ABSTRACT
Ascaris suum
mitochondrial tRNA
Met
lacking the entire T stem was prepared by enzymatic ligation of two chemically
synthesized RNA fragments. The synthetic tRNA could be charged with methionine
by
A.suum
mitochondrial extract, although the charging activity was considerably low
compared with that of the native tRNA, probably due to lack of modification.
Enzymatic probing of the synthetic tRNA showed
a very similar digestion pattern to that of the native tRNA
Met
, which has already been concluded to take an L-shape-like structure [Watanabe
et al
. (1994)
J. Biol. Chem
., 269, 22902-22906]. These results suggest that the synthetic tRNA possesses almost the same
conformation as the native one, irrespective of the presence or absence of
modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too
much difficulty in the amounts necessary for physicochemical studies such as
NMR spectroscopy.
All the prokaryotic and eukaryotic cytoplasmic tRNAs (referred to hereafter as
`usual tRNAs') have a cloverleaf secondary structure possessing three looped
stems (arms), whereas most of the animal mitochondrial (mt) tRNAs have
secondary structures quite different from that of usual tRNAs (
1
). In particular, most nematode mt tRNAs have a TV-replacement loop, which replaces the T arm and the variable loop in usual
tRNAs with a less-structured loop consisting of 4-12 nucleotides (
2
-
5
). The tertiary interactions in these tRNAs have been inferred mainly from their
gene sequences by referring to the tertiary structure of yeast tRNA
Phe
, the crystal form of which has been determined unequivocally by X-ray analysis (
6
), supported by NMR analysis for its solution form (
7
).
Although three sets of binary combinations with regard to the T loop (
4
) common to usual tRNAs (G18-[Psi]55, G19-C56 and T54-A58) are not present in nematode tRNA
Met
, the other five commonly existing sets of binary and ternary combinations [U8-A14-A21, A9-A12-U23, A15-U (L4), A22-G (L3) and G10-U25-G (L2)] are conserved, leading
to the speculation that even such tRNAs lacking the entire T stem can be folded
into an L-shape-like structure roughly similar to that of usual tRNAs (
8
). This assumption has been supported by the enzymatic and chemical probing of
mt tRNAs of several nematode species (
9
).
To date, there have been a number of reports concerning synthetic tRNAs obtained
chemically and/or enzymatically.
Escherichia coli
tRNA
fMet
(
10
,
11
) and yeast tRNA
Ala
(
12
) have been prepared by enzymatic ligation of chemically synthesized
oligoribonucleotides, and
E.coli
tRNA
fMet
(
13
), yeast tRNA
i
Met
(
14
) and
E.coli
tRNA
Ala
(
15
) have been fully synthesized chemically. As for the aminoacylation activity of
these tRNAs, those synthesized so as to include modified bases (
12
,
15
) had 42-90% activity compared with that of the native tRNAs, whereas tRNAs
synthesized only with unmodified nucleotides showed only 3-28% the activity. The low aminoacylation activity observed for the
unmodified tRNAs is thus assumed to be due to a lack of modified residues which
are involved in recognition by the cognate aminoacyl-tRNA synthetases. However, these experiments have been performed only with usual tRNAs possessing the
normal cloverleaf structure; unusual tRNAs with incomplete cloverleaf structures have yet to be investigated.
In order to ascertain if the same assumption can also hold for nematode mt tRNAs
lacking the entire T stem, an attempt was made to synthesize mt tRNA
Met
from
Ascaris suum
(a parasite worm living in pig intestine) by enzymatic ligation of chemically
synthesized RNA fragments. The aminoacylation activity and the enzymatic
probing of the synthetic tRNA both provide good evidence that the synthetic
tRNA possesses the same tertiary structure as that of the native
A.suum
mt tRNA
Met
, both of which seem to be folded into an L-shape-like structure basically similar to the normal L-shape structure of usual tRNAs.
