From the
Two lysine isoacceptor tRNAs corresponding to the codons AAA and
AAG, respectively, were isolated from squid ( Loligo bleekeri),
and their nucleotide sequences were determined. During this analysis,
we discovered that the tRNA with the anticodon CUU was efficiently
cleaved at a specific site in the presence of magnesium ions, whereas
the tRNA with the anticodon UUU was not. Cleavage occurred almost
exclusively at the phosphodiester linkage between G
The reaction of yeast tRNA
Although this seemingly anomalous reaction is not known to have
biological relevance, the discovery of catalytic RNAs or ribozymes
placed this reaction in a broader mechanistic context
(8, 9) . In the first step of the self-splicing reaction
of the Tetrahymena ribozyme, a Mg
On the bases of the above described
findings, yeast tRNA
In the present report, we provide another
example of cation-mediated cleavage of tRNA, in which a Mg
Bulk tRNA was
obtained by stepwise elution with buffer B that contained 0.6
M NaCl in place of 0.2 M NaCl. The unfractionated
tRNA (2,000 A
The tRNA fraction
containing the tRNA
The fractions containing tRNAs
DNAs were
amplified by polymerase chain reaction, using M4
(5`-GTTTTCCCAGTCACGAC-3`) and Reverse (5`-CAGGAAACAGCTATGAC-3`) as
primers and pC60K DNA and pU60K DNA, respectively, as templates. The
resultant DNA was digested with MvaI to generate the CCA end
of tRNA and was transcribed in vitro in a reaction mixture of
100 µl that contained 2 mM of each NTP, 5 mM
dithiothreitol, 10 mM MgCl
For analysis of the terminal structure
produced by cleavage with Pb
The plasmid was
transcribed in the presence of [
To confirm that the
Mg
The T7 transcript that contained the same
sequence as that of tRNA
In another experiment, as shown in Fig. 8, the
cleavage activity was disrupted by a one-nucleotide deletion from the
5`- or 3`-end, respectively, of squid tRNA
Pan and Uhlenbeck
(16) reported a particular variant of the
``leadzyme'' that not only catalyzes the cleavage of a
phosphodiester bond at a specific site but also promotes the hydrolysis
of 2`,3`-cyclic phosphate to produce specifically a 3`-monophosphate.
Our present findings provide the second example of such a two-step
reaction. These two examples seem to be unique to date, but they have
features in common with reactions catalyzed by many protein
ribonucleases. Whereas the ``leadzyme'' is a product of
in vitro selection experiments, squid tRNA
It is
becoming evident that the transesterification reaction involved in the
self-splicing in group I or II introns and possibly in the splicing of
pre-mRNA is really catalyzed by metal ions such as
Mg
The tRNA sequences reported in the present study will appear in
the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with the
accession numbers D45190 and D45191.
We thank Drs. T. Ueda and H. Himeno for useful
discussions and C. Takemoto for providing the aminoacyl-tRNA
synthetase.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
D
(p16). The most remarkable feature of this cleavage
reaction is that the end product was not a 2`,3`-cyclic phosphate but
was mainly a 3`-phosphate. Thus, this reaction was distinct from the
well characterized cleavage of yeast tRNA
by lead and
from reactions catalyzed by various other metalloribozymes. The
presence of a cytidine residue at position 60 was required for
efficient cleavage but was not crucial for the reaction, and the entire
tRNA molecule had to be intact for this specific and efficient cleavage
reaction. The present study provides evidence that there exists a new
catalytic mechanism for cleavage of tRNA that exploits biologically
ubiquitous ions rather than toxic, nonessential ions such as lead.
with Pb
ions at pH 7.0 was the first well characterized example of a
metal-promoted site-specific cleavage reaction involving RNA
(1, 2, 3) . Cleavage occurs almost exclusively
at the phosphodiester linkage between D
and G
(p18)
(1, 2, 3, 4) , resulting in
the formation of a 2`,3`-cyclic phosphate at D
(2, 3) . The reaction involves the initial
abstraction of a proton from a sugar 2`-OH group at the cleavage site
by the ionized lead hydrate, which acts at the metal-binding pocket
formed by the D and T loops
(2, 3) . Other cations, such
as Zn
and Eu
, are also capable of
cleaving purified tRNAs at precise locations to yield 2`,3`-cyclic
phosphates and 5`-hydroxyl termini
(5, 6, 7) .
or
Mn
ion contributes directly to the reaction by
coordination with the 3` oxygen atom in the transition state,
presumably stabilizing the developing negative charge on the leaving
group
(10) . Other ribozymes, such as hammerheads and hairpins,
have also been proposed to be essentially metalloribozymes
(11, 12) .
