From the Skaggs Institute for Chemical Biology, The
Scripps Research Institute, Beckman Center, La Jolla, California 92037 and the
Département de Biochimie, Université de
Montréal, Montréal, Québec, H3C 3J7 Canada
Received for publication, September 12, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Mutations in human mitochondrial
isoleucine tRNA (hs mt tRNAIle) are
associated with cardiomyopathy and opthalmoplegia. A recent study
showed that opthalmoplegia-related mutations gave rise to severe
decreases in aminoacylation efficiencies and that the defective mutant
tRNAs were effective inhibitors of aminoacylation of the wild-type
substrate. The results suggested that the effectiveness of the
mutations was due in large part to an inherently fragile mitochondrial
tRNA structure. Here, we investigate mutant tRNAs associated with
cardiomyopathy, and a series of rationally designed second-site
substitutions introduced into both opthalmoplegia- and
cardiomyopathy-related mutant tRNAs. A source of structural fragility
was uncovered. An inherently unstable T-stem appears susceptible to
misalignments. This susceptibility sensitizes both domains of the
L-shaped tRNA structure to base substitutions that are deleterious.
Thus, the fragile T-stem makes the structure of this human
mitochondrial tRNA particularly vulnerable to local and distant mutations.
The canonical tRNA cloverleaf folds into a two-domain L-shaped
structure. One domain contains the amino acid attachment site and the
12-base pair acceptor-T Within the gene for hs mt tRNAIle, eight different point
mutations are correlated with pathologies (3-10). The genetic errors identified are generally associated with two diseases, cardiomyopathy (3-7) and opthalmoplegia (8-10). The mutations can be classified according to the types of structural defects induced. The
opthalmoplegia mutations are associated with one specific type of
structural defect, CA mispairs, whereas the cardiomyopathy mutations
are more varied.
The opthalmoplegia-related mutants of tRNAIle are poor
substrates for aminoacylation (11). Studies of the reactivity of these molecules with the hs mt isoleucyl-tRNA synthetase (IleRS) revealed kinetic defects that impeded the aminoacylation reaction. Because the
binding affinities of the mutated tRNAs for the enzyme were not
affected, the mutants were effective inhibitors of the charging of
wild-type tRNA. The aminoacylation of these mutant tRNAs was restored
with compensatory mutations that reintroduced base pairing. These
results prompted the proposal that the structure of the hs mt
tRNAIle was inherently fragile and, thus, small
perturbations introduced by the pathogenic mutations were magnified
into large losses in function and the creation of effective inhibitors.
Here, using aminoacylation to probe structure-function properties of hs
mt tRNAIle, the effects of a subset of
cardiomyopathy-associated mutations are investigated. An A59G mutation
in the T Preparation of tRNA Transcripts--
Plasmids encoding tRNA
transcripts were generated by ligating overlapping oligonucleotides
into a pUC18 plasmid (12) between a BamHI and
PstI cleavage site. The tRNA constructs were placed between
a T7 RNA polymerase promoter sequence and a BstN1 cleavage site and adjoined at the 3'-end with a hammerhead ribozyme derivative to increase the transcription yields obtained with this sequence (13).
T7 RNA polymerase was purified from Escherichia coli as
described (14). Plasmids encoding the tRNA constructs were linearized with BstN1 and incubated with T7 RNA polymerase for 2 h
at 37 °C in the presence of 40 mM Tris, pH 8, 10 mM MgCl2, 5 mM dithiothreitol, 0.01% Triton X-100, 8% polyethylene glycol 8000, 50 µg/ml bovine serum albumin, and 1 mM spermidine. DNase I was added to
digest the template, and reactions were extracted with 25:24:1 phenol, pH 4.5, chloroform, isoamyl alcohol. After ethanol precipitation, reactions were resuspended in 50 mM Tris, pH 8, and 10 mM MgCl2 and incubated at 55 °C for 30 min
to effect ribozyme cleavage. The tRNA transcripts were purified by 12%
polyacrylamide gel electrophoresis and electroeluted. After ethanol
precipitation, tRNAs were desalted using a G-50 spin column (Amersham
Pharmacia Biotech). There were no detectable differences in the
efficiency of transcription or transcript length when mutations were
introduced into hs mt tRNAIle.
