A T-stem slip in human mitochondrial tRNALeu(CUN)
governs its charging capacity
Rui Hao1, Ming-Wei Zhao1, Zhan-Xi Hao1, Yong-Neng Yao and En-Duo Wang1,*
State Key Laboratory of Molecular Biology and1Graduate School of the Chinese Academy of Sciences, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese
Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People’s Republic of China
Received April 7, 2005; Revised and Accepted June 9, 2005
[hmtRNALeu(CUN)] corresponds to the most abund-
ant codon forleucineinhuman mitochondrialprotein
genes.Here, invitro studies revealthatthe U48C sub-
stitution in hmtRNALeu(CUN), which corresponds to
the pathological T12311C gene mutation, improved
the aminoacylation efficiency of hmtRNALeu(CUN).
Enzymatic probing suggested a more flexible sec-
ondary structure in the wild-type hmtRNALeu(CUN)
transcript compared with
Structural analysis revealed that the flexibility of
hmtRNALeu(CUN) facilitates a T-stem slip resulting
in two potential tertiary structures. Several rationally
designed tRNALeu(CUN) mutants were generated
to examine the structural and functional con-
sequences of the T-stem slip. Examination of these
hmtRNALeu(CUN) mutants indicated that the T-stem
slip governs tRNA accepting activity. These results
suggest a novel, self-regulation mechanism of
tRNA structure and function.
the U48C mutant.
In protein biosynthesis, transfer RNAs (tRNAs) play a central
role in gene expression as adaptor molecules of the codons
in mRNA and amino acids (1). The human mitochondrial
translation machinery is dependent on 22 tRNAs, one for
each of 18 amino acids and two for Leu and Ser with different
anticodons (2). All of these tRNAs are encoded by the mito-
chondrial genome. The primary and secondary structures of
human mitochondrial tRNAs (hmtRNAs) differ significantly
from those of canonical bacterial and cytoplasmic tRNAs, and
tRNAs in human mitochondria are less thermodynamically
stable because they generally contain higher numbers of mis-
matched and AU base pairs (3). Therefore, while hmtRNAs
should adopt an L-shape tertiary structure of canonical
tRNA in order to function in ribosomal protein synthesis,
their folded structures may be constructed with different
sets of intramolecular contacts that are mostly unknown.
In the past 15 years, a number of point mutations in
hmtRNA genes have been found to be correlated witha variety
of multi-system diseases (4,5). Although the molecular mech-
anisms of these mitochondrial DNA-mediated diseases remain
unclear, accumulating evidence has shown that severe struc-
tural and functional defects of hmtRNAs are caused by the
pathogenic mutations (6–8). Systematic investigation of the
structure and function of hmtRNAs can, therefore, provide
valuable information about related diseases and potentially
facilitate development of diagnostic tools and therapies for
Among the 22 hmtRNAs, hmtRNALeu(CUN) corresponds
to the most frequently used codon (14.9%) (9). Even a slight
impairment of the function of hmtRNALeu(CUN) can lead to
significant deficiencies in mitochondrial protein synthesis.
Although tRNALeu(CUN) is one of the few mitochondrial
tRNAs that possesses all of the structural features for a
classical cloverleaf structure and 3D folding (3), little infor-
mation is available about the structure and function of
Among the five known pathogenic mutations in the
hmtRNALeu(CUN) gene (see http://www.mitomap.org) is the
T12311C mutation (10) that sparks our interest. This mutation
resides at residue 48, which is the connector between the
variable loop and the T-stem in the tRNA secondary structure
(Figure 1A). It is hypothesized that the tertiary interaction
between nt 15 and 48 plays an important role in the tRNA
3D structure; replacement of either of these 2 nt affects tRNA
conformation (11). In this in vitro study, a U48C substitution
was introduced in the hmtRNALeu(CUN) gene, mimicking
the T12311C mutation, in order to examine changes in struc-
ture and aminoacylation of hmtRNALeu(CUN). Surprisingly,
the tRNA accepting capacity and structural stability were
increased by this substitution. Secondary structure analysis of
hmtRNALeu(CUN) suggested two kinds of pairing alignments
*To whom correspondence should be addressed. Tel: +86 21 54921241; Fax: +86 21 54921011; Email: firstname.lastname@example.org
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Nucleic Acids Research, 2005, Vol. 33, No. 11
in the T-stem that could be formed by a 1 nt slip. We sub-
sequently constructed a series of hmtRNALeu(CUN) mutants
to mimic the different types of tertiary structures resulting
from the T-stem slip and studied their structures and amino-
acylation capacities. Here, we analyze the structural basis of
T-stem slip and the resulting tertiary structures that provide
evidence for a novel, self-regulating acceptance mechanism
MATERIALS AND METHODS
Enzyme purification and tRNA preparation
All chemicals were purchased from Sigma–Aldrich Inc.