, which undergoes intramolecular
cleavage in the presence of Pb
ions, has been
redesigned to perform the intermolecular cleavage of RNA as a true
enzyme
(13, 14) . Furthermore, Pan and Uhlenbeck
(15, 16) have used an in vitro selection
method to produce a series of lead-dependent ribozymes that efficiently
cleave RNA substrates with multiple turnover. One particular variant of
the ``leadzyme'' promotes the formation of 2`,3`-cyclic
phosphate termini and goes further, catalyzing hydrolysis of the cyclic
phosphate to a 3`-terminal phosphate
(16) . This two-step
mechanism is commonly exploited by several protein ribonucleases, but
it is unique among all ribozymes described to date. The many examples
of metal-catalyzed cleavages of tRNA described to date are presumed to
resemble, in terms of mechanism, the reactions catalyzed by various
other metalloribozymes.
ion is involved in hydrolysis of a cyclic phosphate to yield a
3`-terminal phosphate, as in the reaction of the
``leadzyme.'' In the present case, an intact tRNA structure
must be preserved if the cleavage reaction is to proceed.
Materials
RNase T1, RNase PhyM, and T4 RNA
ligase were purchased from Pharmacia Biotech Inc. (Tokyo, Japan);
nuclease P1 was from Yamasa Shoyu (Chiba, Japan); RNase U2, guanosine
2`,3`-cyclic monophosphate 5`-monophosphate (designated pG>p), and
guanosine 2`,3`-cyclic monophosphate (designated G>p) were from
Sigma; and RNase CL3 was from Boehringer Mannheim Yamanouchi (Tokyo,
Japan). Alkaline phosphatase from Escherichia coli and T7 RNA
polymerase were obtained from Takara Shuzo (Kyoto, Japan).
[5`-P]pCp, [
-
P]UTP,
[
-
P]GTP, and
[
-
P]ATP were from Amersham (Tokyo, Japan).
T4 polynucleotide kinase and other chemical reagents were purchased
from Wako Pure Chemical Industries (Osaka, Japan).
Purification and Determination of the Nucleotide Sequence
of Squid tRNAs
700 g of squid ( Loligo
bleekeri) were homogenized in liquid nitrogen. After 3 liters of
buffer A (44% (v/v) phenol, 0.5 M NaCl, 0.25 mM EDTA,
50 mM NaOAc, pH 6.0, 10 mM Mg(OAc), 1.2%
diethylpyrocarbonate) had been added, the mixture was shaken for 12 h.
Total nucleic acids recovered by ethanol precipitation were subjected
to chromatography on a column of DE52-cellulose that had previously
been equilibrated with buffer B (0.2 M NaCl, 10 mM
Mg(OAc)
, 20 mM NaOAc, pH 6.0).
units) was applied to a column
(1.5
45 cm) of DEAE-Sepharose that had previously been
equilibrated with buffer C (0.1 M NaCl, 20 mM
Tris-HCl, pH 7.6, 8 mM MgCl
). The column was
washed with buffer C and then eluted with a gradient obtained by
placing 1 liter of buffer C in the mixing chamber and 1 liter of buffer
C that contained 0.4 M NaCl in place of 0.1 M NaCl in
the reservoir. A total of 150 A
units of the
tRNA fraction that contained tRNA
with the anticodon UUU
(designated tRNA
(UUU)) was obtained. Then, 10
A
units/experiment were further applied to a
column (0.5
10 cm) of RPC-5 that had previously been
equilibrated with buffer D (0.4 M NaCl, 10 mM
Tris-HCl, pH 7.5, 10 mM Mg(OAc)
). The column was
washed with buffer D and then eluted with a gradient obtained by
placing 20 ml of buffer D in the mixing chamber and 20 ml of buffer D
that contained 1 M NaCl in place of 0.4 M NaCl in the
reservoir. From an initial 10 A
units of tRNA
applied to the RPC-5 column, a 0.2 A
unit of
purified tRNA
(UUU) was finally obtained after
electrophoresis in a non-denaturating gel.
with the anticodon CUU (designated
tRNA
(CUU); 1,200 A
units) eluted
from the column of DEAE-Sepharose was further applied to another column
(1
50 cm) of DEAE-Sepharose that had previously been
equilibrated with buffer E (0.4 M NaCl, 10 mM
MgCl
, 20 mM NaOAc, pH 4.0). The column was washed
with buffer E and then eluted with a gradient obtained by placing 500
ml of buffer E in the mixing chamber and 500 ml of buffer E that
contained 0.75 M NaCl in place of 0.4 M NaCl in the
reservoir. A total of 91 A
units of a tRNA
fraction that contained tRNA
(CUU) was obtained from the
column of DEAE-Sepharose (pH 4.0). Next, 10 A
units of this fraction/experiment were subjected to
chromatography on the same column of RPC-5 as that used for
purification of tRNA
(UUU), and the column was eluted with
a gradient of NaCl under the same conditions as described above. From
the 10 A
units of the tRNA fraction applied to
the RPC-5 column, 0.09 A
unit of purified
tRNA
(CUU) was finally obtained after electrophoresis in a
non-denaturing gel.
