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(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
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 C
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 In order to
examine the conformational properties of a 32-mer ribozyme (R32) (
Figure 2:
Results of NOE experiments with R32 in
H
Stem II
of R32 was identified by the sequential NOEs of the imino protons of
G In Fig. 2a,
together with the imino proton signals of G
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
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
Under Mg In contrast to results under
Mg
Figure 5:
Imino proton spectra during the titration
of a mixture of R32 and RdC11 (1:1) with Mg
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
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
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
Figure 8:
Single-turnover reaction in the
R32/R11/Mg
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) In the NMR spectrum of R32, the signals from the imino protons of
G We assigned the signal at 10.88 ppm to the imino proton of
U
Our
proposed secondary structure of the R32 (ribozyme) without RdC11 and
Mg
Our
titration studies suggested that the R32-RdC11 complex was not formed
in the absence of Mg Generally, Mg 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 Fig. 9shows
a schematic representation of the proposed effect of Mg
Figure 9:
Schematic representation of the proposed
effect of Mg
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9447-9454
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
AN INVESTIGATION BY NMR SPECTROSCOPY (*)
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.
U
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
).
-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.
)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).
Preparation of the Ribozyme (R32) and of Substrate RNAs
(R11 and RdC11)
The ribozyme (R32), its substrate (R11), and an
uncleavable substrate (RdC11), in which the sugar of the 5` residue at
the cleavage site in the natural substrate (R11) was changed from a
ribose to a deoxyribose (rC
dC), were
chemically synthesized on a 25-µmol scale with a 1,000 Å
highly cross-linked polystyrene support on a DNA/RNA synthesizer (ABI
model 390Z, Applied Biosystems, Inc., Foster City, CA). The RNA
phosphoramidite nucleoside monomers, A
, G
,
C
and U (Applied Biosystems), that we used gave rapid
deprotection under mild conditions. After the synthesis had been
completed, the base-protecting groups were removed by heating a
solution of ethanol and ammonia (1:3) that contained the
oligonucleotide for 6 h at 55 °C. The 2`-O-silyl groups of
the base-deprotected oligonucleotides R11 or RdC11 were removed by
treatment with a 1 M solution of tetrabutylammonium fluoride
in THF (tetrahydrofuran). The crude product was obtained after
quenching the solution with water, and it was desalted on a column of
G-25 Sephadex. The silyl groups of R32 were removed with an undiluted
solution of triethylamine trihydrofluoride (10 µl/OD) for 24 h at
ambient temperature(21) . The reaction was quenched with water,
and the product was precipitated and desalted by addition of excess
1-butanol to the solution in triethylamine trihydrofluoride. The RNA
was then purified by HPLC on an anion exchange column (NucleoPac PA-100
column, 250 
9 mm i.d., Dionex Co.) under the following
gradient: starting elution with solvent A (20 mM LiClO, 20 mM NaOAc in
H
O:CHCN, 9:1, v/v, pH 6.5) and then gradually
mixing with solvent B (600 mM LiClO, 20 mM NaOAc in HO:CHCN, 9:1, v/v, pH 6.5) to the
final concentration of 70% solvent B in 40 min at a flow rate of 1.0
ml/min. The RNA obtained was desalted and isolated by the addition of
four volumes of 1-propanol to the peak fraction that contained the
product. Oligonucleotides were further purified by reversed-phase HPLC
on a Shim-pack CLC-ODS column (Shimadzu, Kyoto). Elution was performed
with a linear gradient of CHCN (5%-20%) in 0.1 M TEAA (triethylamine acetate) buffer (pH 7.0). The purified
oligonucleotides were desalted on a column of Sephadex G-25 (Fast
Desalting column; Pharmacia Biotech Inc.). After evaporation, all
counter ions were replaced with sodium by successive treatments on
small columns of Dowex 50W-X2 (pyridine form), Dowex 50W-X2 (sodium
form), and Chelex-100 (sodium form) resins. The fraction were collected
and dried by lyophilization.Kinetic Measurements
Kinetics of reaction were
followed basically as described
previously(13, 19, 20, 22) . The 5`
terminus of the substrate was labeled with
[
-
P]ATP using T4 polynucleotide kinase.
Reaction rates were measured in 25 mM MgCl and 50
mM Tris-HCl (pH 8.0, adjusted at each temperature) under
ribozyme-saturating (single-turnover) conditions at 0 or 37.5 °C.