The purification protocol employed produced highly active tRNA
transcripts. Aminoacylation assays performed with high concentrations of enzyme (~ 1 µM) were used to assess the percentage
of tRNAs that could be charged with isoleucine, and for both wild-type and mutant tRNAs, between 80 and 100% of the tRNA quantitated by
uv-vis absorbance as described below could be aminoacylated. These
experiments indicated that the 3'-end of this tRNA was transcribed with
high fidelity.
Expression and Purification of Human Mitochondrial
IleRS--
The gene for human mitochondrial IleRS was cloned as
described (15). The cloned gene was inserted into a yeast expression vector containing a glutathione S-transferase affinity tag
and the GAL4 promoter. The fusion protein was expressed under the control of a GAL4 promoter in Saccharomyces cerevisiae in
strain INVSc (Invitrogen, Carlsbad, CA). After cell lysis performed in a French press, human mitochondrial IleRS·GST was batch
purified by isolation on glutathione-agarose (Amersham Pharmacia
Biotech) and subsequent elution with glutathione. This protocol yielded protein samples with >95% purity, as judged by SDS-polyacrylamide gel
electrophoresis. The cleavage of the GST tag by incubation with
thrombin (16) and subsequent FPLC (Amersham Pharmacia Biotech) purification yielded a protein of activity comparable with that of the
fusion protein.
Aminoacylation Assays--
Aminoacylation assays were carried
out at 37 °C in reaction mixtures containing 10 mM
HEPES, pH 7.5, 75 mM NH4Cl, 0.05 mM EDTA, 10 µg/ml bovine serum albumin, 10 mM
MgCl2, 195 µM isoleucine, 5 µM
[3H]isoleucine, 2 mM ATP, 25 nM
enzyme, and 2 µM tRNA as described (17). Before each
assay, tRNAs were annealed in 10 mM Tris, pH 7.5, 0.5 mM EDTA, and 4 mM MgCl2 by
incubation at 70 °C for 5 min and 25 °C for 15 min.
Concentrations of tRNA stock solutions were determined by quantitation
of absorbance at 260 nm using an extinction coefficient of 9100 M Secondary Structure Calculations--
Secondary structural
analysis was performed using MFOLD (19). Parameters were used in
default settings, except for percent suboptimality, which was varied
from 25 to 50%. Only cloverleaf-like structures were considered.
Aminoacylation Efficiency of a Cardiomyopathy-related Mutant
tRNA--
Mutants of hs mt tRNAIle associated with
cardiomyopathy (Fig. 1) that involve
substitutions in the T-stem and loop were generated by in
vitro transcription and tested as substrates for the cognate recombinant hs mt IleRS. (Previous studies showed that transcripts of
hs mt tRNAIle were relatively robust substrates for the
recombinant enzyme, Ref. 11.) A tRNA containing an A59G mutation that
alters an unpaired base in the T-loop had a significantly decreased
rate of aminoacylation (Fig. 2). A
similar decrease in aminoacylation for this A59G mutant was observed in
a previous study employing a crude mitochondrial extract (20).
Interestingly, the T-loop is generally not a locus for the tRNA
identity determinants (21). Thus, we considered the possibility that
this substitution perturbed the secondary structure by shifting the
base pairing so that the loop was reduced in size by three nucleotides
(Fig. 3).
Aminoacylation of a Second Cardiomyopathy-related Mutant
tRNA--
Another cardiomyopathy-associated mutation, C62U, replaces
the CA pair in the T-stem with a UA pair. Secondary structure
calculations revealed that this substitution decreased the number of
stable alternate folds competing with the canonical cloverleaf
structure. Indeed, increased aminoacylation activity was observed with
the C62U substitution, suggesting that the introduction of an
additional Watson-Crick base pair in the T-stem stabilized the
cloverleaf structure and enhanced aminoacylation by locking out
inactive conformations (Fig. 3). The enhanced charging seen with the
C62U substitution suggests that, in this case, the associated
cardiomyopathy is not because of a defect in aminoacylation.
