From the Laboratoire de Biochimie, Unité Mixte
de Recherche Number 7654, CNRS, Ecole Polytechnique,
F-91128 Palaiseau cedex, France and the ¶ Department of
Chemistry and Biotechnology, Graduate School of Engineering, University
of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8654, Japan
Received for publication, February 2, 2001, and in revised form, March 13, 2001
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ABSTRACT |
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Protein synthesis involves two
methionine-isoaccepting tRNAs, an initiator and an elongator. In
eubacteria, mitochondria, and chloroplasts, the addition of a formyl
group gives its full functional identity to initiator
Met-tRNAMet. In Escherichia coli, it has
been shown that the specific action of methionyl-tRNA transformylase on
Met-tRNA In protein biosynthesis, methionine is universally used as the
starting amino acid, and a particular initiator methionine tRNA ensures
initiation of translation. Cells also possess an elongator methionine
tRNA dedicated to the incorporation of internal methionines. Once
aminoacylated by methionyl-tRNA synthetase, the two methionine tRNAs
have distinct fates. The elongator tRNA is carried by an elongation
factor to the A site of the ribosome, whereas the initiator tRNA enters
the ribosomal P site with the help of several initiation factors.
In eubacteria, a decisive step in the acquisition of an initiator
identity of the tRNA is an N-formylation of the esterified methionine (reviewed in Refs. 1 and 2). The added formyl group
reinforces the binding of the initiator tRNA to initiation factor 2 and
impairs its binding to elongation factor Tu (3).
In mitochondria and chloroplasts, the translational system also uses a
formyl-methionyl-tRNAMet for initiation of translation.
However, the rule of the occurrence of two distinct methionine tRNAs
breaks down in the mitochondria of many animals, such as mammals and
insects. In these organelles, only a single tRNAMet,
believed to participate in both the initiation and the elongation of
protein biosynthesis, has yet been evidenced (4, 5). The current
working hypothesis assumes that this Met-tRNAMet molecule
can either bind elongation factor Tu and further participate in
chain elongation or undergo formylation through the action of
mitochondrial methionyl-tRNAMet transformylase
(FMTmt)1 and then be directed
to the initiation machinery. Thus, competition between elongation
factor Tu and FMT for the binding of Met-tRNAMet would
ensure a correct balance between the elongator and initiator functions
of mitochondrial tRNAMet.
Specificity of formylase toward tRNA Bovine mitochondrial FMT has recently been characterized (10, 11). This
enzyme was found to be able to formylate the E. coli
elongator Met-tRNA Expression and Purification of Bovine Mitochondrial FMT--
The
NdeI-BamHI fragment from pET19b-FMTmt (10)
carrying the gene coding for the bovine mitochondrial formylase was
cloned into the corresponding sites of pET15b. The resulting plasmid, pET15b-FMTmt, expressed a 6-His-tagged version of the formylase with a
thrombin cleavage site. The tagged protein was purified by affinity
chromatography on a nickel Hi-Trap column (Amersham Pharmacia
Biotech), as described (10). After this step, the His tag was
cleaved by using thrombin (0.5 unit per mg of protein). Remaining
tagged protein was removed by a second pass on the affinity column. A
final step of purification consisted of ion exchange chromatography on
SP-Sepharose HP (Amersham Pharmacia Biotech, 1.6 × 20 cm, 0.2 M KCl/h, 2.5 ml/min). The protein was homogeneous as judged
by SDS-polyacrylamide gel electrophoresis. FMTmt was further
concentrated by dialysis against 10 mM Tris-HCl (pH 7.6), 100 mM KCl, 10 mM 2-mercaptoethanol, 55%
glycerol and stored at Production of Mutant tRNAs--
E. coli tRNAs and
their derivatives were expressed in JM101Tr (12) from plasmid pBSTNAV2
(7). The gene coding for tRNA Measurement of Catalytic Parameters of FMT--
Initial rates of
aminoacyl-tRNA formylation in the presence of catalytic amounts of the
studied enzyme (0.01 nM to 1 µM for FMTec,
depending on the studied tRNA, and 0.1 nM to 1 µM for FMTmt, depending on the studied tRNA) were
measured as described (15, 16) in a buffer (20 mM Tris (pH
7.6), 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 150 mM KCl, 7 mM MgCl2) containing 125 µM 10-formyltetrahydrofolate and 0.05-10
µM aminoacyl-tRNA. For the sake of homogeneity, identical assay conditions were used for the two formylases. Note that these conditions differ from those previously used for FMTmt (10 mM KCl, 0.5% CHAPS, 5 mM MgCl2;
Ref. 11). Homogeneous preparations of E. coli M547
methionyl-tRNA synthetase (17), valyl-tRNA synthetase (18), or
isoleucyl-tRNA synthetase (19) were used for the aminoacylation of tRNAs.
