(Received for publication, May 4, 1995; and in revised form, December 20, 1995)
From the
The effects of magnesium ions on a 32-mer ribozyme (R32) were
examined by high resolution NMR spectroscopy. In solution, R32 (without
its substrate) consisted of a GAAA loop, stem II, a non-Watson-Crick
3-base pair duplex and a 4-base pair duplex that included a wobble G:U
base pair. When an uncleavable substrate RNA (RdC11) was added to R32
without Mg ions, a complex did not form between R32
and RdC11 because the substrate recognition regions of R32 formed
intramolecular base pairs (the recognition arms were closed). By
contrast, in the presence of Mg
ions, the R32-RdC11
complex was formed. Moreover, titration of mixtures of R32 and RdC11
with Mg
ions also induced the ribozyme-substrate
interaction. Elevated concentrations (1.0 M) of monovalent
Na
ions could not induce the formation of the
R32-RdC11 complex. These data suggest that Mg
ions
are not only important as the true catalysts in the function of
ribozyme-type metalloenzymes, but they also induce the structural
change in the R32 hammerhead ribozyme that is necessary for
establishment of the active form of the ribozyme-substrate complex.
Self-cleaving hammerhead RNA domains are found in many
virus-like plant pathogens, and they catalyze the sequence-specific
cleavage of RNA(1, 2, 3) . The hammerhead
ribozyme was originally predicted to consist of three base pair stems
(I-III) and a central conserved nucleotide core of two nonhelical
segments (Fig. 1). Many NMR studies have been performed in
successful attempts to reveal the presence of these three base pair
stems(4, 5, 6, 7) , but no
structural information about nonhelical regions has been obtained.
Recently, two crystallographic studies have provided a
three-dimensional structure of the hammerhead
ribozyme(8, 9) . The global three-dimensional
structures of these two ribozymes were nearly identical, and in these
crystal structures, the base pair stems (stems I-III) that form A-type
helices and the central conserved core that has two structural domains
were observed; one domain of the conserved core, consisting of the
sequence CU
G
A
and
located next to stem I, makes a sharp turn identical to the uridine
turn in transfer RNAs(10) , and the other domain, consisting of
conserved nucleotides adjacent to stem II, exists as a
non-Watson-Crick, 3-base pair duplex
(U
-G
-A
:G
-A
-A
).
Figure 1: Sequence, numbering(47) , and secondary structure of the 32-mer ribozyme (R32) and of its uncleavable substrate (RdC11).
A divalent cation is essential for the specific cleavage reaction of
the hammerhead ribozyme, and ribozymes are recognized as metalloenzymes (11, 12, 13, 14, 15, 16, 17) .
The x-ray study by Scott et al. identified five potential
Mg-binding sites in the ribozyme, one of which
positioned near the catalytic pocket(9) . However, the role of
Mg
ions in the establishment of an active form of
hammerhead ribozymes remains obscure, though recent electrophoretic
studies demonstrated that the global conformation of the hammerhead
ribozyme folds in response to the concentrations and types of ions
present(18) . Previously, Heus and Pardi studied the dependence
on Mg
ions of the NMR spectrum of the
ribozyme(4) . However, the addition of Mg
ions did not cause significant spectral changes.
In order to
examine the conformational properties of a 32-mer ribozyme (R32) ()and the further role of Mg
ions, we
analyzed the structure by high resolution NMR spectroscopy. We chose
R32 because it is a well defined ribozyme; unlike many other ribozymes,
R32 does not form any inactive complexes under standard conditions for
kinetic measurements (37 °C, 25 mM Mg
),
a property that is required for analysis by
NMR(13, 19, 20) . We report here that
Mg
ions can induce the structural change in R32 that
is necessary for the interaction between the ribozyme and its substrate
(RdC11).
Figure 2:
Results of NOE experiments with R32 in
HO-D
O (4:1, v/v) that contained 0.1 M NaCl, 10 mM sodium phosphate buffer (pH 7.0) at 5 °C. a, normal spectrum; b-h, NOE difference
spectra. The irradiated imino proton resonance is indicated by irr. Observed NOEs are indicated by asterisks.
