The EMBO Journal Peer Review Process File - EMBO-2010-74478
Manuscript EMBO-2010-74478
Crystal structure of a transfer-ribonucleoprotein particle
that promotes asparagine formation
Mickael Blaise, Marc Bailly, Mathieu Frechin, Manja Annette Behrens, Frédéric Fischer, Cristiano
L. P. Oliveira, Hubert Dominique Becker, Jan Skov Pedersen, Søren Thirup and Daniel Kern
Corresponding author: Daniel Kern, Institut de Biologie Moléculaire et Cellulaire
Review timeline:
Submission date:
1st Editorial Decision:
1st Revision received:
2nd Editorial Decision:
2nd Revision received:
Accepted:
14 April 2010
17 May 2010
01 July 2010
12 July 2010
15 July 2010
15 July 2010
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
17 May 2010
Thank you for submitting your manuscript for consideration by The EMBO Journal. It has
now been seen by three referees whose comments to the authors are shown below. You will see that
the referees are generally positive about your work and that they would support its ultimate
publication in The EMBO Journal after some revision. I would thus like to invite you to prepare a
revised manuscript in which you need to address or respond to the points put forward by the referees
in an adequate manner. I should remind you that it is EMBO Journal policy to allow a single round
of revision only and that, therefore, acceptance of the manuscript will depend on the completeness
of your responses included in the next, final version of the manuscript.
When preparing your letter of response to the referees' comments, please bear in mind that this will
form part of the Peer-Review Process File, and will therefore be available online to the community.
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Thank you for the opportunity to consider your work for publication. I look forward to your
revision.
Yours sincerely,
Editor
The EMBO Journal
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-----------------------------------------------REFEREE COMMENTS
Referee #1 (Remarks to the Author):
This manuscript reports very fascinating stricture of novel tRNP, composed of two GatCABs, two
dimeric AspRSs and four tRNA(Asn)s. Two of the four tRNA(Asn)s are functiona with its CCA
terminus entering the active site of GatB, while the other two tRNAs act as a scaffold RNA to
stabilize the high molecular weight ribonucleoprotein complex. Furthermore, the scaffolding RNA
may enable the release of the functional Asn-tRNA(Asn) without dissociating the complex. The
important evidence is the specific interaction between GatB and ND-AspRS in structural aspect,
while the other important evidence is derived from the kinetic analysis presented by Fig. 3. This
conclusion provides a novel insight into the channeling reaction mechanism by ribonucleoprotein
complex and into the genetic code fidelity. Therefore, this referee strongly recommend that the
paper should be urgently published in EMBO J.
Only one request is that the author should more highlight and discuss the the scaffolding tRNA, by
presenting figures showing the recognition mechanism of overall tRNA(scaf). If possible, it is worth
testing the effect, to calalysis and thermostability, of mutations of Tyr95, Tyr264 and Asp96 in GatB
as well as Ser351 and Tyr353 in GatA, which are conserved and interacts with tNA(scaf) at the
GatA/B interface.
Anyway, without the experiments, the manuscript is very strong, but the above experiments should
strengthen the function of the non-functional scaffolding tRNA and the working hypothesis
presented in the paper.
Referee #2 (Remarks to the Author):
This outstanding paper describes a new ribonucleoprotein structure, that of the "transamidosome"
formed by multiple copies of tRNAAsn, a non-discriminating Aspartyl-tRNA synthetase, and a two
trimeric assemblies of the Glutamyl amidotransferase (GATABC) that catalyzes conversion of
acylated aspartyl-tRNAAsn to asparaginyl-tRNAAsn. The structure has considerable structural
novelty, and represents a new window on the post-acylation chemical transformation that is
characteristic of ~20% of the aminoacyl-tRNAs used in ribosomal translation. The crystallography
appears to have been carefully done, although that was reported previously, almost in its entirety and
is, hence, not directly relevant to this report, which describes the structure itself, and its relevant
functional properties. The paper is well-written and definitely merits publication in the EMBO
Journal.
Perhaps the most interesting aspect of the structure is that two non-functional molecules of
tRNAAsn act as scaffolding to anchor and orient the protein subunits. The catalytically relevant
tRNAs bind across the interface between the tRNA synthetases and the GATABCs, such that their
anticodons are bound to the OB folds of the two synthetases, while the acceptor stems are bound to
the GATB active site. The authors identify several key mechanistic interactions that argue that the
crystal likely represents an intermediate structure along the reaction profile.