that were eluted from columns were assayed by aminoacylation
using an extract S-100 of hen oviduct and hybridization with
oligodeoxyribonucleotide probes specific to the squid genes for
tRNA
(UUU) and tRNA
(CUU) by the previously
published methods
(17, 18) . The sequences of the two
species of tRNAs
were determined by the postlabeling
method of Kuchino et al.
(19) and a partial enzymatic
digestion method
(20) . Construction of Plasmids That Contained Genes for
tRNA
(CUU)(C
) and
tRNA
(CUU)(U
) and Transcription in
Vitro of the DNAs That Included These Genes-Three DNA
fragments, namely Ribo-5`, which contains the promoter sequence of T7
RNA polymerase (5`-TAATACGACTCACTATAGCCCGGCTAGCT-3`), Ribo-3`C
(5`-CCTGGCGCCCAACGTGGGGCTCGA-3`), and Ribo-3`U
(5`-CCTGGCGCCCAACGTGGGACTCGA-3`) were synthesized and used as primers
for polymerase chain reaction
(21) . Plasmid DNA that included
the gene for tRNA
(CUU) isolated from a genomic library of
the squid
(
) was used as a template for polymerase
chain reaction. Using two sets of primers, we amplified and cloned DNAs
in PUC19, obtaining pC60K and pU60K, respectively. The pC60K plasmid
contained a gene for tRNA
(CUU) that had a cytidine
residue at position 60 (designated
tRNA
(CUU)(C
)). It generated the same
sequence as that of the native tRNA
(CUU) of squid except
for U
and A
by in vitro transcription. This U-A pair was changed to a G-C pair to allow
the plasmid to be transcribed more efficiently. The pU60K plasmid
contained the same sequence as that of pC60K except for one nucleotide
at position 60; the transcript had a uridine residue at this position
(designated tRNA
(CUU)(U
)).
, 1 mM
spermidine, 40 mM Tris-HCl, pH 8.1, 50 µg/ml bovine serum
albumin, and 100 units of T7 RNA polymerase
(22) .
Analyses of Terminal Structures Produced by Cleavages
Catalyzed by Metal Ions
For analysis of terminal structures
produced by cleavage with Mgions, the 15-mer
oligonucleotide was recovered from a denaturing polyacrylamide gel. The
15-mer was digested with RNase U2 in a reaction mixture that contained
0.5 A
unit of guanosine 2`,3`-cyclic phosphate
(designated G>p), 10 mM sodium citrate, pH 4.5, and 50
units of RNase U2 in a final volume of 5 µl at 37 °C for 1 h.
G>p was included as an internal control to demonstrate that RNase U2
did not cleave a 2`,3`-cyclic phosphate ring under the conditions of
the reaction. The digest was subjected to two-dimensional thin-layer
chromatography with solvent system A (isobutyric acid, NH
,
and H
O, 66:1:33, v/v) in the first dimension and solvent
system B (60 g of ammonium sulfate dissolved in 100 ml of 0.1
M sodium phosphate buffer (pH 6.8) plus 2 ml of
n-propyl alcohol) in the second dimension
(19) . In the
case of digestion with nuclease P1, the 15-mer was incubated in a
reaction mixture that contained 1 µg of nuclease P1 and 0.25
A
unit of carrier tRNA in a final volume of 10
µl at 37 °C for 3 h.
ions, the 17-mer
oligonucleotide was recovered from a denaturing polyacrylamide gel. The
17-mer was digested with nuclease P1 under the same conditions as used
in the case of the 15-mer. In another experiment, for the assay of the
phosphomonoesterase activity associated with nuclease P1, the 17-mer
was preincubated in a reaction mixture that contained 0.2 N
HCl at 37 °C for 1 h in a final volume of 5 µl to open the
2`,3`-cyclic phosphate ring. After neutralization of the mixture by the
addition of 0.2 N NaOH, the 17-mer was recovered by ethanol
precipitation and was digested with nuclease P1. Solvent systems used
for thin-layer chromatography were the same as those used in the case
of the analysis of the 15-mer.
Optimization of the Cleavage Reaction
The
species of tRNAshaving anticodons CUU and UUU
(designated tRNA
(CUU) and tRNA
(UUU),
respectively) were purified to homogeneity. The sequence was first
analyzed by a partial enzymatic digestion method
(20) and was
later confirmed by the postlabeling method of Kuchino et al.