Stem II
of R32 was identified by the sequential NOEs of the imino protons of
G, G
, G
and
G
, whose chemical shifts indicated Watson-Crick
interactions between bases (Fig. 2, c and d).
Because the signal observed at 10.61 ppm was associated with a NOE on
the imino protons of G
(Fig. 2b), it was
assigned to the imino proton of G
. In Fig. 2, d and e, a NOE involving the imino proton of
G
and the signal at 9.88 ppm and a NOE involving the
signal at 9.88 ppm and the signal at 10.13 ppm were observed. Therefore
the signals at 9.88 ppm and 10.13 ppm were assigned to the imino
protons of G
and G
, respectively. Although the
chemical shifts of these guanosine imino protons indicated that they
did not form hydrogen bonds, sequential NOEs confirmed that these
residues were stacked in a duplex.
In Fig. 2a,
together with the imino proton signals of G,
G
, and G
and those of stem II, some unexpected
signals can be observed. NOE experiments (Fig. 2, f, g, and h) confirmed the existence of a 4-base pair
duplex
(G
-C
-U
-G
:C
-G
-G
-C
)
as shown in Fig. 3. Imino proton signals of U
and
G
resonated upfield of the usual region of the
hydrogen-bonding imino protons of Watson-Crick base pairs and has very
strong NOEs on each other (Fig. 2g). These data
indicate that U
and G
form a wobble G:U base
pair(26) . The other imino protons in the 4-base pair duplex
were assigned by sequential NOEs (Fig. 2, f, g, and h). This 4-base pair duplex, including the G:U
base pair, was formed between four residues in the substrate-binding
region (stem III), and three of the conserved nucleotides in the
catalytic loop and one nucleotide in stem I. It is well known that in a
complementary double-helical oligoribonucleotide, a wobble G:U base
pair is approximately equal to an A:U base pair in stabilizing
efficiency(27, 28) . Thus, an intramolecular 4-base
pair duplex forms within R32.
Figure 3: The proposed secondary structure of R32 in 0.1 M NaCl, 10 mM sodium phosphate buffer (pH 7.0). Watson-Crick base pairings are denoted by solid lines, and non-Watson-Crick base pairings are denoted by outlined lines.
The signal at 10.88 ppm showed no NOE,
so an unambiguous assignment could not be made. It may represent imino
proton of U because (i) the imino protons of
G
, G
and G
, which were
predicted to exist in the flexible single-strand region (Fig. 3)
are unlikely to be observed, (ii) even if they could be observed, 10.88
ppm is rather low field for the nonhydrogen-bonded imino protons of
G
, G
and G
, and, moreover,
(iii) apart from the imino protons of G
, G
and G
, all other imino protons were appropriately
assigned except for U
. The complete assignments of the
imino proton signals of R32, without RdC11 and Mg
ions, from results of one-dimensional NOE experiments are
summarized in Table 1.
Figure 4:
Imino proton spectra during the titration
of the R32 ribozyme with RdC11. The spectra a-f were
recorded in 0.1 M NaCl, 10 mM sodium phosphate buffer
(pH 7.0) at 5 °C, and the spectra g-l were recorded
in 0.1 M NaCl, 10 mM MgCl, 10 mM sodium phosphate buffer (pH 7.0) at 5 °C. a and g, RdC11; b and h, R32; c-f and i-l, titration study (molar ratios of RdC11 to
R32 are given in the figure).