Fully-protected ribonucleoside [beta]-cyanoehyl-phosphoramidites and CPG-packed columns (CPG supports bound with 1 [mu]mol of blocked adenosine) were purchased from
Perceptive Biosystems. T4 polynucleotide kinase was obtained from Toyobo, T4
RNA ligase from Takara Shuzo, RNaseT
1
and RNaseT
2
from Sankyo, RNasePhyM and RNaseV
1
from Pharmacia, RNaseU
2
from Sigma, RNaseCL
3
and
Neurospora crassa
endonuclease from Boehringer Mannheim,
Bst
NI from New England Biolabs, and Sequenase Ver.2/7-deaza-dGTP sequencing kit from USB. [5'-
32
P]pCp (105 GBq/mmol), [[gamma]-
32
P]ATP (105 GBq/mmol) and [L-
35
S]methionine (>35 GBq/mmol) were purchased from Amersham. Solid phase DNA probe was obtained from Sci-Media.
Two RNA fragments (Fig.
1
A and B) were synthesized by an Applied Biosystems 381A DNA synthesizer at a
scale of 1 [mu]mol. Deprotection was performed as described (
16
). Purification was achieved in two steps. First, fully deprotected RNA
fragments were separated from partially deprotected RNAs using a YMC-pack C4-AP HPLC column as described previously (
17
). In the second step, a full-length RNA fragment was separated from other RNA fragments with shorter
chain lengths by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis.
Fragment B or the synthetic tRNA (19 nmol) was incubated in 200 [mu]l reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl
2
, 10 mM 2-mercaptoethanol, 100 nmol ATP, and 44 U T4 polynucleotide kinase
containing 3'-phosphatase activity at 37oC for 1 h. Then, the reaction mixture was loaded on a SuperQ
Toyopearl column (Tosoh). After the column was washed with 400 mM ammonium
acetate to elute ATP and T4 polynucleotide kinase, the product was eluted with
2 M triethylammonium bicarbonate (pH 7.8) and the resulting solution was dried
up.
Fragment B (19 nmol) was incubated in 100 [mu]l 10 mM NaIO
4
at 0oC in the dark for 30 min. To the solution, 2 [mu]l 1 M rhamnose was added and it was incubated at 0oC in the dark for 30 min. After 100 [mu]l 2 M lysine-HCl (pH 8.5) was added to the solution, it was warmed
at 45oC for 90 min. Then, 200 [mu]l water and 1 ml ethanol were added and the product was collected by ethanol precipitation.
Fragments A and B' (19 nmol each) were both heated at 65oC for 5 min in a buffer (400 [mu]l) consisting of 100 mM Tris-HCl (pH 7.5) and 15 mM MgCl
2
and then annealed; 40 [mu]l of an 11x buffer [50 mM Tris-HCl (pH 7.5), 15 mM MgCl
2
, 38.5 mM dithiothreitol, 165 [mu]g/ml bovine serum albumin and 1.76 mM ATP], and 150 U T4 RNA ligase were
then added. After overnight incubation at 11oC, T4 RNA ligase was removed by phenol treatment and the ligated product
(the synthetic tRNA
Met
) was separated from the substrate fragments using a monoQ HR HPLC column
(Pharmacia). Elution was performed by a linear gradient with 0.3-0.55 M NaCl in 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 7 M urea at a flow rate of 1 ml/min.
Ascaris suum
mt tRNA
Met
was synthesized by the scheme shown in Figure
1
. The 5'-fragment [34 mer (A)], and the 3'-fragment [31 mer (B)] containing one additional Ap at
the 3'-end, were chemically synthesized at a scale of 1 [mu]mol using CPG supports bound with 1 [mu]mol of blocked adenosine as a starting substrate. After
purification, 8
A
260
units of each fragment were recovered.