Hs mt tRNAIle Constructs with Altered T-stems--
To
further investigate the role of base-pairing patterns within the T-stem
in the stabilization of the tRNA conformation required for function,
different base pairs were substituted for the wild-type C62:A52 pair.
In addition to the C62U mutant, two additional mutant tRNAs were
synthesized. A C62G/A52C double mutant was made to test the effect of a
GC pair, and an A52G mutant was generated to investigate the effect of
a CG pair in the 52:62 position.
The C62G/A52C construct (with a G62:C52 pair) displayed slightly higher
levels of aminoacylation compared with the C62U mutant tRNAIle (with a U62:A52 pair, Fig.
4). The increase in activity for these two variants over the wild-type tRNA therefore results mainly from the
presence of a base pair at this position. The highest activity is
achieved with the G62:C52 pair that is predicted to be most stable.
That the identity of the Watson-Crick pair at the 62:52 position is not
important for aminoacylation is consistent with this location in the
structure of tRNAIle not being a major contact point for
the synthetase in the bacterial system (22, 23).
Secondary structure calculations revealed that neither the U62:A52 nor
the G62:C52 pairs altered the alignment of bases in the T-stem. The
creation of a C62:G52 base pair is predicted to change the pairing such
that two unpaired bases are introduced between the acceptor- and
T-stems, and the size of the T-loop is reduced by one nucleotide (Fig.
3). Consistent with these predictions, the A52G mutant
tRNAIle (with a C62:G52 pair) was charged at low levels
compared with the substrate containing this same base pair in the
opposite orientation (Fig. 4).
Rescue of Pathology-related tRNA Mutants with a Stabilized
T-stem--
Having established the sensitivity to mutation and the
fragility of the T-stem (particularly the capacity to form alternative structures), we wondered whether a stabilized stem could compensate for
the deleterious effects of other pathology-related tRNA mutations. To
address whether the impact of the A59G mutation on aminoacylation was
related to the lability of the T-stem, this pathogenic mutation was
incorporated into a tRNAIle variant containing a C62U
mutation. The substitution of C62 stabilizes the T-stem (by creating a
U62:A52 pair) and, as described above, increases aminoacylation
relative to the wild-type hs mt tRNAIle.
Indeed, the C62U mutant was more resistant to the negative effect of
the A59G mutation on charging (Fig. 5).
Instead of the 80% decrease observed with the wild-type CA-containing
T-stem, only a 10% decrease in charging was observed with an U62:A52
T-stem. The loss of charging caused by the A59G mutation in the
wild-type construct therefore appears to reflect a greater tendency for the molecule to misfold. This tendency can be counteracted by strengthening the T-stem.
Consistent with the notion of misfolding contributing to the effects of
the A59G mutation, the secondary structure calculated for this mutant
tRNAIle differs from that of the wild-type structure. The
most stable structure resembles the canonical cloverleaf, but the
T-loop is shortened by 4 nucleotides and 2 nucleotides are forced out
of the T-stem into the junction between this helix and the acceptor stem (Fig. 3). With the introduction of the C62U substitution, the
wild-type secondary structure is regained (Fig. 3, far
right).
The ability of a stabilized T-stem to rescue the aminoacylation and
restore the structure of the cardiomyopathy-related A59G mutant
prompted us to examine the effect of this structural element on the
functional defects caused by the opthalmoplegia-related mutations:
U12C, U27C, G40A. Local structural perturbations in AU-rich stems were
previously suggested as the source of attenuated reactivities displayed
by these mutants (11). By reexamining these mutations in constructs
where the T-stem is stabilized, the contribution of distal structural
elements to the deleterious impact of the opthalmoplegia mutations
could be evaluated.