Under Mg-free conditions, the
addition of RdC11 produced additional resonances in the region between
12.5 ppm and 13.5 ppm (Fig. 4, c-f). However, the
chemical shifts and the line widths of the signals from R32 did not
change at all, and additional signals were observed in the spectrum of
RdC11 alone without R32 (Fig. 4a). These results
indicate that R32 did not interact with RdC11 in the absence of
Mg
ions. There are four imino protons in RdC11 (the
imino protons of G
, U
, G
,
and G
). However, in the spectrum of RdC11, more than
four signals were observed (Fig. 4a), including broad
signals. Thus, it seemed that RdC11 adopted a random conformation and
R32 existed as shown in Fig. 3. Considering that the R32
ribozyme can cleave the substrate RNA specifically without forming any
inactive complexes at 37 °C in the presence of Mg
ions(13, 19, 20, 22) , we were
surprised to learn that even at 37 °C, the recognition arms of the
ribozyme were still base paired intramolecularly in the absence of
Mg
ions. In the many other ribozymes that have
previously been studied by NMR
spectroscopy(4, 5, 6, 7) , an
interaction between the ribozyme and the substrate RNA can be observed
without Mg
ions.
In contrast to results under
Mg-free condition, dramatic spectral changes were
observed when MgCl
was added to the solution (Fig. 4, i-l). The imino protons of
U
, G
and G
of R32 were not
observed in the presence of Mg
ions (Fig. 4h). Moreover, with the addition of more and more
RdC11, new broad signals appeared, and the signals due to the imino
protons of G
, U
and G
, which
belonged to 4-base pair duplex that included the wobble G:U base pair,
were gradually lost. The disappearance of signals of the other imino
protons of the 4-base pair duplex (G
and
G
) was not informative because of overlapping of
signals. These spectral changes can be explained by several
possibilities: (i) the 4-base pair duplex of R32 was opened (the
recognition arms were opened); (ii) a complex between R32 and RdC11 was
formed; (iii) an equilibrium existed between the open form of R32 and
the R32-RdC11 complex; and (iv) an equilibrium existed between the open
form of R32, the R32-RdC11 complex, and further configuration. It is
impossible to decide unambiguously among these possibilities because of
line broadening and the overlapping of signals. However, it is likely
that Mg
ions induced the opening of the recognition
arms that is necessary for the recognition of the substrate RNA. Our
kinetic data indicate the formation of a ribozyme-substrate complex in
the presence of Mg
ions (see below).
Figure 5:
Imino proton spectra during the titration
of a mixture of R32 and RdC11 (1:1) with Mg ions in
0.1 M NaCl, 10 mM sodium phosphate buffer (pH 7.0) at
5 °C (a-e) or with Na
ions in 0.1 M NaCl, 10 mM sodium phosphate buffer (pH 7.0) at 5
°C (a, f-i). The concentrations indicated
in the figure correspond to added ions (not free
ions).
It is well known that the concentration of
``salt'' causes dramatic changes in the concentration of
bimolecular versus unimolecular complexes in
self-complementary oligonucleotides. Therefore, in order to distinguish
the Mg-mediated complex formation from the charge
screening salt effect, we then carried out titration experiments with
Na
ions. Fig. 5(a and f-i) shows the titration study of mixtures of R32 and
RdC11 with Na
ions. Although the line broadening of
the imino proton signals was observed with the addition of
Na
ions, the chemical shifts of these signals did not
change, and no additional signals were observed. These results indicate
that elevated concentrations of Na
ions did not induce
the conformational change of R32 ribozyme, and it is clear that the
conformational change induced by Mg
ions originates
from the essential role of Mg
ions and not from the
charge screening effect.
Figure 6: Effects of temperature on the imino proton spectra of R32 in 0.1 M NaCl, 10 mM sodium phosphate buffer (pH 7.0).
The
thermal denaturation profile of R32 was also monitored optically (Fig. 7). One transition was observed with a melting temperature
of 58 °C in the absence of Mg ions (solid
line) and with a melting temperature of 66 °C in the presence
of Mg
ions (dotted line) (in both cases,
calculation was made from a corresponding derivative curve; not shown).
Most probably, this transition corresponded to the melting of the
4-base pair duplex and the non-Watson-Crick 3-base pair duplex.
Clearly, Mg
ions contributed to the stabilization of
duplexes. Melting of stem II was expected to occur at approximately 90
°C(5) . However, because of line broadening above 80
°C, we did not attempt to determine the melting temperature of stem
II.