(USA), except otherwise noted.
T7 RNA polymerase and human mitochondrial leucyl-
tRNA synthetase (hmLeuRS) were purified from Escherichia
coli overproducing strains as described previously in our
laboratory (12,13). In vitro transcription of mitochondrial
tRNA and subsequent refolding of tRNA were performed as
described previously (14). The tRNA concentration was deter-
mined by UV absorbance at 260 nm and the extinction coef-
ficient was calculated from the sequence of each tRNA (15).
Assaying aminoacylation of tRNA transcript using
Accepting activity of the tRNA transcript and the time course
of tRNA aminoacylation were assessed as described previ-
ously (14). The optimized reaction mixture contained 50 mM
HEPES, pH 7.6, 25 mM KCl, 10 mM MgCl2, 2.5 mM ATP,
1 mM spermidine, 100 mg/ml BSA, 20 mM L-[14C]leucine,
0.5 mM hmLeuRS and 1.5 mM hmtRNALeu(CUN) transcript.
The aminoacylation plateau was determined during a 40 min
incubation time. The charging level of each tRNA transcript
was calculated as the percentage of aminoacylated tRNA
versus the total amount of specific tRNA per experiment.
The apparent kinetic parameters, kcatand KM, of hmLeuRS
for hmtRNALeu(CUN) and its mutants were derived from
Lineweaver-Burk plots on experiments performed with
1–50 mM tRNA and 0.15 mM hmLeuRS. Displayed data
were the averages of three independent experiments.
Secondary structure prediction of tRNA
MFOLD 3.1 program (http://www.bioinfo.rpi.edu/~zukerm/)
was used to analyze the secondary structure of tRNA (16).
Parameters were used in default settings, except for percent
suboptimality, which was set as 50%. Only cloverleaf-like
structures were considered.
Nuclease mapping of tRNA structure in solution
tRNA transcripts were 50-end-labeled with32P by the reaction
and [g-32P]ATP as described previously (17). The purified
32P-labeled transcripts were denatured by heating to 60?C
in the absence of Mg2+and then cooled slowly in the presence
of 10 mM MgCl2 (18).
with various nucleases at 25?C for 10 min in 20 ml of
40 mM Tris–HCl, pH 7.5, 10 mM MgCl2and 40 mM NaCl.
For digestion with nuclease S1 (Amersham Pharmacia, UK),
1 mM ZnCl2was added. The digestion reaction mixtures
contained 10000 c.p.m.32P-labeled tRNA, 4 mg non-labeled
carrier tRNA and 0.1 or 2.0 U RNase T1 (Amersham, USA),
0.035 or 0.1 U RNase V1 (Ambion, USA) and 50 or 150 U
nuclease S1, respectively. Reactions were stopped by adding
digested products were recovered by ethanol precipitation.
32P-labeled tRNAs were digested
Figure 1. The effect of pathogenic T12311C (U48C) mutation on the
aminoacylation hmtRNALeu(CUN). (A) Theoretical cloverleaf structure of
tRNALeu(CUN) as deduced from the DNA sequence. The pathogenic point
mutation T12311C (U48C) is indicated with arrow. (B) Aminoacylation of
WT hmtRNALeu(CUN) and U48C mutant transcripts.
Nucleic Acids Research, 2005, Vol. 33, No. 11 3607
The pellets were resuspened in 10 ml of dye mix solution
[0.0125% xylene cyanol and 0.0125% bromphenol blue in
50% formamide]. Samples were run on a 21 cm (width) ·
50 cm (height) · 0.4 mm (diameter) 12% polyacrylamide
gel containing 8 M urea buffered with 1· TBE, pH 8.3).