Limited Proteolysis of FMTmt--
FMTmt (2 mg/ml,
i.e. 50 µM) was digested at 37 °C in 50 µl of buffer (100 mM Tris-HCl (pH 7.6), 100 mM KCl, 10 mM MgCl2, 1 mM 2-mercaptoethanol) by adding 1 µl of protease V8
(0.125 mg/ml from Roche Molecular Biochemicals). Aliquots were analyzed
by electrophoresis on a 12% polyacrylamide SDS gel following the time
course (5-50 min). A 35-kDa peptide fragment accumulated, as the
result of cleavage at the position 52LE/VV55,
as previously demonstrated (11). To assay a protection from proteolytic
cleavage by bound tRNA, the experiment was repeated in the presence of
a saturating concentration (65 µM) of E. coli f-Met-tRNA Influence of the Nucleotidic Composition of the Acceptor Stem of
tRNA on the Formylation Reaction Catalyzed by FMTmt--
The sequence
of tRNAMet from bovine mitochondria is shown in Fig.
1, together with that of E. coli tRNA
As previously reported (11), FMTmt can formylate E. coli
elongator Met-tRNA The Important Role of the
Puo11-Pyd24 Pair in the D-stem of
tRNA--
Like most initiator tRNAs, tRNAMet from bovine
liver mitochondria possesses a purine-pyrimidine pair at position
11-24. Instead, elongator tRNAs usually have a
pyrimidine11-purine24 pair. As previously
demonstrated, the purine-pyrimidine pair contributes to the recognition
of its substrate by the bacterial formylase (6, 9). Mutation of the
A11-U24 pair of
tRNA
With FMTmt as the formylating enzyme, the above-mentioned mutation in
tRNA Specificity toward the Aminoacyl Group Esterified to tRNA--
In
the case of the FMTec, the side chain of the aminoacyl group attached
to tRNA modulates the efficiency of the formylation reaction.
Methionine provides the highest efficiency of formylation, as compared
with the other tested amino acids, Gln, Phe, Val, or Lys (7, 20,
21).
To study the importance of the amino acid moiety in the case of FMTmt,
we used two derivatives of tRNA
When using FMTmt as the formylating enzyme, the changes in catalytic
efficiency upon changing methionine to valine or isoleucine resembled those measured with FMTec (Table I). However, the relative consequences of these two changes on the FMTmt reaction are greater than any of those measured upon varying the tRNA nucleotidic structure. We suspected, therefore, that the presence of a cognate methionyl group
could be the predominant parameter specifying formylability of its
substrate by FMTmt. To probe this idea, we used an E. coli tRNA Mild Proteolysis of FMTmt--
In the E. coli system,
the opening of the acceptor stem of tRNAMet involves an
enzyme loop, called loop 1 (see Fig. 3). In the free enzyme, loop 1 is
disordered and highly sensitive to trypsin cleavage, with a cut between
Arg42 and Gly43. In the presence of bound
f-Met-tRNA Discrimination among tRNAs by FMTec or FMTmt--
Fig.
2 summarizes the relative effects of the
various changes performed in tRNA
The results obtained in this study show that FMTmt is much less
sensitive than its bacterial counterpart to the nucleotides composing
the tRNA substrate. This difference enables the side chain of the
methionyl group attached to tRNA to become a dominant identity element
for mitochondrial formylation (Fig. 2). In agreement with this
conclusion, once aminoacylated with methionine, E. coli tRNA
Bovine mitochondria contain 22 tRNA species, all encoded by the
mitochondrial genome, among which a single tRNAMet appears
to be responsible for both initiation and elongation of translation.