(19) for identification of modified nucleotides in the tRNAs.
The sequences of the two tRNAs are shown in Fig. 1, in which
differences between the two tRNAs are shaded.
Figure 1:
Sequences
of two species of tRNAs from squid with the anticodons
CUU ( A) and UUU ( B), respectively. The nucleotides
that differ between these two tRNAs are shaded. N,
U*, and A* are unidentified modified nucleotides.
N in tRNA
(CUU) is probably a derivative of C.
The nucleotide at position 54 was shown not to be Um
(2`- O-methyluridine) but rather to be Tm (2`- O-methyl
ribothymidine). The nucleotide at position 55 is probably a
pseudouridine but was not unambiguously identified in the present
study. The site of cleavage in the presence of Mg
ions is indicated by an
arrow.
Fig. 2
shows autoradiograms of sequencing polyacrylamide gels for
the two tRNAs after partial digestion with base-specific RNases. As
shown in Fig. 2( A and B, lanes 1),
tRNA(CUU) was cleaved at a specific site even in the
absence of any enzyme, whereas tRNA
(UUU) was not cleaved
under the same conditions. This cleavage reaction was presumed to be
due to incubation of the tRNA with a buffer that contained 10
mM Mg
ions for a certain period of time
before sequencing.
Figure 2:
Autoradiograms of sequencing
polyacrylamide gels for analysis of tRNA(CUU)
( A) and tRNA
(UUU) ( B) from squid. After
treatment with bacterial alkaline phosphatase of the two tRNAs
(about 0.02 A
unit of each), each was
labeled at its 5`-end by [
-
P]ATP and
polynucleotide kinase. An aliquot of the labeled tRNA was incubated in
the absence ( lane 1) or in the presence of
Na
CO
buffer (pH 9) ( lanes 2 and
7), RNase T1 ( lane 3), RNase U2 ( lane 4),
RNase Phy M ( lane 5), or RNase CL3 ( lane 6) according
to the protocol described elsewhere (20). The cleavage product of
tRNA
(CUU) in lane 1 is indicated by a black
arrow, and the corresponding position in tRNA
(UUU)
is indicated by a white arrow. Electrophoresis was performed
in 15% polyacrylamide gels that contained 7 M urea and 10%
glycerol for 3 h.
We examined the kinetics and the optimum
conditions for the cleavage reaction. Fig. 3 A shows the
time course of the reaction in the presence of 30 mM
MgCl(pH 7.5). In this experiment, native squid
tRNA
(CUU) labeled with [5`-
P]pCp
at the 3` terminus was used as a substrate to determine whether there
might be another cleavage site in the 3` portion of the tRNA. After
incubation for 12 h, about 75% of the tRNA had been cleaved. As shown
in lanes 1 and 2 of Fig. 3 B,
Mg
ions were required for the cleavage reaction, and
spermidine could not take the place of Mg
ions. In
the absence of Mg
ions and spermidine, slight
cleavages were observed due to degradation of the tRNA
(Fig. 3 B, lane 1). However, when spermidine was
present, no such degradational cleavages occurred
(Fig. 3 B, lane 2). Spermidine may possibly
contribute to the maintenance of a stable tertiary structure of the
tRNA. The optimal concentration of Mg
ions was found
to be 30 mM, and almost the same extent of cleavage was
observed at concentrations of 30 and 50 mM
(Fig. 3 B, lanes 7-10). In the presence of
Mg
ions, spermidine slightly enhanced the reaction
(about 10%) (Fig. 3 B, lanes 3-10), a
result that suggests that spermidine can assist in the proper folding
that is required for generation of a cleavable tRNA molecule, as in the
case of hepatitis delta virus RNA
(23) . These experiments
(Fig. 3 B), in which the native tRNA
(CUU)
that had been labeled at the 5`-end was used, also showed that no
cleavage sites other than p16 were present in the 5` portion of the
tRNA. Fig. 3 C shows the effects of temperature on the
reaction. In the presence of Mg
ions, the cleavage
reaction proceeded efficiently at 37 °C but not at 10 °C. At 55
°C, the tRNA was nonspecifically degraded.
Figure 3:
Determination of the optimal conditions
for the cleavage of the native tRNA(CUU) from squid.
A, the cleavage reaction was performed in a reaction mixture
that contained 30 mM MgCl
, 2 mM
spermidine, and 50 mM Tris-HCl (pH 7.5) at 37 °C in a
final volume of 77 µl. At each time point, 11 µl of the
reaction mixture were withdrawn and analyzed. B, the reaction
mixture with different concentrations of Mg
ions was
incubated at 37 °C for 3 h with or without spermidine. C,
the reaction mixture was incubated for 5 h at the temperatures
specified. In each group, the concentration of Mg
ions was varied. Electrophoresis was performed in 15%
polyacrylamide gels that contained 7 M urea and 10% glycerol.