Alkaline ladders and G-ladders were obtained as described
Aminoacylation capacity of tRNALeu(CUN) was
increased by the pathogenic T12311C (U48C)
In the hmtRNALeu(CUN) gene, the pathogenic T12311C
mutation changes the U to C at position 48, which is
the connecting base between the variable loop and the
T-stem (Figure 1A). We constructed the wild-type (WT)
hmtRNALeu(CUN) and the U48C mutant in vitro and tested
their aminoacylation efficiency by the cognate hmLeuRS.
Interestingly, the accepting activity of the mutant tRNA
with the U48C substitution was increased compared with
the WT (Figure 1B). Under optimal conditions, 40% of the
WT transcript was aminoacylated (compared with the cal-
culated theoretical accepting activity of the transcript, see
Materials and Methods), while the accepting activity of
the U48C mutant was >60%. Kinetic aminoacylation assays
were further performed with 1–50 mM tRNA and 0.15 mM
hmLeuRS. The kinetic parameters kcatand KMwere deter-
mined for both the mutant and WT transcripts. Establishment
of these parameters enables a valuable comparison of
aminoacylation efficiency (kcat/KM). The increase of charging
capacity was further evidenced by the finding that the U48C
mutant was leucylated with a 1.31-fold higher efficiency than
the WT hmtRNALeu(CUN) (Table 1).
Because magnesium ions may affect tRNA folding,
we assayed the denaturing/renaturing processes with several
concentrations of magnesium (from 3 to 20 mM) for each
transcript. Similar aminoacylation levels of WT and mutant
tRNA were obtained under all of these conditions (data not
shown), indicating that magnesium ion concentration had
little effect on hmtRNALeu(CUN) folding.
Structure analysis of hmtRNALeu(CUN)
In order to understand the relatively lower aminoacylation
efficiency of the WT transcript, we examined the secondary
structure of hmtRNALeu(CUN) using the MFOLD program
(16) and enzymatic probing (20).
types of T-stem base pair alignments in the hmtRNALeu(CUN)
transcript. In one alignment (Ta alignment), the U48 was
located between the T-stem and variable loop as an unpaired
(Tb alignment), the U48 paired with A65 in the T-stem and
the enlarged T-loop contained 8 nt. Thus, the Ta alignment
could become the Tb alignment by a 1 nt slip, which formed
a base pair between 48 and 65 bases (Figure 2A). Because
the U48C substitution decreased the tendency to form a base
pair between C48 and A65, thus prohibiting the T-stem slip,
most of the U48C transcript was stabilized in Ta alignment.
Considering the limitations of the MFOLD prediction
(e.g. its ignorance of RNA 3D structure), we carried out an
enzymatic probing experiment to detect the solution structure
of the WT and mutant transcripts. The results of the enzymatic
probing of hmtRNALeu(CUN) were in agreement with
the MFOLD prediction described above. Nuclease S1 and
RNase T1 (guanine specific) were used to probe the unpaired
nucleotides, and RNase V1 was used to monitor base-paired or
stacked nucleotides. The 50-end-labeled cleaved fragments
were separated by PAGE/urea. As the nonspecific cleavage of
the transcripts at certain weak points could affect the results,
each of these enzymes was used with different concentrations.
Only dose-dependent enzymatic cleavages were considered.
The autoradiograms are presented in Figure 3A and results
of enzymatic mapping are summarized in Figure 3B.
Most of the nucleotides within the theoretical stem regions
of the WT hmtRNALeu(CUN) were cleaved by RNase V1 and
most of those within the canonical loop regions were recog-
nized by nuclease S1. The hmtRNALeu(CUN) transcript
appears to fold into the classical cloverleaf structure despite
the absence of post-transcriptional modification. The general
cleavage patterns of the WT and U48C mutant were similar to
each other except for some minor differences in their T-loops.