Figure 7:
Changes in absorbance (260 nm) with
temperature of R32 in 10 mM phosphate buffer (pH 7.0) that
contained 0.1 M NaCl (solid line) or in 10 mM phosphate buffer (pH 7.0) that contained 0.1 M NaCl and
10 mM MgCl (dotted line). The transition
temperature was calculated to be 58 or 66 °C, respectively
.
Figure 8:
Single-turnover reaction in the
R32/R11/Mg system when reactions were initiated by
adding the following components last: substrate R11 (open
circles), ribozyme R32 (open squares), or Mg
ions (open diamonds). Reactions were carried out with
3.8 µM ribozyme and 0.11 µM substrate at 0
°C in the presence of 25 mM MgCl
. Relative
amounts of product were determined after electrophoresis of reaction
mixtures.
Katahira et al.(37) studied the
structure of an oligoribonucleotide that contained an adjacent G:A
mismatch as a model of the hammerhead ribozyme. The sequence of the
oligomer was r(GGAC GAGUCC). Four different types of base
pairing have been observed for G:A mismatches in both the crystal and
solution states: (i) head to head G(anti):A(anti); (ii) sheared (side
by side) G(anti):A(anti); (iii) G(anti):A(syn); and (iv)
G(syn):A(anti). However, it was revealed that the
5`-PyGAPu-3`:5`-PyGAPu-3` (Py = pyrimidine, Pu = purine)
sequence formed a sheared G:A base pair. This result corresponds to the
results of x-ray studies(8, 9) . In this model
oligomer(37) , the signal from the imino proton of the
guanosine in the sheared G:A base pair was observed at 10.29 ppm,
because the rate of exchange with water protons is restricted by the
neighboring base pairs, in spite of the absence of hydrogen bonding.
In the NMR spectrum of R32, the signals from the imino protons of
G and G
were observed at 9.88 and 10.13 ppm,
and sequential NOEs (G
G
G
) were also observed (Fig. 2, d and e). These data support the hypothesis that
A
:G
and G
:A
formed
sheared G:A base pairs. Many structural studies of ribozymes by NMR
spectroscopy have been
reported(4, 5, 6, 7, 39) ,
but the assignments of the imino protons of G
and
G
, as well as data that support the existence of sheared
G:A base pairs in the ribozyme, have not previously been published.
We assigned the signal at 10.88 ppm to the imino proton of
U, although no apparent sequential NOE could be detected
(see ``Results''). In the x-ray crystallographic structure,
U
forms a non-Watson-Crick base pair with A
,
and hydrogen bonds are formed between 6NH
of adenosine and
2CO of uridine and between 1N of adenosine and 2` OH of uridine,
whereas the imino proton of U
is involved in the hydrogen
bonding to O6 of G
, the 3`-neighboring residue(8) .
We could not clarify the base-base interaction between U
and A
, but the presence of a signal at 10.88 ppm in
the NMR spectrum suggests that U
and A
do not
form a Watson-Crick base pair and that the rate of exchange of the
imino proton of U
with water protons is low. This result is
not inconsistent with the non-Watson-Crick A
:U
base pair in the x-ray structure.
Our
proposed secondary structure of the R32 (ribozyme) without RdC11 and
Mg ions is shown in Fig. 3. In this model,
there are seven Watson-Crick G:C base pairs, three sheared G:A base
pairs, one wobble G:U base pair, and one non-Watson-Crick
A
:U
base pair. We could not clarify the
base-base interaction between A
and A
,
because neither residue has an imino proton and the base protons of
A
and A
could not be assigned. However, it
is likely that the adenine rings of A
and A
stack in the duplex and adopt a base pair-like configuration,
because an A:A base pair has been observed in several
RNAs(40, 41) . If the A
:A
base pair exists, R32 would have a long duplex with 13 continuous
base pairs. Existence of the 4-base pair duplex and the 3-base pair
non-Watson-Crick duplex may reflect the observed melting temperatures
of 58 and 66 °C, respectively, in the absence and the presence of
Mg
ions (Fig. 7).
