ABSTRACT
In order to understand the catalysis mechanism of the hairpin ribozyme, mutant
ribozymes were constructed. The distance between the loop A domain and the loop
B domain was extended by inserting various lengths of nucleotide linkers at the
hinge region in
cis
mutants, or the domains were separated physically in a
trans
mutant. All the mutant ribozymes, including the
trans
mutant, could cleave substrate RNA at the predicted site. A
cis
mutant with a single nucleotide insertion exhibited cleavage activity about
twice as high as that of the wild-type (wt) ribozyme. The insertion of 2-5 nucleotides (nt) gradually reduced the activity to the level of
the wt ribozyme. Insertion of a longer linker, up to 11 nt, resulted in the
reduction of activity to one half of that of the wt ribozyme. The ribozyme with
a single nucleotide insertion at the hinge region seems to form a more suitable
conformation for catalysis by three-dimensional fold-back of the loop B to loop A containing the cleavage site. The
trans
mutant, in which the A and B domains were physically separated, maintained a
significant level of activity, suggesting that both domains are necessary for
catalysis, but separable. These results demonstrate that interaction between
the A and B domains results in catalysis.
RNA catalysts, termed ribozymes, present possibilities for the alteration of
gene expression. The hairpin ribozyme derived from the negative strand of a
satellite RNA of tobacco ringspot virus [(-)sTRSV] catalyzes efficient cleavage of an external RNA substrate in
trans
(
1
,
2
). The reaction proceeds via a transphosphorylation mechanism, yielding products
with 2',3'-cyclic phosphate and 5'-hydroxyl termini at the -N <=> GUC- sequence (
3
,
4
). This cleavage mechanism is shared by two other small RNA enzymes: the
hammerhead and hepatitis delta ribozymes (reviewed in
5
).
The active conformation of the hairpin ribozyme is thought to consist of two non-base paired bulge loops, A and B, flanked by two helix stems each in the A
and B domains respectively of the ribozyme-substrate complex (
4
,
6
-
9
). Mutagenesis, photo-cross-linking, chemical modification and
in vitro
selection experiments have revealed some of the essential features of the
hairpin ribozyme (
6
-
8
,
10
-
17
). Nucleotide sequences in the non-base paired bulge loops A and B are highly conserved (
6
,
7
). The guanosine residue at the -N <=> GUC- cleavage site seems to be absolutely conserved and it might
interact with the opposite substrate binding strand (SBS) in the bulge loop A
of the ribozyme-substrate complex (
11
,
15
-
17
). Bulge loops A and B might be coordinated by Mg
2+
ion and the interaction between them seems to be responsible for the catalysis
(
8
,
13
,
14
,
18
-
21
). However, the nature of the interaction and the function of the loop B in the
catalytic cleavage of the substrate are not clear.
As part of a strategy toward producing virus-resistant garlic plants, a hairpin-type ribozyme derived from (-)sTRSV which cleaves at the middle of RNA genome of the
garlic latent virus was designed (
22
,
23
). In the wild-type (wt) (-)sTRSV-derived hairpin ribozyme, there was an adenosine `A' residue
at the hinge region between the A and B domains, which is weakly involved in
the formation of the helix 3 structure (
6
,
7
). The `A' residue seems to function as a flexible hinge to fold the two domains
back together which results in the interaction between them. Enforcement of the
interaction between the two domains by inserting various lengths of
oligonucleotide or propanediol phosphate linkers at the opposite strand of the
hinge region resulted in the increase of the ribozyme activity (
19
-
21
). To understand the spatial interaction between the two domains of the hairpin
ribozyme, various lengths of nucleotide linker from 1 to 11 nucleotides (nt)
were inserted between them in
cis
mutants in this study, which impart varying degrees of flexibility to the
hinge. Furthermore, we separated the two domains physically in the
trans
mutant without losing too much activity.
T7 RNA polymerase and RNase-free DNase were purchased from Promega. [[alpha]-
32
P]UTP and rNTPs were from Amersham International plc.
The secondary structures of RNA with the lowest possible free energy on the
basis of
T
m
were calculated with the program PCFOLD which makes use of the secondary
structure prediction algorithm developed by Zuker (
24
).
Construction of the transcription templates for the ribozyme and the substrate
were described previously (
22
,
25
). The substrate construct was derived from the coat protein gene of the garlic
latent virus, which gave a 148 nt long RNA transcript (
23
). DNA manipulations and cloning techniques were performed as described by Sambrook
et al
. (
26
). For
cis
mutant constructs, 26 bp synthetic double-stranded oligodeoxyribonucleotides (5'-ACCTTTGAAGAAGTCATTGTTAACTG-3') containing SBS and a linker sequence with the
Hpa
I site were annealed and ligated into pRIB as described (
22
). Linker sequences were designed so that no additional secondary structure
formation was possible. The plasmid was digested with
Hpa
I, treated with S1 nuclease and blunt-end ligated. Deletion mutants were identified by determining nucleotide sequences.