The WT transcript was cleaved by RNase T1 at G53, which is
thought to be paired with C61 in the Ta alignment. This indic-
ated that at least some proportion of the WT transcript existed
in the Tb alignment, in which G53 remained unpaired in the
in three separate experiments (data notshown),suggesting that
the result is not related to the quality of the gel. However, in
the case of U48C mutant, no corresponding cleavage was
observed. The differences in the enzymatic digestion patterns
kcat/KM(10?3s?1mM?1) Relative kcat/KM
52.4 – 1.8
112.3 – 1.0
115.5 – 3.2
110.3 – 2.2
123.1 – 4.7
53.8 – 3.2
56.2 – 4.9
23.9 – 3.9
39.1 – 5.7
36.3 – 6.9
37.2 – 5.3
34.9 – 2.6
11.5 – 2.5
8.8 – 1.6
Aminoacylation reactions were as described in Materials and Methods. Data presented here were the average of three independent experiments.
Nd, not determined.
3608 Nucleic Acids Research, 2005, Vol. 33, No. 11
of the WT and U48C mutant are summarized in Figure 3B.
It appears that the fragment indicated with a pentacle in
Figure 3B was due to the U48 shift in the T-stem.
Manipulation of the T-stem slip of hmtRNALeu(CUN)
To investigate the effect of the different T-stem base
pair alignments onthe structure
hmtRNALeu(CUN) that would stabilize in either the Ta or
Tb alignment depending on the T-stem structure (Figure 2B).
Similar to U48C mutant, U48A and U48G mutants were cre-
ated to restrain the T-stem slip by prohibiting the base pairing
between 48 and 65 residues. These two mutants would theor-
etically stabilize in the Ta alignment. Two double mutations,
U51A/A63U and G52A/C62U, were designed to prevent
the T-stem from slipping by introducing an inside bolt in
the T-stem. In the U51A/A63U mutant, the U50?A63 base
pairbecameunpaired U50?U63sothatthe Tbalignmentofthe
T-stem was unfavorable and the Ta alignment dominated.
Similarly, in G52A/C62U mutant the unpaired U51?U62
was unstable, favoring the Ta alignment of T-stem. Another
mutant, U51G, was constructed to facilitate a T-stem slip
by forming a base pair between G51 and C62, thus favoring
a Tb alignment. In the U51G mutant, the T-stem slip reduced
the size of the variable loop 1 nt (from 4 to 3) and enlarged the
T-loop by 1 nt (from 7 to 8) relatively. To ascertain the effect
of the alteration on aminoacylation of hmtRNALeu(CUN),
mutants were constructed with a 3 nt variable loop or 8 nt
T-loop. The D48 mutant had a deleted nucleotide at position
48 to test the effect of a reduced variable loop and the
U48C/+G54 mutant contained 8 nt in its T-loop.
The design for each tRNA variant was confirmed by
secondary structure prediction (data not shown). Enzymatic
probing was further performed with the representative tRNA
transcripts. Although the digestion signal at the T-loop was
weak, probably due to the tertiary structure protection, the
expectedalignment patterns werestilldetectable.Aspresented
in Figure 4A, no dose-dependent RNase T1 cleavage site at
G53 was detected in either the U51A/A63U or the D48 mutant,
which indicated that the T-stem slip was restrained by these
mutations. Meanwhile in the U51G and U48C/+G54 mutants,
RNase T1 cleaved at G53 and G54, respectively, which was
consistent with the T-loop enlargement. Moreover, some other
characteristic differences in the structures of tRNA variants
were detected. For example, the nucleotides in the T-stem
were cleaved by RNase V1 in the 7 nt T-loop variant (D48,
U48C and U51A/A63U), whereas T-stem cleavages did not
occur in either of the mutants with an enlarged T-loop (U51G
and U48C/+G54). Moreover, G10 and G11 in the D-stems of
both the U51G and U48C/+G54 mutants were cleaved by
RNase T1, suggesting that the T-loop enlargements induced
changes in their D-loops. These differences most likely reflect
domain–domain interactions between the D- and T-loops in
Figure 2. Predicated T-stem secondary structures of WT hmtRNALeu(CUN) and the designed variants. Numbering of the nucleotides is according to Sprinzl et al.
basepairinTbalignmentwith8ntT-loop;(B)varioushmtRNALeu(CUN)constructswithdesignedT-stem.TheU48N(N = A,G,C),G52A/C62UandU51A/A63U
mutantsweredominatedintheTaalignment;the U51Gmutationwasdominatedinthe Tbalignment;theD48 andU48C/+G54mutantsweredesignedto mimicthe
effect of T-stem slip on V-loop and T-loop, respectively.