Our
titration studies suggested that the R32-RdC11 complex was not formed
in the absence of Mg ions and that Mg
ions provided the properties in R32 necessary for the
ribozyme-substrate interaction ( Fig. 4and Fig. 5).
Titration studies of ribozymes by other groups indicated that
Mg
ions did not induce essential conformational
changes in ribozymes(4, 7) . However, there is a
significant difference between such ribozymes and our R32 ribozyme. The
ribozymes previously studied formed a complex with the substrate RNA
without Mg
ions. Under the same conditions, R32
cannot form a complex with the substrate RNA (RdC11), an event that is
prerequisite for the specific cleavage by R32 with Mg
ions. Due to the existence of the unfavorable conformation within
the R32 ribozyme, we could detect an additional structural role of
Mg
ions, properties that could not be detected in
previous studies(4, 7) .
Generally, Mg ions have a function to facilitate the intermolecular duplex
formation because of the charge screening effect. Therefore, our
finding that Mg
ions induce the interaction between
the ribozyme (R32) and the substrate (RdC11) may be taken as one of
those examples. However, our NMR studies indicated that this
intermolecular interaction was not induced by elevated concentrations
of Na
ions (even in the presence of 1.0 M NaCl) (Fig. 5, a and f-i).
Therefore, it is clear that the conformational change of our ribozyme
was not caused by the charge screening effect and it is due to the
essential role of Mg
ions.
Because our R32
ribozyme forms the intramolecular base pairs that could not recognize
the substrate RNA, it may be thought that our experiment is a
particular system. However, many other ribozymes suffered from
formation of inactive structures, resulting in incomplete cleavage of
substrates. Ribozymes studied by Sarma et al.(39) also showed intramolecular base pairs in the absence
of Mg ions, although they also catalyzed the
sequence-specific cleavage of the substrate RNA. Many other ribozymes
expressed in vivo have a higher chance to form intramolecular
base pairs because of their extra flanking sequences, leading to
reduced catalytic activities (42, 43, 44, 45, 46) .
Therefore, the intramolecular base pairing is not particular to our R32
ribozyme but rather is general for many ribozymes. In fact, in terms of
its kinetic behavior, our R32 ribozyme is one of the better ones: there
has been no indication of formation of any inactive structure of R32
under standard conditions for kinetic measurements (in the presence of
25 mM Mg
ions). Lilley's group clearly
demonstrated global conformational changes of the hammerhead ribozyme
in response to the concentrations of Mg
ions(18) . Our finding complements that of Lilley's
group: if a ribozyme forms an unfavorable conformation, Mg
ions might help establish correct a ribozyme-substrate complex
especially for trans-acting ribozymes.
Fig. 9shows
a schematic representation of the proposed effect of Mg ions on the R32 ribozyme. Under Mg
-free
conditions, R32 does not interact with RdC11 because the substrate
recognition region of R32 forms intramolecular base pairs. The binding
of Mg
ions to the central conserved nucleotide core
of the R32 ribozyme induces a conformational change in R32 and,
probably, the substrate recognition regions of R32 can now interact
with RdC11. Although the exact binding site of the divalent ion was not
revealed, it could involve the region of the 3-base pair
U
-G
-A
:G
-A
-A
duplex because signals from the region disappeared upon addition
of Mg
ions. It is well known that a divalent cation
is essential for the specific cleavage reaction of the ribozyme, but it
was not previously clear whether the catalytic Mg
ions stabilize the tertiary structure of the active form of the
ribozyme. In the cleavage reaction of the R32 ribozyme, at least,
Mg
ions act to induce changes in its property that
are favorable for recognition of the substrate RNA. It remains to be
determined whether the same Mg
ions have a catalytic
as well as a structural function.
Figure 9:
Schematic representation of the proposed
effect of Mg ions on the R32 ribozyme and the
reaction that it catalyzes. In the absence of Mg
ions, R32 forms intramolecular duplexes as shown in Fig. 3. Formation of the active ribozyme-substrate complex is
possible only in the presence of Mg
ions.