For the 3'-fragment to serve as a donor for the ligation reaction with the 5'-fragment as well as to prevent its self-ligation, both the 5'- and 3'-ends must be
phosphorylated. For this purpose, Fragment B was first phosphorylated at the 5'-end with T4 polynucleotide kinase and ATP, and then the 3'-end was treated with NaIO
4
followed by amine treatment so as to remove the 3'-terminal adenosine, leading to exposure of the 3'-phosphate. The resulting Fragment B' and Fragment A were annealed and ligated with
T4 RNA ligase giving a ligated product (Fig.
2
) with ~80-90% yield.
The product was purified by a monoQ HR HPLC column and then treated with T4
polynucleotide kinase to add a phosphate group at the 5'-end and to remove a phosphate group from the 3'-end. Finally, 7
A
260
units of the synthetic tRNA
Met
were obtained, the nucleotide sequence of which was confirmed by the Donis-Keller's method (
18
) using RNases T
1
, U
2
, PhyM and CL
3
(
19
,
20
) to be the same as that of the native tRNA
Met
except for the modified residues (Fig.
3
).
Ascaris suum
total tRNAs were prepared from the body wall muscle of
A.suum
as described (
9
).
Ascaris suum
mt tRNA
Met
was isolated from total tRNAs in two steps by the selective hybridization method [according to ref.
21
, except that Streptavidin agarose (Gibco-BRL) was used instead of the magnetic resin] using a solid-phase DNA probe possessing a sequence complementary to the residue
number U8-C38 of the tRNA and further purification by 10% denaturing polyacrylamide
gel electrophoresis. The yield of mt tRNA
Met
was 0.2
A
260
unit from 300
A
260
units of
A.suum
total tRNA. The nucleotide sequence of the tRNA was confirmed by Donis-Keller's method (
18
).
Various oligodeoxyribonucleotides were synthesized with an Applied Biosystems
381A DNA synthesizer for the construction of tRNA genes. The oligonucleotides
were ligated with one another and the resultant DNA was inserted into the multicloning sites of pUC19
by the method of Sampson and Uhlenbeck (
22
). The nucleotide sequence of the plasmid thus constructed was checked by the
dideoxy-termination method (
23
) using Sequenase Ver.2. The template DNA was prepared from
E.coli
JM109 cells cultivated on a large scale and completely digested by
Bst
NI. The transcription reaction was performed in 4 ml of a reaction mixture
containing 800 [mu]g template DNA as described (
24
) and the products were purified by 10% denaturing polyacrylamide gel
electrophoresis. From the gel, a 0.25
A
260
unit transcript one base longer than the native tRNA
Met
and one of 0.05
A
260
unit the same length as tRNA
Met
were recovered. The transcript having the additional base at the 3'-end was treated with NaIO
4
to remove the base and then dephosphorylated at the 3'-end with T4 polynucleotide kinase. After confirming that the
product was the same length as tRNA
Met
by polyacrylamide gel electrophoresis, it was combined with the other
transcript having the same original length as tRNA
Met
. The total yield of the two transcripts thus obtained was 0.26
A
260
unit.
Ascaris suum
mt extract was prepared as described (
9
). Aminoacylation of tRNAs was carried out at 37oC in 20 [mu]l of a reaction mixture containing 100 mM Tris-HCl (pH 8.7), 15 mM MgCl
2
, 4 mM ATP, 20 mM KCl, 0.8 mM spermidine, 0.5 mM spermine, 5% polyethylene
glycol (PEG 6000), 25 [mu]M [
35
S]methionine (500 Bq/pmol), and
A.suum
mt extract. To stop the reaction, 6 [mu]l aliquots of the reaction mixture were mixed with 7 [mu]l of a cold dye solution containing 0.02% bromphenol blue, 0.02% xylene
cyanol, 0.1 M sodium acetate (pH 5.0) and 8 M urea. The mixture was loaded onto 10% polyacrylamide gel containing 0.1 M sodium acetate (pH 5.0), and 8 M urea (
25
). After electrophoresis, the gel was stained with toluidine blue and dried. The
radioactivity corresponding to each tRNA band was estimated by an imaging
analyzer (BAS 1000; Fuji Photo).