Details of the anticodon interactions rationalize the inability of the Non-discriminating AspRS to
distinguish between the third bases, U for tRNAAsn and C for tRNAAsp. Similarly, the GATBacceptor stem interactions rationalize the ability of GATB to convert the aspartate only when it is
attached to tRNAAsn and not to tRNAAsp. Complementary to this interaction with the acceptor
stem, a disordered domain apparently forms a cavity complementary to and clamping the D-loop,
effectively measuring the length of the acceptor stem.
An additional appeal of the paper is that it addresses an unexpected discrepancy between the crystal
and solution structures. The solution structure apparently differs from the crystal structure in having
one less ND AspRS dimer and two fewer tRNAAsn. To clarify this situation, the authors carried out
SAXS measurements to distinguish between possible models derived from the crystal structure. The
SAXS measurements and analysis not only confirm the previously determined stoichiometry of the
solution structure, it also confirms that the two tRNAAsn molecules bound to the AspRS diimer
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disporportionate, such that one resembles the catalytic, while the other resembles the scaffolding
tRNAAsn from the crystal structure.
Pre-steady state and steady-state kinetic experiments document an important difference between the
free AspRS dimer, in which both tRNAAsn molecules react at equal rates, and the transamidosome
complex, in which the structural disporportionation is reflected in strict half-site reactivity.
Finally, the authors provide evidence that formation of the transamidosome stabilizes the GATABC
subunits against thermal denaturation, which is important for the thermophilic bacterium from which
it was isolated.
This is an altogether exciting paper, in which diverse experimental approaches shed new light on
several important phenomena related to the genetic code. However, the following points should be
addressed by the authors to improve the impact of this paper.
The complexity of the structure has created problems for the authors in illustrating the overall
architecture of the molecule. While Figures 1 and 2 have solved some of these problems, it comes at
the expense of such intense miniaturization that the important messages are hard to extract. This
figure likely could benefit from reworking, using other features of PYMOL. In particular,
cylindrical helices would significantly clarify the tiny structures represented in all parts of the figure,
without appreciable loss in information. A schematic diagram might also significantly improve
comprehension.
The problem discussed in (1) is especially severe in dealing with the differences between the
catalytically active and scaffolding tRNAs. The structure contains a single, approximately two-fold
symmetry axis. The two ND AspRS dimers participate in the ribonucleoprotein complex formation
by employing only the four anticodon-binding domains. How the symmetry axes of the synthetases
relate to that of the complex is of considerable potential interest, particularly in view of the
differences between the stoichiometries of the solution and crystal structures. However, it is next to
impossible to infer anything about this question from the figures.
The reference on page 9, par 1 to Figure 4G is probably to Figure 5G as there is neither a figure nor
a legend for Figure 4G. Figure 4 refers to the catalytic tRNA, Figure 5 to the scaffolding tRNA.
Referee #3 (Remarks to the Author):
This manuscript describes the crystal structure of a large complex consisting of two molecules of a
dimeric AspRS, two molecules of GatCAB, and four molecules of tRNAAsn, which are the
components necessary to synthesize Asn-tRNAAsn in T. thermophilus, where the normal AsnRS is
absent. The manuscript also describes the work on SAXS, which reveals a smaller complex of only
one molecule of the dimeric AspRS, and two molecules of tRNAAsn, but with two molecules
GatCAB. In both cases, only half of the tRNA molecules are in the functional state, while the other
half are in a non-functional state, which may serve as a scaffold to hold the complex together and to
prevent the premature release of the Asp-tRNAAsn intermediate. Although the formation of such
large complexes has been previously suggested, the elucidation of the structural details for these
complexes is potentially important for shedding light on how the complexes are formed, how they
stay together, and how they prevent the premature release of the mis-matched intermediate. These
structures (when interpreted properly) can make an important contribution to the understanding of
the so-called "indirect" pathway of synthesis of matched aa-tRNA via a mis-matched intermediate.
One concern for the structural work is that it is not clear how the SAXS analysis could differentiate
between the two tRNAAsn molecules bound in the complex? While crystal structural analysis
clearly showed that half of the tRNA molecules are in the functional state, while the other half are
not, how does the SAXS analysis distinguish between the functional and non-functional states?