The cleavage product is indicated by an arrow in each
case.
The Cleavage of tRNA
We examined the
cleavage of squid tRNA(CUU) Can
Be Potentiated by Several Metal Ions
(CUU) in the presence of other
metal ions. Both Ca
and Pb
ions
potentiated cleavage of squid tRNA
(CUU) at specific sites
(see below). To compare the sites of cleavage in the presence of
various metal ions, we incubated 5`-labeled tRNA
(CUU)
with Mg
, Ca
, and Pb
ions separately. In the presence of Ca
and
Mg
ions, the tRNA was cleaved at the same site.
However, the efficiency and specificity of the reaction with
Ca
ions were lower than with Mg
ions (data not shown). By contrast, in the presence of
Pb
, the tRNA was mainly cleaved at the phosphodiester
linkage between C
and G
(p18), namely two
nucleotides downstream from the cleavage site found with Mg
and Ca
ions, confirming the previous data
obtained with yeast tRNA
(GAA) (data not shown).
Characterization of the End Products of Cleavage in
the Presence of Mg
To characterize the end
products of cleavages that occurred in the presence of metal ions, we
constructed the pC60K plasmid, which can be transcribed by T7 RNA
polymerase to generate the same sequence as that of the native squid
tRNA, Ca
, and
Pb
Ions
(CUU) with the exception of U
and
A
. This U-A pair was changed to a G-C pair to allow the
plasmid to be transcribed more efficiently (the product was designated
tRNA
(CUU)(C
)).
-
P]UTP, and
the resultant labeled transcript was cleaved in the presence of
Mg
ions. The 15-mer cleavage product was isolated by
electrophoresis and was further digested with RNase U2 for analysis of
the 3` terminus of the oligonucleotide by thin-layer chromatography.
Because RNase U2 recognizes purines predominantly, adenosine residues
to produce a 2`,3`-cyclic phosphate, this enzyme appears unlikely to
modify the 3`-end structure of the 15-mer generated in the presence of
Mg
ions. To our surprise, as shown in
Fig. 4A, the major terminus after cleavage was found to
be guanosine 3`-phosphate (designated G3`p) and not guanosine
2`,3`-cyclic phosphate (designated G>p), which is a common end
product of cleavage in several ribozyme systems
(24, 25, 26) . In addition to G3`p, small
amounts of G2`p and G>p were also detected. Because RNase U2 cannot
cleave a 2`,3`-cyclic phosphodiester bond under the conditions used (as
confirmed by the result that an excess of G>p in the reaction
mixture could not be cleaved under these conditions; see
``Experimental Procedures''), the result suggests that
tRNA
was cleaved intramolecularly in the presence of
Mg
ions at the phosphodiester linkage of p16 with
opening of the 2`,3`-cyclic phosphate ring to give rise mainly to G3`p
and also to a small amount of G2`p.
Figure 4:
The
cleavage product generated by Mg ions has mainly a
3`-phosphate and not a 2`,3`-cyclic phosphate ring at its 3`-end.
A, analysis of products of digestion with RNase U2 of the
15-mer generated by Mg
ions by thin-layer
chromatography. X and X` are unidentified
oligonucleotides produced from internal positions within the 15-mer.
B, analysis of products of digestion with nuclease P1 of the
same 15-mer by thin-layer chromatography. C, analysis of
products of digestion with RNase U2 of 15-mer generated by
Ca
ions by thin-layer chromatography. D, analysis of products of digestion with nuclease P1 of the same
15-mer by thin-layer chromatography. E, analysis of products
of digestion with nuclease P1 of the 17-mer generated by Pb
ions by thin-layer chromatography. F, analysis of
products of digestion with nuclease P1 of the same 17-mer after
treatment of the 17-mer with hydrochloric acid. See ``Experimental
Procedures'' for details of these analyses.