Nucleic Acids Research, 2005, Vol. 33, No. 113609
hmtRNALeu(CUN). The patterns of enzymatic digestion of
each tRNA mutant are given in Figure 4B.
The charging efficiency of hmLeuRS for each designed
tRNA variant was examined. The accepting activities of
these mutants are shown in Figure 5 and the kinetic parameters
are presented in Table 1. The aminoacylation of the three
U48N (N = A, G, C) mutants increased similarly compared
with the WT transcript. The results indicate that the increased
accepting activity of the U48C mutation is due to the second-
ary structure rather than a specific residue substitution.
The two T-stem double mutants showed greater increases
(2.91-fold for U51A/A63U and 2.13-fold for G52A/C62U
as compared with that for WT transcript) in charging effici-
ency (kcat/KM) than those of U48 mutants, suggesting that the
stability of Ta alignment and instability of Tb alignment is
very important for the accepting activity. The D48 mutant,
which mimicked the shortened variable loop resulting from
a T-stem slip, also presented increased plateau level (65%)
and charging efficiency (1.61-fold increase relative to WT).
It is possible that the shortened variable loop inhibited
T-stem slip, favoring the Ta alignment. Conversely, the
U51G mutation that promoted the T-stem slip, favoring the
Tb alignment, dramatically decreased the charging activity
of hmtRNALeu(CUN). Less than 0.5% of the U51G mutant
was aminoacylated following a 40 min incubation under the
experimental conditions. The U48C/+G54 mutation with the
8 nt T-loop, which mimicked the T-loop induced by a T-stem
slip, also showed decreased aminoacylation. Neither of these
two mutants displayed sufficient aminoacylation to determine
the kinetic parameters. This result suggests that the size of
the T-loop is critical for the tRNA charging activity in that
charging capacity is decreased by the addition of bases into
the T-loop, even when T-stem is unchanged. The data suggest
that the negative effect of a T-stem slip on aminoacylation of
hmtRNALeu(CUN) is due to the increased size of the T-loop
induced by the slip.
Comparison of the variable loops and T-stems of E.coli (21),
Aquifex aeolicus (22) and human cytoplasmic (23) tRNALeus
with those of hmtRNALeu(CUN) indicates that there are sev-
eral features that determine whether a tRNALeus structure is
conducive to a T-stem slip. All 5 bp in the T-stem of E.coli
tRNALeu(CUG) and A.aeolicus tRNALeu(CUC) and the 4 bp in
the T-stem of E.coli tRNALeu(CUC) and human cytoplasmic
tRNALeu(CUU) are GC pairs. This high level of GC base
pairing in the T-stem stabilizes the Ta alignment and thereby
inhibits a T-stem slip in the above tRNALeus. A T-stem slip is
further disabled by the inability of the base at position 48 in
the variable loop to form a base pair with the base at position
65 in these tRNALeus. Finally, the large size of the variable
loops in these Ta stable tRNALeus (12–16 nt and 2–4 bp) is a
hindrance to a T-stem slip. In contrast, because only 2 of the
5 bp of the T-stem are GC pairs in hmtRNALeu(CUN), the Ta
structure configuration is less stable than that in the above
tRNAs. Moreover, in hmtRNALeu(CUN) U48 exactly pairs
with A65 following a T-stem slip, and the variable loop is
smaller containing only 4 nt and no base pair. These unique
structural features of hmtRNALeu(CUN) enable a T-stem slip.
The inherently fragile structure of hmtRNA has been dis-
cussed previously (24–26). In this work, the structural fragility
of hmtRNALeu(CUN) is specified as a mechanism of T-stem
slip. The T-stem slip can result in a mixture of Ta and Tb
Figure 3. Comparative enzymatic probing of in vitro transcribed human
mitochondrial tRNALeu(CUN) and the U48C mutant. (A) Autoradiograms
of the various cleavage products of 50-end-labeled tRNA transcripts separated
on denaturing 12% polyacrylamide gels. Lane C, control incubations without
probe; lane L, alkaline ladder; lane G, G ladder. The tRNA was 50-labeled
to increasing concentrations of nuclease. The RNase T1 cleavage product
can be easily seen in the dashed-line squares. (B) Result of the enzymatic
probing of WT and U48C mutant hmtRNALeu(CUN) transcripts. Intensities
of cuts are proportional to the darkness of the symbols. The pentacle denotes
the specific RNase T1 cleavage at G53 on WT transcript.