Because the asymmetry is an important part of the authors' model, it needs to be explicitly
explained.
The major weakness of the work is the lack of convincing support for the catalytic cycle proposed in
Figure 3c.
First, the authors drew the dimeric AspRS as asymmetrically binding to two tRNA molecules, one in
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the active state while the other in the inactive state (step 1). There is no evidence presented in the
work for this asymmetry. While the asymmetry has been shown for other class II aaRSs, has it been
clearly demonstrated for the ND-AspRS? In fact, the authors had suggested themselves that it is the
binding of GatCAB that changes the ND-AspRS from a symmetric structure to an asymmetric
structure. The inconsistency needs to be resolved.
Second, the authors suggest a dynamic equilibrium between two complexes, where one complex
contains one ND-AspRS bound to two GatCAB molecules, while the other complex contains two
ND-AspRSs bound to two GatCAB. The existence of these two types of complexes is supported by
SAXS and by crystal structure, respectively. The problem is that the authors further suggest that the
active-state tRNA bound to the first dimeric ND-AspRS does not get aminoacylated until the second
dimeric ND-AspRS bound (step 2). What is the evidence for this ordered sequence? In fact, the
kinetic data is more consistent with the alternative sequence, where the tRNA in the first ND-AspRS
gets aminoacylated with Asp and directly modified to Asn and the product stays bound to the
complex. The release of the Asn-tRNAAsn product occurs only when the second ND-AspRS enters
the complex. In this alternative sequence, the product release is slow of the first ND-AspRS and is
activated only when the second ND-AspRS is bound.
Third, the authors suggested that the rate constant of the slow phase in the burst kinetics of Fig.3b
represents the aminoacylation of the non-functional state tRNA bound to the dimeric ND-AspRS.
This may not be correct and there is no evidence for this interpretation. In fact, a more likely
interpretation is that the slow phase represents the slow release of the Asn-tRNAAsn of the
functional state of tRNA in the first ND-AspRS (see the above comment).
Fourth, the authors suggest that after the first ND-AspRS complexed to Asn-(cat)-tRNAAsn and
(scaf)-tRNAAsn leaves the tRNP, the released dimeric ND-AspRS will then catalyze
aminoacylation of the (scaf)-tRNAAsn following rearrangement of the complex (page 6, bottom of
the second paragraph). This suggestion is too hypothetical without the support of solid data. How
can the released dimeric ND-AspRS catalyze aminoacylation of the non-functional state tRNA in
the absence of the GatCAB complex? Doesn't this generate the mis-charged Asp-tRNAAsn
intermediate in free solution? The same problem occurs in Discussion (page 10 bottom 6 lines),
where the discussion of the order of the catalytic cycle is troubling and lacks kinetic support.
1st Revision - authors' response
01 July 2010
Answer to Referee 1
“Only one request is that the author should more highlight and discuss the scaffolding tRNA, by
presenting figures showing the recognition mechanism of overall tRNA(scaf).”
ANSWER : We have described the mechanism of recognition of the scaftRNAAsn on end page 10 and
page 11. Further, the mechanism is illustrated by the additional Figures 6A,6B, 6C, 6D and 6E.
Answer to Referee 2 :
“The complexity of the structure has created problems for the authors in illustrating the overall
architecture of the molecule. While Figures 1 and 2 have solved some of these problems, it comes at
the expense of such intense miniaturization that the important messages are hard to extract. This
figure likely could benefit from reworking, using other features of PYMOL. In particular, cylindrical
helices would significantly clarify the tiny structures represented in all parts of the figure, without
appreciable loss in information. A schematic diagram might also significantly improve
comprehension. The problem discussed in (1) is especially severe in dealing with the differences
between the catalytically active and scaffolding tRNAs. The structure contains a single, approximately
two-fold symmetry axis. The two ND AspRS dimers participate in the ribonucleoprotein complex
formation by employing only the four anticodon-binding domains. How the symmetry axes of the
synthetases relate to that of the complex is of considerable potential interest, particularly in view of
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the differences between the stoichiometries of the solution and crystal structures. However, it is next
to impossible to infer anything about this question from the figures.”
