimicks a tRNA. It is also
difficult to rationalize the genetic and biochemical data from Ito et al.6 on the basis of
the RF2 structure.
One suggestion that may resolve these
apparent paradoxes is suggested by the
structure of eRF1 (Fig. 1). eRF1 may be
sufficiently flexible that the relative orientations of domain I, which contains the
NIKS motif, and domain II, which contains the GGQ motif, could be adjusted so
that the distance between these two motifs
becomes 75 Å upon eRF1 binding to the
ribosome. For RF2, it is less clear how this
may be achieved. However, it may be significant that the structure of a ribosomal
protein S5, which contains a loop structurally homologous to the GGQ loop of
RF2, is radically different in isolation compared to that when integrated in the small
subunit of the ribosome3. Therefore, docking of the crystal structures of isolated
translation factors to the ribosome could
be very misleading with respect to how
they actually look in ribosomal complexes.
Knowing the exact functions of the different domains of RF2 and where these
domains interact with the ribosome is crucial for how we interpret the structure of
yet another important protein, the ribosome recycling factor (RRF). This is an
essential protein responsible for recycling
ribosomes from their post-termination
state to a new round of initiation11,12. For
RRF, it has been proposed that the threehelix domain mimics the anticodon loopstem of tRNA13, but this argument is
considerably weakened by the fact that the
peculiar RNA helix-like charge distribution, which is observed in domain IV of
EF-G14, is absent in the three-helix domain
of RRF. Now, the work by Vestergaard
et al.3 reveals that the three-helix domain
of RF2 is remarkably similar to that of RRF
(Fig. 1). If their docking model of RF2 to
the ribosome, with the three-helix domain
in the decoding center is correct, this may
suggest that the three-helix domain of RRF
binds in a similar way and would support
the idea that this domain of RRF indeed
mimics the anticodon loop-stem of tRNA.
If, instead, domain II in RF2 with the
SPF motif is in contact with the decoding
center of the small ribosomal subunit6, the
three-helix domain will be excluded from
this site. This would suggest that the threehelix domain of RRF may not mimic the
nature structural biology • volume 9 number 2 • february 2002
© 2002 Nature Publishing Group http://structbio.nature.com
news and views
anticodon loop-stem region of tRNA, but
perform a completely different task. For
eRF1, it has been shown that, for a single
round of termination, domain III is not
needed15, and that this domain interacts
with the GTPase eRF315, the homolog of
which is essential for recycling of the
class 1 release factors in E. coli2,16. Therefore the function of the three-helix
domain of RF2 could be to interact with
RF3, whereas that of RRF may be to interact with EF-G, an elongation factor (and
GTPase) involved in the ribosome recycling function of RRF11,12,17.
Perspectives
The crystal structure of RF2 sheds some
light on the process of termination of protein synthesis. However, the most important aspects of termination by peptide
release factors — that is, codon recognition and removal of the peptide from peptidyl-tRNA — remain obscure. One
obvious reason for this is that the tasks of
peptide release factors are carried out
when the RFs are associated with ribosomes. Ultimate understanding of the
function of RFs will most likely require
determination of the structure of the
release factor–ribosome complexes, and
here the prospects look very bright.
In the last two years, high resolution
structures were obtained from crystals of
both large and small ribosomal subunits18–21; these were followed by the crystal
structure of the whole ribosome at 5.5 Å ed to M.E. email: [email protected].
resolution22. In addition, major break- uu.seor T.T. email: [email protected]
throughs in electron microscopy have
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Acknowledgments
We thank V. Ramakrishnan, R. Buckingham,
J. Frank and U. Rawat for helpful comments on the
manuscript. This work was supported by the
Swedish Foundation for Strategic Research, the
Swedish Research Council, the Wenner-Grenska
Samfundet Foundation and the Estonian Science
Foundation.
Måns Ehrenberg is at ICM, BMC, Box 596,
S-75124 Uppsala, Sweden, and Tanel Tenson
is in the Institute of Molecular and Cell
Biology, Tartu University, Riia 23, Tartu,
Estonia. Correspondence should be address-
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comments
Revisiting the allosteric mechanism of
aspartate transcarbamoylase
In an elegant work, Macol et al.1 claim to
prove that the binding energy of a single
bisubstrate molecule (PALA) to one of the
six active sites of Escherichia coli aspartate
transcarbamoylase (ATCase) is sufficient
to entirely shift the T to R quaternary
structure equilibrium2 into the R state. To
this aim, they designed a hybrid enzyme
containing a single functional active site,
with the other five sites rendered incapable of binding PALA by a point mutation. Although the authors’ conclusion is
reasonable for the hybrid enzyme, we
believe that its extension to the wild type
enzyme, the real issue at stake, is not warranted.
The authors content themselves with a
qualitative interpretation of their experimental data, equating a change in the
small-angle X-ray scattering pattern
(SAXS) upon PALA binding to the T to R
transition. They thereby overlook that in
the absence of substrate neither the scattering pattern of the R105A mutant nor
that of the hybrid enzyme is identical to
that of the wild type T state (Fig. 1). The
differences in the SAXS patterns can result
from the existence of different quaternary
structures or from a shift in the T-R equilibrium as compared to the wild type
enzyme. The observation that the crystal
structure of the mutant is identical to the
nature structural biology • volume 9 number 2 • february 2002
wild type T structure (for which the crystal and solution structures have been
shown to be identical3) supports the second possibility, the major conformation
present in solution being selectively incorporated into the crystal. This is further
confirmed by the fact that both patterns
can be approximated by a linear combination of 62% Twt-curve/38% Rwt-curve
(R105A) and 68% Twt-curve/32% Rwtcurve (hybrid, red curve in Fig. 1), respectively. This means that the unliganded
hybrid enzyme is in a 68% T-32% R equilibrium, corresponding to an allosteric
equilibrium L (L = T0 / R0) value of 2.1
(68/32) (∆GT-R = 0.44 kcal mol–1), much
87