To confirm this result, another
experiment was performed. The 15-mer cleavage product was digested with
nuclease P1 and the digest was analyzed by TLC. As shown in
Fig. 4B, a considerable amount of inorganic phosphate
was detected in addition to pU derived from internal nucleotides of the
oligonucleotide, pG2`p and pG>p. Our interpretation of this result
is that the labeled 3`-phosphate in pG3`p that was generated by the
cleavage reaction was released by 3`-phosphomonoesterase activity
associated with nuclease P1. Because this activity releases the
monophosphate of N3`p more efficiently than that of N2`p (3,000-fold
preference for N3`p as compared with N2`p; Ref. 27), the presence of a
small amount of pG2`p can be explained by this difference in
efficiency. The same result was found in the case of the cleavage
reaction that occurred in the presence of Caions
(Fig. 4, C and D).
ions cleave the phosphodiester linkage to form a
2`,3`-cyclic phosphate that is then further opened to the 3`-phosphate,
we performed a kinetic experiment as shown in Fig. 5. The amounts
of G3`p and G2`p increased with time, whereas the amount of G>p
decreased concordantly. The result suggests that the cleavage actually
goes through the cyclic intermediate.
Figure 5:
Time course
of the generation of G>p, G3`p, and G2`p in the cleavage reaction by
Mg ions. The experimental conditions are as described
in the legend to Fig. 4. At times specified, an aliquot was taken from
the reaction tube and was subjected to electrophoresis. The 15-mer was
digested with RNase U2, followed by TLC. The radioactivity
corresponding to G>p, G3`p, and G2`p was analyzed by an image
analyzer (bas2000).
As a control experiment, an
end product of cleavage in the presence of Pbions
was also analyzed. The pC60K plasmid was transcribed in vitro in the presence of [
-
P]GTP by T7 RNA
polymerase, and the labeled transcript was incubated with
Pb
ions. The resultant 17-mer oligonucleotide was
digested with nuclease P1, and the digest was analyzed by TLC as shown
in Fig. 4 E. In this case, only pC>p and not P
was detected, confirming the data obtained in several earlier
studies of the cleavage of yeast tRNA
in the presence of
lead ions. When the 17-mer oligonucleotide was pretreated with
hydrochloric acid to open the 2`,3`-cyclic phosphate ring, nuclease P1
released free phosphate groups from pCp (Fig. 4 F),
confirming the release of the 3`-phosphate from pGp in the experiment
for which results are shown in Fig. 4( B and
D). These results unambiguously demonstrated that the cleavage
of the tRNA in the presence of Mg
ions generates
mainly a 3`-phosphate terminus and not a 2`,3`-cyclic phosphate ring.
Effect of C
Under the optimal ionic conditions, determined as
described above (30 mM MgClon the Cleavage
Reaction
, 2 mM
spermidine, 50 mM Tris-HCl, pH 7.5) at 37 °C, the
efficiency of the cleavage reaction was compared between the
tRNA
(CUU) and tRNA
(UUU), as shown in
Fig. 6
. After 8 h of incubation, 48% of tRNA
(CUU)
had been cleaved, whereas only 3% of tRNA
(UUU) had been
cleaved. Thus the ratio of the efficiencies of the cleavage reactions
was 16 to 1.
Figure 6:
Comparison of the efficiency of the
cleavage of the two native species of squid tRNAs in the
presence of Mg
ions. The reaction conditions were the
same as described in the legend to Fig. 3 A. The cleavage
product is indicated by an arrow.
As shown in Fig. 1, the sequences of the D-loop
and D-stem regions of these two tRNAs are the same, suggesting that the
T-loop region, which interacts in the tertiary structure with the
D-loop, might be responsible for the difference in the efficiencies of
the cleavage reactions. In tRNA(CUU) the nucleotide at
position 60 is cytidine, whereas in tRNA
(UUU) the same
position is occupied by uridine. Therefore, we next examined the effect
of C
on the cleavage reaction by constructing a T7
transcript that contained U
in the sequence background of
tRNA
(CUU).
(CUU) with the exception of one
base pair, U
-A
, and U at position 60 in
place of C (designated as tRNA
(CUU)(U
)) was
constructed. The efficiency of the cleavage reaction was compared
between tRNA
(CUU)(C
) and
tRNA
(CUU)(U
). Fig. 7 A shows
the results of the experiment, in which the 5`-labeled transcript was
used. tRNA
(CUU)(U
) was found to be less
effectively cleaved in the presence of Mg
ions than
tRNA
(CUU)(C
) (about 30% efficiency),
indicating that C
might be responsible for efficient
cleavage. Because tRNA
(CUU)(U
) was cleaved
to some extent (Fig. 7 A, lanes 10-11),
the nucleotide at position 60 is not the sole constituent required for
cleavage. A similar result was obtained in the case of the 3`-labeled
transcript, as shown in Fig. 7 B. In both experiments,
each transcript was found to be cleaved at a rate severalfold lower
than that of the respective native tRNA. Several additional cleavages
other than that between G
and D
were observed
(these additional cleavages occurred at the anticodon loop) (data not
shown). However, because the additional products were also detected in
control assays in the absence of Mg
ions
(Fig. 7, A, lanes 6 and 12 and
B, lanes 6 and 12), incubation at 37 °C
was presumed to degrade the transcripts. Because both transcripts could
be aminoacylated to the same extent by using an S-100 extract of bovine
liver (data not shown), they may remain the native conformation of tRNA
molecule. These results suggest that the stable tertiary structure of
the native tRNA
(CUU) molecule might be responsible for
the highly specific and efficient cleavage that occurs in the presence
of Mg
ions, although there remains the possibility
that the alteration of a nucleotide pair of U
-A
to G
-C
is responsible for this change of
the specificity and efficiency of the reaction.