ANSWER : Yes the complexity of the structure created problems for an accurate representation. We
therefore reworked Figure 1, where we show the helices in cylindrical forms and represented different
orientations of the structure. Moreover, as suggested by the referee to facilitate the interpretation of
the structure we added schematic representations of the structures shown in Figures 1A, 1B and 1D.
Answer to Referee 3 :
“One concern for the structural work is that it is not clear how the SAXS analysis could differentiate
between the two tRNAAsn molecules bound in the complex? While crystal structural analysis clearly
showed that half of the tRNA molecules are in the functional state, while the other half are not, how
does the SAXS analysis distinguish between the functional and non-functional states? Because the
asymmetry is an important part of the authors' model, it needs to be explicitly explained.”
ANSWER : We did additional SAXS experiments and performed a rigid body modeling in order to
fit the GatB C-t to the scattering data. The results suggest that in the complex the C-t of one GatB is
flexible and therefore not bound to tRNAAsn while the C-t of the other GatB is rigid since bound to
tRNAAsn. This is described at the end of the second paragraph page 6 of the manuscript and
illustrated by the additional Figures 2G and 2H.
“The major weakness of the work is the lack of convincing support for the catalytic cycle proposed
in Figure 3c. First, the authors drew the dimeric AspRS as asymmetrically binding to two tRNA
molecules, one in the active state while the other in the inactive state (step 1). There is no evidence
presented in the work for this asymmetry. While the asymmetry has been shown for other class II
aaRSs, has it been clearly demonstrated for the ND-AspRS? In fact, the authors had suggested
themselves that it is the binding of GatCAB that changes the ND-AspRS from a symmetric structure
to an asymmetric structure. The inconsistency needs to be resolved.”
ANSWER: ND-AspRS binds the two tRNA Asn molecules symetrically when GatCAB is absent as
demonstrated by kinetic experiments with ND-AspRS alone (Fig. 3, A, performed at 25°C). Presteady state kinetics (performed at 25°C) show a homogenous hyperbolic curve demonstrating that
the two tRNAs are aminoacylated with the same rate on the AspRS with a limiting step that should
be the transfer (Fig. 3A). When aminoacylation is monitored in the transamidosome (Fig 3B), an
initial burst does appear, interpreted as an initial rapid aminoacylation step. The second slow phase
gets extrapolated for one aa-tRNA formed by one dimeric ND-AspRS, that is to say, only one aatRNA for two subunits, leading to the conclusion that GatCAB takes control over the dimer to lay
down a new half-of-the site mechanism allowing only one tRNA to be loaded and processed. The
asymmetry only appears within the complex. Further, the steady state rate (slow phase) is much
slower than the first step, suggesting that the structural rearrangement of the complex strongly
favours the first transfer of Asp onto tRNA(Asn) when GatCAB is present. As the steady state rate
of AspRS within the complex (0,0018 s-1) reaches the value measured in the absence of GatCAB in
the same conditions (0,0015 and 0,0017 s-1), it could logically be inferred that the steady state rate
observed with the transamidosome may be explained by a slow dissociation of [AspRS/AsptRNA(Asn)][/AspRS/tRNA(Asn)] dimeric half-loaded-and-processed complex, in which the
constraints induced by GatCAB are released that leads to a slow aminoacylation of the uncharged
tRNA(Asn) with a similar rate as by AspRS alone. The interpretation of the GatCAB-induced half
of the sites reactivity agrees well with the structural informations of the complex showing non
equivalent binding of the two tRNA Asn on the dimeric AspRS. This is discussed in the first
paragraph page 7.
“Second, the authors suggest a dynamic equilibrium between two complexes, where one complex
contains one ND-AspRS bound to two GatCAB molecules, while the other complex contains two
ND-AspRSs bound to two GatCAB. The existence of these two types of complexes is supported by
SAXS and by Crystal structure, respectively. The problem is that the authors further suggest that the
active-state tRNA bound to the first dimeric ND-AspRS does not get aminoacylated until the second
dimeric ND-AspRS bound (step 2). What is the evidence for this ordered sequence? In fact, the
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kinetic data is more consistent with the alternative sequence, where the tRNA in the first ND-AspRS
gets aminoacylated with Asp and directly modified to Asn and the product stays bound to the
complex. The release of the Asn-tRNAAsn product occurs only when the second ND-AspRS enters
the complex. In this alternative sequence, the product release is slow of the first ND-AspRS and is
activated only when the second ND-AspRS is bound.”