Consequently, to ensure a correct formylation reaction, recognition of
the methionyl group carried by this single tRNAMet species
may be sufficient. Moreover, in the organelle, 12 tRNA species of 22 lack a GC or CG pair at the top of their acceptor stem. This may be
another reason to explain why the base pair at position 1-72 has become
a marginal determinant for the selection by the mitochondrial
formylating enzyme. More important for the recognition by FMTmt is the
purine-pyrimidine pair at position 11-24. Only two tRNA species (Leu
and Trp), in addition to tRNAMet, display this feature in
bovine mitochondria. We therefore conclude that the combination of a
methionyl group and the 11-24 pair is enough to unambiguously direct
specific formylation in animal mitochondria. Notably, mutations in the
nucleotidic part of the tRNA substrate mainly affect the
Km of the formylation reaction, whereas the changing
of esterified amino acid influences the catalytic rate (Table I). The
use of the 11-24 pair as an identity element can thus prevent excessive
competition of non-methionine tRNAs for the binding to FMTmt. Finally,
some FMTmt molecules can be assumed to accidentally escape the
mitochondrial import system and promote erroneous formylation of
cytoplasmic tRNAs. However, the occurrence of a pyrimidine-purine pair
at position 11-24 of all mammalian cytoplasmic tRNAMets
could help them to escape such an undesired action of FMTmt.
Mechanism of Action of FMTmt--
The crystallographic structure
of FMTec complexed with
formyl-methionyl-tRNA
The present study establishes that a
Puo11-Pyd24 pair is important for the
formylation reaction in the mitochondrial system. Therefore, the
initial positioning of the tRNA substrate at the surface of the
C-terminal domain of FMTmt is likely to occur in the same way as in the
bacterial system. This idea is favored by the conservation in the FMTmt
sequence of residues of FMTec involved in tRNA binding, like
Lys291, Lys292, and Asn301 (11). In
the E. coli system, Asn301 binds the
O2 atom of U24 of
tRNA
In the bacterial enzyme, loop 1 establishes many contacts with the
acceptor stem of bound fMet-tRNA
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloverleaf representations of bovine
mitochondrial tRNAMet (left) and E. coli initiator tRNA
Catalytic parameters of the studied E. coli tRNA derivatives in the
formylation reaction catalyzed by methionyl-tRNA transformylases of the
indicated origins
6 s
1
M
1). With the bacterial enzyme,
Met-tRNA
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Comparison of the weight of substrate
determinants in the formylation reaction catalyzed by either E. coli or bovine mitochondrial formylase. The
bars represent the logarithm of the ratio of the
kcat/Km parameter for the
studied tRNA over that for the authentic
tRNA
-barrel (Fig.
3). The tRNA substrate binds on its
D-stem side at the surface of the enzyme. This allows the formation of
a base-specific interaction of the A11-U24 base
pair with the C-terminal domain. Close to the active site, a loop of
the enzyme makes numerous interactions in the major groove of the
acceptor stem. Such a positioning results in the splitting of the
C1-A72 mismatch, with typical bending of the 3'
arm toward the active center, where the methionyl moiety fits in a
specific pocket.
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Fig. 3.
Top, schematic representation of the
three-dimensional structure of FMTec complexed to
formyl-met-tRNA helices and
arrows representing
strands. The position of loop 1 is
indicated. Two conserved sequences bordering loop 1 are shaded in
gray.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: University of Tokyo, Dept. of Integrated Biosciences, Graduate school of Frontier Sciences, 7-3-1, Hongo, Bunkyo, Tokyo 113-8656, Japan.
To whom correspondence should be addressed. E-mail:
yves@botrytis.polytechnique.fr.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M101007200
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ABBREVIATIONS |
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The abbreviations used are: FMT, methionyl-tRNAMet transformylase; FMTmt, mitochondrial FMT; FMTec, Escherichia coli FMT; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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