Aminoacylation efficiencies were compared for wild-type and mutant
tRNAs by calculating the ratios of initial rates for tRNAs containing a
C62:A52 (wild-type) versus an U62:A52 base pair. In all
cases, the decreases in aminoacylation efficiency seen with the U12C,
U27C, G40A, and A59G mutant tRNAs are less severe with a secondary C62U
mutation. As previously discussed, even the charging of wild-type tRNA
is enhanced. For three of the pathology-associated mutants (U12C, U27C,
A59G), the context provided by the U62:A52 substitution enhanced
aminoacylation even more than when the U62:A52 substitution was placed
in the wild-type tRNA. Positions 12 and 27 lie in the second domain of
the L-shaped tertiary structure, whereas position 59 is in the first,
acceptor-T In the case of the opthalmoplegia-related mutations, previous work
showed that these mutant hs mt isoleucine tRNAs were severely defective
in aminoacylation (11). The effects of the mutations on the tRNA
structure left the mutant tRNAs sufficiently ordered to be excellent
inhibitors of aminoacylation of wild-type hs mt tRNAIle.
Thus, in the potentially heteroplasmic environment of the mitochondria (24, 25) with mixtures of wild-type and mutant tRNAs, the mutant tRNAs
could in principle arrest protein synthesis by virtue of their ability
to act as inhibitors.
In contrast, whereas the A59G cardiomyopathy-associated mutation
attenuates significantly aminoacylation, the C62U substitution actually
improves the efficiency of charging. This improvement is plausible
because the substitution strengthens the proper alignment of the T-stem
by replacing an A-C pair with A-U (Fig. 3). That the aminoacylation
efficiency with this mutant is actually increased through the
stabilization of a canonical tRNA structure emphasizes that the nature
of the connection between a pathology and a specific mutation
may be different for each mutation. The higher activity of the more
stable C62U mutant tRNAIle raises the possibility that the
inherent fragility of the wild-type hs mt tRNA structure may be
important for function. For example, among other possibilities, a
flexible, relatively loose structure may be required for optimal
activity with the mt translation apparatus (23).
The inherent weakness of the structure hs mt tRNAIle
gives rise to a significant degree of domain-domain communication.
Because of the capacity for "slippage" in the secondary structure
caused by base pair misalignments (Fig. 3), the stability of one domain is reliant on the integrity the other. (Whereas interdomain
communication is prevalent in this tRNA possessing a minimized
structure, it has also been detected in other tRNAs, Ref. 26.) In
particular, the slipped secondary structures can disrupt loose tertiary
interactions and thereby make one domain particularly sensitive to
perturbations in the other.
Although the cause-and-effect basis for the opthalmoplegias and
cardiomyopathies associated with mutations in hs mt tRNAs has not been
conclusively established, these pathologies have nonetheless provided
the motivation and rationale for investigating in more depth the
structural features of hs mt tRNAs. In particular, were it not for the
cardiomyopathy-associated mutations studied here, the fragility of the
T-stem of hs mt tRNAIle might not have been appreciated.
This fragility is manifested by the ease with which single point
mutations can lead to serious realignments of the T-stem loop (Fig. 3)
that, in turn, have consequences that are global (Fig.
6). It is these global effects that may be most significant for pathology because, for example, they may influence not only aminoacylation, but also the ability of the tRNA to
function in subsequent steps of protein synthesis or to be a substrate
for processing or modification enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C minihelix. The other domain of 10 base
pairs contains the dihydrouridine and anticodon-stem and -loop. The
primary structures of hs mt
tRNAs1 differ significantly
from cytoplasmic tRNAs (1). Despite their analogous function in the
decoding of genetic information, mt tRNAs are generally shorter than
cytoplasmic tRNAs and feature higher numbers of AU pairs. In some
cases, entire structural elements are deformed or even missing from the
canonical cloverleaf, although a truncated L-shaped structure can still
be made. The minimized structures of hs mt tRNAs appear to be
susceptible to point mutations, as errors in the corresponding mt genes
are associated with disease. Over 70 pathology-related mutations in mt
tRNA genes are known (2). The impact of the base substitutions on the
structure and function of hs mt tRNAs is difficult to predict a
priori, given the limited information available concerning the
properties of these molecules.