ANSWER : Formation of the larger complex and association/dissociation of AspRS complexed to
tRNAs from this complex should not be considered as an equilibrium. Indeed, as suggested by
kinetic experiments, we propose that this complex represents an intermediate state necessary for
expulsion of the first dimeric AspRS/tRNA complex when properly aminoacylated and
transamidated. As this intermediate would totally control the steady-state rate of the transamidosome
in our model, its amount in solution would not depend on equilibrium between all complexes, but
would be determined by the existence of a stationary non-equilibrium state, which means that the
intermediate's concentration remains constant over time during the time-range used in the
experiments, laying down a constant rate when this state is reached, e.g. after the first cycle. This is
discussed in the first paragraph page 7.
"The problem is that the authors further suggest that the active-state tRNA bound to the first dimeric
ND-AspRS does not get aminoacylated until the second dimeric ND-AspRS bound (step 2). What is
the evidence for this ordered sequence?"
ANSWER : The model proposed suggests that the first dimeric AspRS/tRNA binds to two GatCAB
and that it is first aminoacylated asymmetrically before this half-charged complex is excluded from
the complex through the association of the second uncharged dimeric AspRS/tRNA. The only
uncertainty encountered would be when the Asp-tRNA gets processed by GatCAB. The structure
suggests that Asp-tRNA(Asn) acceptor arm points toward ammonia channel, leading to the
hypothesis that the large complex could catalyze the transamidation step. Since at the steady-state
asparatylation and asparaginyltion by the transamidosome occur with similar rates both are
determined by the same step that is probably the exchange of the AspRS dimers. See discussion in
the second paragraph page 7.
“Third, the authors suggested that the rate constant of the slow phase in the burst kinetics of Fig.3b
represents the aminoacylation of the non-functional state tRNA bound to the dimeric ND-AspRS.
This may not be correct and there is no evidence for this interpretation. In fact, a more likely
interpretation is that the slow phase represents the slow release of the Asn-tRNAAsn of the
functional state of tRNA in the first ND-AspRS (see the above comment).”
ANSWER : We agree with the reviewer. In our previous work (Bailly et al, 2007), we showed that
the transamidosome remains stable through the entire catalytic process. Here, we only propose that
the aminoacylated and processed tRNA (Asn-tRNAAsn) leaves the complex bound to its AspRS
dimer when a new dimer fits. This is the only possibility explaining why the complex appears to be
stable while it catalyzes more than one catalytic cycle, and why it is protected against denaturation at
85°C, a fact that requires the two GatCAB to remain bound within the transamidosome, taking into
account the thermolability of the free form. Furthermore, the 3rd curve on Fig. 3, B was measured
with stoichiometric amounts of AspRS and tRNA, thus, as only one tRNA could be aminoacylated
and transamidated within the complex, the second slow phase could only be due to the presence of
the (second) scaffold tRNA molecule. When free tRNA(Asn) was added (curve 4), the slow phase
increased. This could be explained by exchange of Asn-tRNA(Asn) and/or Asp-tRNA(Asn)
exchange on AspRS with free tRNA(Asn), leading to a new transamidosome cycle. This exchange
could only occur when AspRS is released from the complex, otherwise the transamidosome would
dissociate to take a new tRNA molecule, a fact that is definitely excluded as demonstrated before
through its great stability (Bailly et al, 2007). See discussion page 7.
“Fourth, the authors suggest that after the first ND-AspRS complexed to Asn-(cat)-tRNAAsn and
(scaf)-tRNAAsn leaves the tRNP, the released dimeric ND-AspRS will then catalyze aminoacylation
of the (scaf)-tRNAAsn following rearrangement of the complex (page 6, bottom of the second
paragraph). This suggestion is too hypothetical without the support of solid data. How can the
released dimeric ND-AspRS catalyze aminoacylation of the non-functional state tRNA in the
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absence of the GatCAB complex? Doesn't this generate the mis-charged Asp-tRNAAsn intermediate
in free solution? The same problem occurs in Discussion (page 10 bottom 6 lines), where the
discussion of the order of the catalytic cycle is troubling and lacks kinetic support.”