Enzymatic probing of tRNAs was performed as described (
26
) with the following modification: 5'- or 3'-end-labeled tRNAs were digested with RNaseT
2
(0.00006 or 0.00002 U) or RNaseV
1
(0.045 or 0.015 U) in 50 mM sodium acetate (pH 6.0), 20 mM MgCl
2
. The digestion was performed at 37oC for 7 min.
The synthetic tRNA
Met
was shown to have methionine-accepting activity as catalyzed by the
A.suum
mt extract (see Fig.
4
). The activity was ~1/5 that of the native tRNA
Met
. To ascertain whether the low activity of the synthetic tRNA
Met
was due to some form of side reaction during the chemical synthesis (incomplete
deprotection, etc.) or to a lack of the modified residues, the tRNA
Met
transcript synthesized with T7 RNA polymerase was used as a reference. The tRNA
transcript had the same curve in the [
35
S]-labeled methionine accepting reaction (Fig.
4
) and showed similar
K
m
and
V
max
values to those of the synthetic tRNA
Met
(Table
1
). The
K
m
and
V
max
values of both the synthetic and transcribed tRNAs
Met
were almost 40-fold larger and 40-50% lower respectively, than the corresponding values of the native
tRNA
Met
, resulting in a
V
Table 1
To compare the tertiary structures of the synthetic and native tRNAs
Met
, enzymatic probing was performed using single strand-specific RNaseT
2
and double strand-specific RNaseV
1
. The limited digestion patterns obtained by polyacrylamide gel electrophoresis
for both tRNAs are shown in Figure
5
and the summarized results are illustrated in Figure
6
. The patterns of RNaseT
2
digestion were exactly the same for the two species, while those of RNaseV
1
digestion were almost the same except that sites 9 and 26 were both cleaved in
the synthetic tRNA
Met
, but not in the native one. These results clearly demonstrate that the
secondary as well as the tertiary structures of the synthetic and native tRNA
Met
are almost the same.
Recently, methods for the artificial preparation of RNA molecules have made
great progress. The
in vitro
transcription method which utilizes T7 RNA polymerase to transcribe any RNA
gene with the T7 promoter sequence upstream of it on a plasmid (
22
) is very easily utilized, and any RNA with a relatively long chain length can
be synthesized. One of the main drawbacks of this method, however, is that the
transcription yield is dependent on the sequence of the RNA to be synthesized;
in particular, the enzyme prefers G at the start point of the transcription and
it appears that the yield may be related to the structural stability of the RNA
product. Another disadvantage is that modified residues cannot be introduced
directly into the transcript.
On the other hand, the merits of the chemical synthesis method which has
recently become available are that any RNA can be synthesized independently of
the nucleotide sequence and that modified residues can be introduced into the
RNA. However the yield seems to be inversely dependent on the length of the
RNA; for example, the yield of Fragment A (34mer) is 8
A
260
units using a 1-[mu]mol CPG column, while that of the full-length tRNA (64mer) is 0.3
A
260
unit. Thus, we attempted to combine the chemical synthesis of short RNA
oligomers and their enzymatic ligation. Although such a strategy has already
been reported with usual tRNAs possessing the normal cloverleaf structure (10-12), it has not been adapted to the preparation of mt tRNAs with unusual
secondary structures.
In the present work, we have focused on the synthesis of
A.suum
mt tRNA. It is especially difficult to isolate the native tRNA because of the
difficulty of collecting sufficient numbers of the
A.suum
worm, as well as the very low yield in the preparation of a specific mt tRNA.
The yield of the native tRNA
Met
is only 0.2
A
260
unit from 300
A
260
units of unfractionated
A.suum
total tRNA, which itself is isolated from ~200-300 g (wet weight) of
A.suum
body wall muscle (
9
).