C-loop (hereafter called the T-loop) significantly decreased
the aminoacylation efficiency of this molecule. In contrast, a C62U
mutation (that introduced an AU pair in place of a CA pair within the
T-stem), increased the aminoacylation efficiency. Because the C62U
mutation both stabilized the T-stem and improved aminoacylation, we
considered the possibility that the T-stem had an inherent fragility
that affected the entire molecule. With this in mind, we set out to study systematically the effects of specific, rationally designed manipulations of the T-stem to elucidate structural properties that
cause the hs mt tRNAIle to be susceptible to pathogenic
mutations. These investigations were aimed at not only exploring the
local consequences of mutations in the T-stem and loop, but also at
seeing whether these mutations affected distant parts of the tRNA.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (mononucleotide) cm
1 (18).
Assays were otherwise performed as described (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Location of pathogenic mutations within the
cloverleaf secondary structure of human mitochondrial
tRNAIle.
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Fig. 2.
Aminoacylation at pH 7.5, 37 °C of human
mitochondrial tRNAIle transcripts containing
cardiomyopathy-associated mutations. Data shown correspond to
wild-type ( ), A59G (
), or C62U (
) tRNAs. Assays contained 2 µM tRNA and 25 nM IleRS.
View larger version (10K):
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Fig. 3.
Schematic of alternative secondary structures
caused by the introduction of mutations into hs mt tRNAIle
as predicted by MFOLD. The introduction of the pathogenic A59G
mutation (right) and the rationally designed A52G mutation
(far left) produces structures with misaligned T-stems. A
tRNA containing a C62U mutation (middle) retains the
wild-type structure. For a construct containing the A59G mutation, the
introduction of the C62U mutation restores the wild-type structure
(far right). Mutated bases are italicized.
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Fig. 4.
Variation of 62:52 base pair.
Aminoacylation was monitored at pH 7.5, 37 °C in reactions
containing 2 µM tRNA and 25 nM IleRS for
tRNAs containing G62:C52 ( ) or C62:G52 (
) mutations. For
comparison, data corresponding to aminoacylation of wild-type tRNA
(----) or a C62U tRNA mutant are shown (-
-).
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Fig. 5.
Functional rescue of the A59G
tRNAIle mutant by incorporation of a compensatory C62U
mutation that repairs the T-stem. Data corresponding to the
aminoacylation of the C62units/A59G mutant ( ) are compared with
those corresponding to the aminoacylation of the single C62U mutant
(
). The inset shows the more dramatic effect of the A59G
substitution when in the context of the wild-type sequence.
Aminoacylation was monitored at pH 7.5, 37 °C in reactions
containing 2 µM tRNA and 25 nM IleRS.
C minihelix domain. Thus, these results show that the
consequence of a fragile T-stem extends beyond the minihelix to the
opposite domain, and thereby suggest an important domain-domain communication.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 6.
Comparison of relative initial rates of
aminoacylation for tRNAs containing pathogenic mutations in the context
of T-stems featuring a C62:A52 (wild-type) or U62:A52 base
pair.
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FOOTNOTES |
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* This work was supported in part by the National Institutes of Health (NIH) Grant GM15539 and by a fellowship from the National Foundation for Cancer Research.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.
§ Recipient of an NIH postdoctoral fellowship. Present address: Dept. of Chemistry, Boston College, Chestnut Hill, MA 02467.
** Recipient of an operating grant from the Medical Research Council of Canada and a fellowship from le Fonds de la Recherche en Santé du Québec.
¶ To whom correspondence may be addressed. E-mail: schimmel@scripps.edu (to P. S.) or shana.kelley{at}bc.edu (to S. O. K.).
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M008320200
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ABBREVIATIONS |
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The abbreviations used are: hs mt tRNA, human mitochondrial tRNA; IleRS, Ile-tRNA synthetase; GST, glutathione S-transferase.
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REFERENCES |
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