3610 Nucleic Acids Research, 2005, Vol. 33, No. 11
on autoradiograms of 12% polyacrylamidegels. C standsforH2Oladder; GforG ladder;L foralkaline ladder;T1 forRNase T1 digestionladder;V1 forRNase V1
solid-line square indicates the enlarged T-loop. The band difference can be easily seen in the dashed-line squares. (B) Location of enzymatic cleavage sites on
cloverleaf diagrams of the transcripts. Specifications and intensities of cuts are as indicated in the key. Nucleotides that could not be tested because of technical
denote the RNase T1 cleavage specific for the enlarged T-loop.
Nucleic Acids Research, 2005, Vol. 33, No. 113611
alignments among the WT hmtRNALeu(CUN). Mutation ana-
lysis indicates that the T-stem slip, especially the varied size
of the T-loop, correlates with the charging capacity. When its
T-stem presents a Ta alignment and the T-loop contains 7 nt,
the tRNA can be charged. While the T-stem slips to the Tb
alignment and the T-loop is enlarged to 8 nt, the accepting
capacity is lost. The T-stem slip appears to function as a
switch for the charging capacity of hmtRNALeu(CUN).
As the T-stem is generally not a locus for tRNALeus
recognition in systems including E.coli, Haloferax volcanii,
Saccharomyces cerevisiae and human mitochondria (27–32),
it is highly probable that the T-stem slip in hmtRNALeu(CUN)
governs its charging capacity by inducing a change in
tRNA tertiary structure. Enzymatic probing experiments
reveal the D-stem relaxation in mutants with an enlarged
T-loop (Figure 4), indicating domain–domain interactions
between the D- and T-loops in hmtRNALeu(CUN). Consider-
ing the interdomain interactions found in other hmtRNAs
(25,26,33), the prevalence of intramolecular communication
could be expected in the tRNA possessing a fragile structure.
The generation of a functional tRNA is a complicated pro-
cess with many steps, including transcription, end-processing,
post-transcriptional modification, aminoacylation and parti-
cipation in ribosomal protein synthesis. Previous work has
shown that a tRNA molecule can assume different confor-
mations to interact with different partners (34–36). The altern-
ative conformations of hmtRNALeu(CUN) that result from
the T-stem slip may be required for its optimal interaction
with the processing enzymes or the translation apparatus in
human mitochondria. It is possible that the conformations
in equilibrium may change according to the requirements of
the mitochondrion. Because a mitochondrion is a primitive
organelle, such a minute structural change may provide an
economical mechanism for functional regulation of hmtRNA.
However, once the T12311C mutation was introduced into
the U48C mutant, the T-stem slip ‘switch’ was destroyed,
preventing the change from the Ta alignment to the Tb align-
ment and inhibiting the regulation of the charging capacity.
Thus, we hypothesize that it is either the fixed charging
capacity or the frozen structure of the U48C mutant that
may be the cause of the related disease.
Usually, in vitro transcription is an efficient approach to
study the contribution of an individual nucleotide to tRNA
structure and function (6,8,24–26). Owing to the absence
of post-transcriptional modifications, different properties
between the transcripts and their native counterparts were
discovered (18,27,37,38). Based on this knowledge, although
the hmtRNALeu(CUN) transcript appears to fold correctly
and is aminoacyable, it mimics the native tRNA mainly at
an immature level. Further in vivo study will be helpful to
elucidate the mechanism of the related diseases.
This work was funded by the Natural Science Foundation
of China (Grant 30270310 and 30330180), Committee of
Science and Technology in Shanghai (Grant 02DJ140567)
and the 863 projects of China (Grant 2004AA235091).
Funding to pay the Open Access publication charges for this
article was provided by the National Science Foundation of
China (Grant 30270310).
Conflict of interest statement. None declared.
1. So ¨ll,D.(1993)TransferRNA:anRNAforallseason.InGestelandm,R.F.
and Atkins,J.F. (eds), The RNA World. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, pp. 157–183.