ANSWER : Free ND-AspRS is able to aminoacylate both tRNAs Asp and Asn (Fig 3. A, B, curves
1, 2). It is limited to one of its tRNA substrates only when complexed with GatCAB (curves 3, 4).
This could actually generate a misacylated Asp-tRNA(Asn). However, it is worth to remind that EFTu is a discriminating molecule in Thermus thermophilus. In addition GatCAB binds strongly to
AspRS. tRNA Asn complex (see second paragraph page 8, and end of the discussion). We did not
examinate the possibility that this Asp-tRNA(Asn) could be processed otherwise by GatCAB
through a distinct derived mechanism implying another alternative model, e.g rapid exchange of
Asn-tRNA(Asn) by a free tRNA(Asn) while Asp-tRNA(Asn) remains bound to AspRS to be
corrected within a new transamidosome. We cannot infere any model as it was not the purpose of
this work, but this mechanistic failure could explain why such a discriminating EF-Tu has been
selected during evolution and conserved in Thermus despite the transamidation mechanism within
the transamidosome that should prevent such problems by itself.
2nd Editorial Decision
12 July 2010
Thank you for sending us your revised manuscript. Our original referee 3 has now seen it again.
He/she is still in favour of publication of the study here. Still, he/she feels strongly that the catalytic
cycle that you are putting forward is not validated sufficiently by kinetic data and therefore suggests
to remove the model (see below). I would thus like to ask you to tone down the part of the study that
refers to this model as suggested before we will ultimately accept the manuscript.
Please let us have a suitably amended manuscript as soon as possible. I will then formally accept the
manuscript.
Yours sincerely,
Editor
The EMBO Journal
-----------------------------------------------REFEREE COMMENTS
Referee #3 (Remarks to the Author):
While I appreciate the significance of the crystal structure of the complex of (GatCAB)2-(dimeric
ND-AspRS)2-(tRNA)4, I still have reservation of the proposed catalytic cycles of how this complex
synthesizes 4 molecules of Asn-tRNAAsn. The main concern is that the proposed cycle is largely
based on structure, but little kinetic data are provided to give enough insight into the individual steps
as proposed in the model. A kinetic model needs to be tested and evaluated by kinetic methods.
Because of the lack of sufficient information, many features of the model remain highly speculative
and even troubling. The most troublesome is the notion that the released half-processed complex
consists of one AspRS-asn-tRNAAsn and one AspRS-tRNAAsn, the latter of which then
synthesizes the mischarged asp-tRNAAsn. The authors argue that the mischarged asp-tRNAAsn will
be discriminated by EF-Tu, but this has not been directly tested and it is not known if the
discrimination by EF-Tu is sufficient. Even if EF-Tu does provide some discrimination, the release
of a mis-charged aa-tRNA defeats the purpose of the complex and raises serious issues with most
biologists. This example illustrates the danger of proposing a detailed kinetic model without detailed
kinetic data.
The authors should consider dropping the kinetic model from the manuscript, because this model
needs substantial more work for it to stand on a firmer ground. The structural aspects of the
manuscript are very strong and interesting and would be appropriate for EMBO J.
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2nd Revision - authors' response
15 July 2010
Answer to the referee
In the first version the reviewer has expressed several comments and criticisms to which
we answered point per point. In the revised version he asks to remove the description of the
mechanism of transamidation. We cannot agree with this proposition, since without the mechanistic
model, the 3D structure will loose a great part of its interest. I will try to convince you that
publication of the 3D structure without the catalytic process has no sense.
We report the structure of a transfer ribonucleoprotein particle that promotes
asparaginylation of tRNA Asn in Thermus thermophilus. It constitutes the first structure described so
far of a tRNP catalyzing non conventional tRNA aminoacylation.
In previous reports we have shown that the partners of this pathway e. g. the tRNAAsn
substrate, the dimeric AspRS that charges Asp on tRNAAsn, and GatCAB, the enzyme that promotes
conversion of the tRNA-bound Asp into Asn form in solution a complex we called transamidosome.
The cristalline structure of a tRNP complex we describe here reveals a higher complexity
than the particle characterized in solution since it includes a second dimeric AspRS bound on two
tRNAAsn. The structure shows that only one tRNA Asn of each dimeric AspRS interacts functionally.