Cis
mutants having a linker varying in length from 1 to 11 nt, except 8 nt, were
obtained (Fig.
1
). For the
trans
mutant ribozyme, RNAs were transcribed separately from pRIB for the B domain
and pBIND for SBS.
RNAs were prepared by
in vitro
transcription of the linearized plasmid with T7 RNA polymerase as suggested by
the manufacturer. The labeled RNA was quantitated by Cerenkov counting. RNA
products were analyzed by electrophoresis in 5-7% polyacrylamide-urea gels and autoradiography.
Ribozyme cleavage reaction
Ribozyme cleavage reactions with
cis
mutants were carried out as described previously (
22
). Cleavage reaction with the
trans
mutant was carried out as follows. A mixture of substrate (148 nt long) and SBS
RNA (27 nt) was heated for 2 min at 95oC and cooled slowly to room temperature over a period of 30 min. The B
domain RNA (54 nt) was denatured by heating at 95oC for 2 min and cooling. The mixture for cleavage reactions contained 40 mM
Tris-HCl (pH 8.0), 4 mM MgCl
2
, 2 mM spermidine, 10 nM each of
32
P-labeled substrate RNA and the loop B domain RNA, and 10 or 50 nM SBS RNA
as indicated in a total volume of 5 [mu]l. Reactions were carried out at 37oC and products were denatured by heating in loading mixture (50%
formamide, 89 mM Tris-borate-EDTA buffer and 0.1% of xylene cyanol and bromophenol blue) and
separated in 7% polyacrylamide-urea gel. The gel was dried and autoradiographed.
Ribozymes having various lengths of nucleotide linkers from 1 to 11 nt were
maintained at 10 nM and the substrate concentration was varied from 10 to 320
nM. The reaction was carried out at 50oC for 1 h in 4 mM MgCl
2
, 2 mM spermidine and 40 mM Tris-HCl (pH 8.0).
It is known that interaction between non-base paired bulge loop A in the substrate binding domain and the loop B
domain of the hairpin ribozyme leads to the catalysis (Fig.
1
;
5
-
7
,
13
,
14
,
19
-
21
). The interaction could be modulated by the flexibility of the hinge between
the two domains. To increase the flexibility of the hinge, various lengths of
nucleotide linkers from 1 to 11 nt were inserted between SBS and the B domain
in
cis
mutants (N1-N11; Fig.
1
). The
cis
mutants with insertion of various length linkers were tested for cleavage activity. All of the mutant ribozymes (69-79 nt) could cleave the substrate RNA of 148 nt to the products of 44 and 104
nt long. A single nucleotide `A' insertion resulted in the highest activity and
then the activity gradually decreased to the level of the wt ribozyme as the
length of the linker was increased to 5 nt (Fig.
2
A and B). The activity decreased further with the increase of the length of
nucleotide linker. The observed catalytic reaction constant (
k
cat
) of the mutant with a single nucleotide insertion (N1) was about twice as high
as that of the wt ribozyme. With the insertion of a linker >5 nt,
k
cat
decreased to ~1/2 of the wt ribozyme. The effect of the nucleotide sequence of the linker
on the activity was tested with mutant ribozymes having 2 and 10 nt-long linkers (N2 versus N2' and N10 versus N10' in Fig.
1
). However, no significant difference in the activity between them was observed
(data not shown).
To relieve structural constraint at the hinge between the substrate binding
catalytic domain and the B domain, the two domains were separated physically
(Fig.
3
). The substrate, SBS, and the B domain RNAs transcribed separately
in vitro
were mixed together in the reaction buffer. Cleavage reaction occurred at the
predicted site only if all three components were present (Fig.
4
B). The
trans
mutant showed lower activity than that of the wt ribozyme but a significant
fraction (3.1% in Fig.
4
A) of the substrate was cleaved. When an equal molar concentration of the
substrate and SBD RNA were added, the activity of the
trans
mutant was ~1/10 of that of the wt ribozyme at 37oC (Fig.
4
A, lane 4 versus lane 5). However, when the amount of SBS RNA was increased 5-fold compared with that of the substrate RNA, the cleavage reaction
activity increased 2.5-fold (Fig.
4
B, lane 4 versus lane 5); ~1/4 of the wt ribozyme.