In vitro
transcription using T7 RNA polymerase for synthesizing this tRNA
Met
species also gave a very low yield; 0.26
A
260
unit of tRNA
Met
was recovered from 4-ml of reaction mixture, which is known to yield several
A
260
units of
E.coli
tRNAs in the normal case (
22
). By a combination of chemical synthesis and enzymatic ligation, 7
A
260
units of tRNA
Met
were finally recovered from a 1-[mu]mol-scale column as the starting reaction vessel, indicating the
apparent suitability of this method for preparation of tRNAs with unusual
secondary structures, whose tertiary structure needs to be elucidated by physicochemical analysis such as NMR spectroscopy.
The
K
m
value for aminoacylation of the native mt tRNA
Met
(0.12 [mu]M) is similar to those of other mt tRNAs such as bovine mt tRNA
Ser
GCU
(0.178 [mu]M), tRNA
Ser
UGA
(0.046 [mu]M) (
27
), tRNA
Phe
(0.19 [mu]M) (
28
), and hen mt tRNA
Phe
(0.15 [mu]M) (
29
). If this means that the aminoacylation reaction occurs in a similar way in
animal mitochondria, it may be said that the incomplete cloverleaf structure of
A.suum
mt tRNA
Met
is not directly related with the affinity toward the aminoacyl-tRNA synthetase.
The finding that the synthetic tRNA
Met
and tRNA
Met
transcripts had similar
K
m
and
V
max
values provides good evidence that the synthetic tRNA has no artificial modification on the normal residues. The large
K
m
and low
V
max
values of these tRNAs compared with those of the native tRNA
Met
strongly suggest that the modified residues in the native tRNA are responsible
for recognition with methionyl-tRNA synthetase. By taking it into consideration the fact that the
synthetic tRNA
Met
can be charged with
E.coli
synthetase (preliminary result), whose recognition sites lie mainly in the
anticodon (
30
-
32
) and the acceptor stem (
33
,
34
) regions, it seems that the modified residues of f
5
C34, [Psi]3 and [Psi]71 are involved in recognition with mt methionyl-tRNA synthetase. Site-specific introduction of these modified nucleosides into
the synthetic tRNA will clarify this possibility, and this is now under way.
Enzymatic probing of the synthetic and native tRNAs
Met
gave almost the same patterns in sensitivity toward RNases T
2
and V
1
, strongly suggesting that the synthetic tRNA without modified residues has a
similar conformation to that of the native tRNA. This provides a rationale for
the project to determine the tertiary structure of the bizarre tRNA using
chemically synthetic tRNA, in which we are currently involved. In the probing, little difference was observed in the sensitivity-only at positions 9 and 26 (Fig.
6
), which may have been arisen from the presence or absence of the modification.
The synthetic tRNA
Met
contained no modified residue, whereas the native tRNA
Met
contained m
1
A9 and m
2
G26 at both positions. Since these sites are sensitive toward RNaseV
1
in unmodified synthetic tRNA
Met
, the residues A9 and G26 are considered to be involved in base-pairs or in the stacking between bases. This has been proved to be the
case in the other
A.suum
tRNAs (
9
). Therefore, it is very likely that m
1
A9 and m
2
G26 are not cleaved by RNases T
2
and V
1
, not because they are uninvolved in the binary or ternary combinations of bases but because the modifications
themselves are resistant against RNase attack. The results in Figure
3
clearly support this conclusion, showing that both the RNases U
2
and PhyM did not cleave the native tRNA
Met
at m
1
A9 and RNaseT
1
only weakly cleaved it at m
2
G26.
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (to G.K, K.K. and K.W.) from the Ministry of
Education, Science and Culture of Japan, and by the Human Frontier Science
Program Organization (to K.W.), for which the authors are thankful.
tRNA
Met
K
m
([mu]M)
V
max
(relative)
V
max
/
K
m
(relative)
Native
0.12
1
1
Synthetic
4.9
0.59
0.014
Transcript
5.6
0.72
0.015
REFERENCES
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