However a structure, including a tRNA substrate bound in a non functional state, could be
interpreted as artifactual since various ribonucleoprotein complexes crystallized in a non functional
state. To analyze whether the non functional binding of the two tRNA Asn is relevant, we investigated
the presteady-state and steady-state kinetic properties of the transamidosome. We show that only
two tRNAs of the four which are present are aminoacylated in the first turnover as a consequence
from distinct modes of binding of the tRNAs inside the complex and not from a intrinsic
anticooperativity of the dimeric aspartyl-tRNA synthetase since in free form the enzyme charges the
two tRNAs with the same rate constant. The kinetic results which agree with the 3D structure
indicate that only two tRNAs are bound functionally whereas the two other ones are scaffold
tRNAs . Further the role of the scaffolding tRNAs is revealed by the increased thermostability
acquired by the GatCAB inside the complex. Indeed, we show that the thermal stability of this
protein increases from 50 to 85 °C (the optimal growth temperature of T. thermophilus) when
assembled with the other partners of the transamidosome.
To reconcile the properties of the transmidosome observed in solution (one dimeric AspRS
bound to two tRNAsAsn and two GatCABs) and in the crystalline structure (two GatCABs bound to
two dimeric AspRSs and four tRNAsAsn) we propose that the 3D structure reflects a transient state of
the catalytic process in which binding of a novel dimeric AspRS saturated with tRNAAsn promotes
dissociation of the first complex where only one tRNA Asn has been asparaginylated. This labile
complex not seen in solution is stabilized in the crystal structure.
The major criticism of the reviewer concerns the happening of the scaffold tRNA Asn
which escaped to aminoacylation. While the asparaginylated tRNA will bind the EF-Tu factor to be
carried on the ribosome, the non aminoacylated tRNAAsn can be exchanged with another tRNA
molecule or it can be aspartylated by the AspRS before binding to GatCAB. The possibility that
tRNAAsn is aspartylated in the absence of GatCAB exists also before formation of the
transamidosome. What happens when tRNAAsn is aspartylated in the absence of GatCAB ?
Referee : « The authors argue that the mischarged Asp-tRNAAsn will be discriminated by EF-Tu,
but this has not been directly tested »
« Even if EF-Tu does provide some discrimination, the release of a mis-charged aa-tRNA defeats
the purpose of the complex and raises serious issues with most biologists »
Answer : We have tested the binding capacity of aspartylated tRNA Asn on EF-Tu and found that it
is deprived of measurable affinity. Therefore it do not bind EF-Tu as demonstrated in Becker HD
and Kern D (1998) Thermus thermophilus: a link in evolution of the tRNA-dependent amino acid
amidation pathways. Proc Natl Acad Sci USA 95: 12832-12837 and Roy H, Becker HD, Mazauric
MH and Kern D (2007) Structural elements defining elongation factor Tu mediated suppression of
codon ambiguity. Nucleic Acids Res 35: 3420-3430
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In contrast aspartylated tRNA Asn exhibits a strong affinity for GatCAB when bond on AspRS as
shown in : Bailly M, Blaise M, Lorber B, Becker HD, and Kern D (2007) The transamidosome: a
dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine
biosynthesis. Mol Cell 28: 228-239.
Therefore if tRNAAsn is aspartylated by AspRS prior binding to GatCAB, the AspRS.Asp-tRNAAsn
complex will be introduced in a preexisting transamidosome or it will form a new transamidosome
by binding GatCAB.
In conclusion the model described agrees well with the structural and functional data. It
gives a relevance to the tRNAs not functionally bound in the transamidosome and establishes that
the 3D structure is not artifactual. Therefore according to your suggestion and since all steps are not
firmly demonstrated we have clearly indicated in the paper that the mechanism we propose is a
model and toned down the description of the mechanism. Following modifications have been made :
In the results
Page 7 second part of paragraph one line 14 ‘With respect to the structure of the
transamidosome… » is altered
The second paragraph has been deleted.
In the discussion
Page 12 the end of the second paragraph line 11 « Our study suggests… » has been modified
The third paragraph has been deleted.
Legend to Figure 3B is modified « A proposed model of the dynamic of the presteady-state and
steady-state functioning of the transamidosome ».
© European Molecular Biology Organization
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