2. Anderson,S., Bankier,A.T., Barrell,B.G., de Bruijn,M.H., Coulson,A.R.,
Drouin,J., Eperon,I.C., Nierlich,D.P., Roe,B.A., Sanger,F. et al. (1981)
Sequence and organization of the human mitochondrial genome.
Nature, 290, 457–465.
3. Helm,M., Brule,H., Friede,D., Giege ´,R., Putz,D. and Florentz,C. (2000)
Search for characteristic structural features of mammalian
mitochondrial tRNAs. RNA, 6, 1356–1379.
4. Pulkes,T. and Hanna,M.G. (2001) Human mitochondrial DNA diseases.
Adv. Drug Deliv. Rev., 49, 27–43.
5. Florentz,C. and Sissler,M. (2001) Disease-related versus polymorphic
mutations in human mitochondrial tRNAs. Where is the difference?
EMBO Rep., 2, 481–486.
6. Kelley,S.O., Steinberg,S.V. and Schimmel,P. (2000) Functional
defects of pathogenic human mitochondrial tRNAs related to
structural fragility. Nature Struct. Biol., 7, 862–865.
7. Wittenhagen,L.M. and Kelley,S.O. (2003) Impact of disease-related
mitochondrial mutations on tRNA structure and function.
Trends Biochem. Sci., 28, 605–611.
metabolism and human disease. Nucleic Acids Res., 32, 5430–5441.
from international DNA sequence databases: status for the year 2000.
Nucleic Acids Res., 28, 292.
Point mutations in mitochondrial tRNA genes: sequence analysis of
chronic progressive external ophthalmoplegia (CPEO). J. Neurol. Sci.,
11. Dirheimer,G., Keith,G., Dumas,P. and Westhof,E. (1995) Primary,
secondary and tertiary structures of tRNAs. In So ¨ll,D. and
RajBhandary,U.L. (eds), tRNA Structure, Biosynthesis and Function.
American Society for Microbiology, Washington DC, pp. 93–126.
12. Li,Y., Wang,E.D. and Wang,Y.L. (1999) A modified procedure for
fast purification of T7 RNA polymerase. Protein Expr. Purif., 16,
13. Yao,Y.N., Wang,L., Wu,X.F. and Wang,E.D. (2003) Human
mitochondrial leucyl-tRNA synthetase with high activity produced from
Escherichia coli. Protein Expr. Purif., 30, 112–116.
Figure 5. Effects of the designed mutations on aminoacylation capacity of
hmtRNALeu(CUN). All values were the average of three experiments. The
standard errors were <5%.
3612Nucleic Acids Research, 2005, Vol. 33, No. 11
14. Hao,R., Yao,Y.N., Zheng,Y.G., Xu,M.G. and Wang,E.D. (2004) Download full-text
Reduction of mitochondrial tRNALeu(UUR) aminoacylation by some
MELAS-associated mutations. FEBS Lett., 578, 135–139.
15. Puglisi,J.D. and Tinoco,I.,Jr (1989) Absorbance melting curves
of RNA. Methods Enzymol., 180, 304–325.
16. Zuker,M. (2003) Mfold web server for nucleic acid folding and
hybridization prediction. Nucleic Acids Res., 31, 3406–3415.
17. Silberklang,M., Gillum,A.M. and RajBhandary,U.L. (1979)
Use of in vitro 32P labeling in the sequence analysis of
nonradioactive tRNAs. Methods Enzymol., 59, 58–109.
18. Helm,M., Brule,H., Degoul,F., Cepanec,C., Leroux,J.P., Giege ´,R. and
Florentz,C. (1998) The presence of modified nucleotides is required
for cloverleaf folding of a human mitochondrial tRNA. Nucleic
Acids Res., 26, 1636–1643.
19. Peattie,D.A. and Gilbert,W. (1980) Chemical probes for higher-order
structure in RNA. Proc. Natl Acad. Sci. USA, 77, 4679–4682.
20. Krol,A. and Carbon,P. (1989) A guide for probing native small nuclear
of tRNALeuisoacceptors by the insertion mutant of Escherichia coli
leucyl-tRNA synthetase. Biochemistry, 38, 9084–9088.