Figure 7:
Comparison of the efficiency and
specificity of the cleavage of tRNA(CUU)(C
)
and tRNA
(CUU)(U
). The two tRNAs were labeled
at the 5` terminus ( A) or at the 3` terminus ( B) and
used as substrates for the cleavage reaction. The cleavage products,
namely the 15-mer ( A) and the 61-mer ( B), are
indicated by arrows. The reaction conditions were the same as
described in the legend to Fig. 3 A. In the case of lanes 6 and 12, the reactions were performed for 5 h in the
absence of Mg
ions and in the presence of 1
mM EDTA. Electrophoresis was performed in 15% polyacrylamide
gels that contained 7 M urea and 10% glycerol for 1 h.
Integrity of the tRNA Molecule May Be Required for
Cleavage in the Presence of Mg
In order to investigate the structural constraints
required for the cleavage reaction, native tRNAIons
(CUU)
labeled at the 5` or the 3` terminus was partially hydrolyzed with
alkali. The hydrolysates were separated by electrophoresis on a
polyacrylamide gel, and from each ladder the partially hydrolyzed tRNA
molecules were extracted. Each molecule lacking a part of the tRNA
sequence was incubated under the conditions of the cleavage reaction
for 3 h, as described in the legend to Fig. 3 A.
Fig. 8A shows the results of the reactions with, as
substrates, the hydrolysates of tRNA
(CUU) labeled at its
5`-end by [
-
P]ATP. The intact
tRNA
(CUU) of 76 nucleotides that was extracted from the
denaturing gel was a good substrate for the cleavage reaction
(Fig. 8 A, lane 1), indicating that the
denaturation and renaturation procedures in this experiment did not
substantially change the specificity and efficiency of the cleavage, as
compared with the cleavage of the initially purified
tRNA
(CUU). As shown in lane 2 of
Fig. 8A, the specificity and efficiency of the cleavage
were severely disrupted in the case of tRNA
(CUU) with the
one-nucleotide deletion of A at the 3`-end. With progressive loss of
the 3` portion of the tRNA, a new cleavage site emerged in the extra
loop region. After deletion of several nucleotides, the specific
cleavage at the position of p16 tended to disappear. In the case of the
tRNA
(CUU) labeled with [5`-
P]pCp
at the 3`-end, a similar result was obtained (Fig. 8 B).
These results suggest that intact tRNA
(CUU) is a
prerequisite for the specificity and efficiency of the cleavage between
G
and D
.
Figure 8:
Integrity of
conformation of native tRNA(CUU) is required for the
efficient cleavage. A and B, analyses of the cleavage
of partially hydrolyzed tRNA molecules that lacked a part of the
sequence and had been labeled at the 5` terminus ( A) and the
3` terminus ( B), respectively. Electrophoresis was performed
in 15% polyacrylamide gels that contained 7 M urea and 10%
glycerol. The product of cleavage at a specific site (p16) is
indicated by an arrow. The number above each lane indicates the length of the fragment in that lane.
Lane M shows an RNase T1 ladder. Lane C shows a
labeled RNA sample without treatment of
alkali.
Highly Specific and Efficient Cleavage of Squid
tRNA
In this report, we have demonstrated that
Mgin the Presence of Mg
Ions
ions promote the cleavage of squid
tRNA
. As described in the Introduction, the specific
cleavage of yeast tRNA
between D
and
G
in the presence of Pb
ions has been
studied extensively as a model for the role of metal ions during the
chemical step of RNA catalysis
(2, 3) . The cleavage is
highly efficient and specific for this site, providing a good tool for
deduction of the tertiary structures of tRNA by the analysis of the
positions of metal ions in tRNAs
(4, 7, 28) . In
addition to Pb
ions, several other ions, such as
Zn
and Eu
ions, are capable of
potentiating the cleavage of purified tRNAs at precise locations
(5, 6, 7) . Mg
ions can also
potentiate reactions of purified tRNAs, such as elongator tRNA
and tRNA
of yeast and lupin
(7, 28, 29) . However, in these cases, the
efficiency and specificity of the cleavage in the presence of
Mg
ions are not as great as in the case presented
here, and all of the Mg
-promoted cleavage reactions
reported to date occur only under alkaline conditions (pH
8.5-9.5). In our experiments, about 50% of substrates were
cleaved in 8 h, and, moreover, the cleavage occurred not only in the
presence of Mg
ions but also in the presence of
Ca
ions at neutral pH. Accordingly, the cleavage
reaction presented here is unique in that a tRNA other than
tRNA
can be cleaved in the presence of Ca
ions or Mg
ions at neutral pH.