To study the catalysis mechanism of the hairpin ribozyme,
cis
mutant ribozymes in which various lengths of nucleotide linkers were inserted
at the hinge between the A and B domains, and a
trans
mutant in which the two domains were separated physically from each other, were
tested. Until now, the bulge loops A and B have been the focus of study into
the catalytic mechanism of the hairpin ribozyme (
4
,
8
,
11
,
15
-
17
). Mutagenesis approaches have also been adopted to study mechanism of the
hairpin ribozyme (
6
-
8
,
10
). Much attention has been paid to search for tertiary interactions between non-base paired bulge loops A and B (
6
,
7
,
13
,
14
,
19
-
21
). A bent structure at the hinge between the two domains which gives `paperclip
structure' seems to be the essential feature for the ribozyme activity (
14
,
19
-
21
). It was reported that `A' at the hinge might be involved in the ribozyme fold-back for catalysis which results in the interaction between bulge loops (
6
,
7
).
The fold-back structure could be coordinated by Mg
2+
(
8
,
13
,
14
,
18
). It was reported that the hairpin ribozyme contains at least two cation
binding sites essential for catalysis (
14
,
18
), but, one of these motifs could be involved in Mg
2+
-binding only when the B domains fold back (
14
). Point mutation analyses confirmed that most nucleotides at the bulge loops A
and B of the hairpin ribozyme are highly conserved for cleavage reaction in
addition to 5'-N <=> GUC-3' in the substrate (
6
-
8
,
10
). It has been proposed recently that the loop B in the hairpin domain could
interact with the cleavage site of the substrate, 5'-N <=> GUC-3', in the ground state (
14
).
We tested the possibility of ribozyme fold-back by inserting nucleotide linkers at the hinge region between the A and
B domains. The mutant ribozyme N1 which has a single nucleotide insertion at
the hinge region showed the highest efficiency in cleaving the substrate. This
mutant ribozyme could form a more suitable conformation for catalysis due to
the fold-back of the loop B over the bulge loop A. As the length of linker
increased to 5 nt, proper fold-back conformation might still be accommodated. When the linker length
increased >5 nt, however, bulkiness of the linker seemed to be rather
inhibitory to maintain the proper fold-back conformation, thereby making the cleavage reaction less efficient.
Even though there was no significant effect by nucleotide sequence differences
of linkers, we cannot exclude the possiblity that longer nucleotide linkers may
bring up unfavorable secondary structure for the ribozyme.
The phosphate backbone of a single `A' at the hinge of the wt ribozyme does not
seem to be flexible enough to allow efficient interaction between the bulge
loops A and B. When those two domains were pushed back by connecting the
opposite strand with nucleotide or propanediol phosphate linkers, increases in
the catalytic activity were noticed (
19
,
21
). Even though the joint was disconnected to relieve the structural stress at
the site, the ribozyme was still active as far as where the opposite strand is
connected by an oligocytidylate linker which holds the two domains together (
20
). Ribozyme activity of their reversely-joined ribozyme also increased with the length of oligocytidylate linker.
These results are consistent with our observation that the increase in
flexibility of the joint, due to the addition of a 1-5 nt linker, resulted in higher activity.
The concept of modular assembly in RNA molecules could be supported by these
results (
27
,
28
). The structural motif present in loop B which is common to eukaryotic 5S rRNA
and the sarcin/ricin loop of 26S rRNA, might dock with loop A in the hairpin
ribozyme. In docking of the two loop domains, spatial arrangement for
productive interaction was controlled with various efficiencies by the inserted
linker in this study. This modular assembly concept was supported by the
activity of the
trans
mutant.
To relieve the structural constraint at the hinge region further, the A and B
domains were physically separated, which allowed free interaction between the
two domains. In the
trans
mutant, we showed that the A domain and the B domain were necessary for
catalysis. Our results demonstrate, however, that two domains were separable
physically into two molecules without losing too much activity. The observed
reaction velocity of the
trans
mutant was 1/10 of that of the wt ribozyme as shown in Figure
4
. It has been reported by Butcher
et al.
(
27
) in which
k
cat
of the reconstituted
trans
ribozyme was ~1/2-1/10 of the corresponding intact wt ribozyme depending on the
relative concentration of SBS and the loop B domain to the substrate. The
K
m
value for the reconstituted reaction increased 10
3
-10
4
compared with that of the intact wt ribozyme. These results support the
possible interaction between the loops A and B. From a photo-cross-linking study, it was suggested that domain B could fold into a similar
tertiary structure in the presence or absence of domain A (
12
). This result suggests that the
trans
mutant ribozyme studied in this work may function by the same mechanism as the
wt hairpin ribozyme. Further studies by mutagenesis with the
trans
mutant could reveal the regions responsible for the interaction between loops A
and B and thus catalysis mechanism.
The present investigation was supported by a grant from the Genetic Engineering
Program of the Ministry of Education and in part by a grant from the Ministry
of Science and Technology of Korea.
REFERENCES
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