22. Xu,M.G., Chen,J.F., Martin,F., Zhao,M.W., Eriani,G. and Wang,E.D.
(2002) Leucyl-tRNA synthetase consisting of two subunits from
hyperthermophilic bacteria Aquifex aeolicus. J. Biol. Chem., 277,
23. Chang,Y.N., Pirtle,I.L. and Pirtle,R.M. (1986) Nucleotide sequence
and transcription of a human tRNA gene cluster with four genes.
Gene, 48, 165–174.
(2003) Towards understanding human mitochondrial leucine
aminoacylation identity. J. Mol. Biol., 328, 995–1010.
25. Kelley,S.O., Steinberg,S.V. and Schimmel,P. (2001) Fragile T-stem in
disease-associated human mitochondrial tRNA sensitizes structure to
local and distant mutations. J. Biol. Chem., 276, 10607–10611.
26. Roy,M.D., Wittenhagen,L.M., Vozzella,B.E. and Kelley,S.O. (2004)
Interdomain communication between weak structural elements within a
disease-related human tRNA. Biochemistry, 43, 384–392.
27. Sohm,B., Sissler,M., Park,H., King,M.P. and Florentz,C. (2004)
Recognition of human mitochondrial tRNALeu(UUR) by its cognate
leucyl-tRNA synthetase. J. Mol. Biol., 339, 17–29.
28. Larkin,D., Williams,A., Martinis,S. and Fox,G. (2002) Identification
of essential domains for Escherichia coli tRNALeu aminoacylation and
amino acid editing using minimalist RNA molecules. Nucleic Acids
Res., 30, 2103–2113.
29. Tocchini-Valentini,G., Saks,M.E. and Abelson,J. (2000) tRNA leucine
identity and recognition sets. J. Mol. Biol., 298, 779–793.
30. Asahara,H., Nameki,N. and Hasegawa,T. (1998) In vitro selection of
RNAs aminoacylated by Escherichia coli leucyl-tRNA synthetase.
J. Mol. Biol., 283, 605–618.
31. Soma,A., Uchiyama,K., Sakamoto,T., Maeda,M. and Himeno,H. (1999)
synthetase. J. Mol. Biol., 293, 1029–1038.
32. Soma,A., Kumagai,R., Nishikawa,K. and Himeno,H. (1996) The
anticodon loop is a major identity determinant of Saccharomyces
cerevisiae tRNALeu. J. Mol. Biol., 263, 707–714.
33. Watanabe,Y., Tsurui,H., Ueda,T., Furushima,R., Takamiya,S., Kita,K.,
Nishikawa,K. and Watanabe,K. (1994) Primary and higher order
structures of nematode (Ascaris suum) mitochondrial tRNAs lacking
either the T or D stem. J. Biol. Chem., 269, 22902–22906.
34. Ishitani,R., Nureki,O., Nameki,N., Okada,N., Nishimura,S. and
Yokoyama,S. (2003) Alternative tertiary structure of tRNA for
recognition by a posttranscriptional modification enzyme.
Cell, 113, 383–394.
35. Randau,L., Schauer,S., Ambrogelly,A., Salazar,J.C., Moser,J.,
Sekine,S., Yokoyama,S., So ¨ll,D. and Jahn,D. (2004) tRNA
recognition by glutamyl-tRNA reductase. J. Biol. Chem., 279,
36. Hauenstein,S., Zhang,C.M., Hou,Y.M. and Perona,J.J. (2004)
Shape-selective RNA recognition by cysteinyl-tRNA synthetase.
Nature Struct. Mol. Biol., 11, 1134–1141.
Suzuki,T. (2004) Codon-specific translational defect caused by
a wobble modification deficiency in mutant tRNA from a human
mitochondrial disease. Proc. Natl Acad. Sci. USA, 101, 15070–15075.
38. Sissler,M., Helm,M., Frugier,M., Giege ´,R. and Florentz,C. (2004)
Aminoacylation properties of pathology-related human mitochondrial
tRNA(Lys) variants. RNA, 10, 841–853.
39. Sprinzl,M., Horn,C., Brown,M., Ioudovitch,A. and Steinberg,S. (1998)
Compilation of tRNA sequences and sequences of tRNA genes.
Nucleic Acids Res., 26, 148–153.
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