A Cytidine Residue at Position 60 Is Required for
Efficient Cleavage
An analysis of the cleavage of the yeast
tRNAin the presence of Pb
ions was
made using the T7 RNA polymerase-generated transcript with the same
sequence as that of the native molecule
(30) . The rate of
cleavage of this transcript was about half that of the native tRNA.
Furthermore, mutations that disrupted tertiary interactions between the
T- and D-loops reduced the cleavage rate, and some mutants, in
particular U
C, in which U
was replaced by C,
or C
U, in which C
was replaced by U, reduced
the cleavage rate 10-fold
(30) . Crystallographic data indicate
that three Pb
ions bind to tRNA
and
that one particular Pb
ion (designated
Pb
(1) ) binds tightly to U
and
C
in the T-loop
(2, 3) . Thus, it seems
that the tertiary interactions of tRNA are important both in
positioning the lead-coordinating nucleotides of U
and
C
and in keeping the 2`-hydroxyl of the substrate near the
Pb
ion. In the present study, we determined the
nucleotide sequences of two isoacceptor tRNAs
.
tRNA
(CUU) was cleaved very efficiently in the presence of
Mg
ions, whereas tRNA
(UUU) was not
cleaved. The influence of C
in tRNA
(CUU) on
the cleavage reaction was confirmed by the experiment with T7 RNA
polymerase-generated transcripts that contained C
and
U
, respectively, with the sequence background of squid
tRNA
(CUU) (Fig. 7). In yeast tRNA
,
Mg
-binding sites were identified by x-ray
crystallography
(31, 32, 33, 34, 35) . One of
the Mg
binding sites (designated Mg
(3) ) makes
contact with C
, and, therefore, our single-base
substitution is likely to have changed the position or the affinity of
an Mg
ion in such a way that the cleavage was less
efficient.
(CUU).
Recently, Limmer et al. (36) reported that the
single-stranded 3`-terminal end (5`-NCCA-3`) of tRNA influences the
structure and stability of the acceptor stem via an effect on stacking.
Therefore, it is likely that our deletion mutants had lost the
stability of their acceptor stems, with subsequent destabilization of
the entire tRNA structure that is required for the specific and
efficient cleavage of squid tRNA
(CUU) in the presence of
Mg
ions.
The Predominant Product of Cleavage Terminates with a
3`-Phosphomonoester
The most remarkable finding of the present
study was that the cleavage product terminated mainly with a
3`-terminal phosphate and not with a 2`,3`-cyclic phosphodiester.
Except for a simple example reported by Pan and Uhlenbeck (Ref. 16; see
Introduction), all ribozymes, such as hammerhead, hairpin, and
hepatitis delta ribozymes, seem to be unable to catalyze hydrolysis of
the 2`,3`-cyclic phosphate produced from the transesterification
reaction
(24, 25, 26) . By contrast,
self-splicing group I and group II introns and the RNA subunit of
ribonuclease P catalyze the attack of an exogenous nucleophile,
yielding 3`-OH and 5`-phosphate groups
(8, 9, 37) . Our finding is not the first
example of cleavage of a tRNA in the presence of Mgions. However, because the structure of the end product has not
previously been rigorously examined, it is unclear whether the
3`-terminal phosphate structure is generated specifically in the case
of squid tRNA
(CUU) or is a general feature of such
cleavage of all tRNAs in the presence of Mg
ions.
is
not an artificial product but a native molecule that exists in
vivo. It is of interest, also, that the two-step cleavage of squid
tRNA
is catalyzed by biologically ubiquitous ions such as
Mg
and Ca
at neutral pH and not by
toxic, nonessential cations such as Pb
.
. The RNAs themselves are not involved directly in
the chemistry of the catalysis but are necessary to maintain a specific
tertiary structure that binds metal ions within a precise configuration
(10, 38, 39) . The cleavage of squid
tRNA
(CUU) described herein required the entire tRNA
structure for effective cleavage in the presence of Mg
ions. The finding that an Mg
ion situated
within a certain configuration can cleave RNA in the same way as
present day ribonucleases suggests various roles for metal ions during
evolution in the RNA world
(40) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
Journal of Lipid Research
Biochemistry and Molecular Biology Education