In all cases, kinetic measurements were made under conditions such that
all the available ribozyme or substrate was expected to form a
Michaelis-Menten complex. These conditions were achieved by employing
high concentrations of the ribozyme (3.8 µM). The
concentration of the substrate was 0.11 µM. The K
value of the ribozyme for its substrate was 0.02
µM at 37 °C under the present
conditions(19, 20, 22) . Reactions were
stopped by the removal of aliquots from the reaction mixture at
appropriate intervals and mixing them with an equal volume of a
solution of 100 mM EDTA, 9 M urea, 0.1% xylene
cyanol, and 0.1% bromphenol blue. Substrates and 5` cleaved products
were separated by electrophoresis on a 20% polyacrylamide/7 M urea denaturing gel and were detected by autoradiography. The
extent of cleavage was determined by quantitation of radioactivity in
the bands of substrate and product with a Bio-Image Analyzer (BA100 or
BAS2000, Fuji Film, Tokyo).Measurement of Melting Temperature of R32
In order
to determine the T of the 4-base pair duplex and
the non-Watson-Crick 3-base pair duplex regions of R32 (see
``Results''), thermal denaturation of the ribozyme was
monitored with a UV spectrophotometer (model 2100S, Shimadzu, Kyoto). A
sample of R32 of about 1 OD/ml (total 3 ml) was prepared (i) in a
phosphate buffer (pH 7.0) that contained 0.1 M NaCl and (ii)
in a phosphate buffer (pH 7.0) that contained 0.1 M NaCl and
10 mM MgCl. The sample was preheated at 80 °C
and cooled gradually to about 10 °C. The absorption of the sample
at 260 nm was measured continuously at 10 °C for 10 min, and then
the temperature was raised from 10 to 80 °C at a rate of 1
°C/min. The T was determined by plotting the
second derivative of the thermal denaturation curve (not shown).NMR Spectroscopy
All NMR experiments were
performed with a model ALPHA-500 spectrometer (JEOL, Tokyo, 500 MHz for H). The H chemical shifts were determined
relative to the internal standard, 2-methyl-2-propanol (1.23 ppm).
One-dimensional NMR spectra were collected in
HO-DO (4:1, v/v) that contained 0.1 M NaCl and 10 mM phosphate buffer (pH 7.0) using a
1-1 solvent suppression sequence(23) . The results of
nuclear Overhauser effect (NOE) experiments were recorded at 5 °C.
For titration studies with RdC11, a concentrated stock solution of
RdC11 was added directly to the sample tube for NMR. After each step in
the titration, the sample was heated to 90 °C and slowly cooled
over 30 min to allow formation of the ribozyme-pseudosubstrate
(R32-RdC11) complex. For the titration experiments with Mg and Na ions, concentrated solutions of
MgCl and NaCl were added directly to the sample tube, but
the preheating to 90 °C of the oligomers was not performed because
high concentration of Mg ions can destroy RNA
oligomers at high temperature.
Assignment of Imino Proton Resonances of R32 (Ribozyme)
without RdC11 (a Pseudosubstrate) and Mg
The imino proton resonances of R32 without RdC11 and
Mg Ions ions were assigned. In terms of its general
structure, the hammerhead ribozyme consists of three base pair stems
(stems I-III) and a central conserved nucleotide core of two nonhelical
segments (Fig. 1). In the absence of RdC11 (a pseudosubstrate),
we expected that R32 would have stem II and the GAAA loop. Fig. 2a shows the NMR spectrum of R-32 in the low field
region at 5 °C. Because the imino protons can exchange with the
solvent water, resonance of an imino proton is observed in the NMR
spectrum only if the exchange is slow on the NMR time scale (>msec).
In this spectrum, many hydrogen-bonded and nonhydrogen-bonded imino
protons were observed. These imino protons were assigned by
one-dimensional NOE experiments (24, 25) .
O-DO (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.
, 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.,
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.
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.
Titration of R32 with RdC11
Fig. 4shows
the spectra of imino protons during titration of R32 with RdC11.
Because the chemical shifts of protons are extremely sensitive to
conformational changes, they can provide information about interactions
between R32 and (pseudo)substrates. The spectra in Fig. 4(a-f) were recorded in 0.1 M NaCl,
10 mM phosphate buffer (pH 7.0) at 5 °C, and the spectra
in Fig. 4(g-l) were obtained under the same
conditions except for the addition of Mg ions. After
each step in the titration, the sample tube was heated to 90 °C and
slowly cooled over 30 min to induce formation of the ribozyme-substrate
(R32-RdC11) complex. NMR studies of R32 without RdC11 indicated that
the recognition stems of R32 formed a 4-base pair duplex (Fig. 3). In order for R32 to recognize RdC11, it is necessary
for this 4-base pair duplex
(G-C-U-G:C-G-G-C)
to be opened.
, 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).
-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.-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).Titration of the Mixtures of R32 and RdC11 with Either
Mg
The titration
of mixtures of R32 and RdC11 with Mg or Na Ions or Na ions was studied (Fig. 5). A divalent cation is essential
for the specific cleavage reaction of the hammerhead
ribozyme(1, 2, 3, 12, 13, 15, 17, 29) .
Because the ribooligonucleotide has many phosphate groups that are
potential binding sites for Mg and Na ions, the concentrations indicated in Fig. 5are not
mostly of free ions but only the concentrations of added ions. In this
figure, significant spectral changes are clearly visible. When
Mg ions were added to the mixture of R32 and RdC11,
some new broad signals appear, and most of the imino protons of
G, U, U, G, G and G are lost (Fig. 5, a-e).
Because the disappearing imino protons belong to the 4-base pair duplex
and the 3-base pair non-Watson-Crick duplex, we can assume that the
intramolecular base pairing of these duplexes was disrupted. This
result is similar to the results of the titration of R32 with RdC11 in
the presence of Mg ions (see Fig. 4, i-l).
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).
-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.Melting of R32
Fig. 6shows the changes in
imino proton spectra with changes in temperature. An imino proton
resonance is observed only if the lifetime of the imino proton is long
compared with the rate of exchange with the solvent. As the temperature
is increased, base pairs begin to denature and imino protons become
better able to exchange with solvent water. This phenomenon is observed
in the NMR spectrum as the broadening and eventual disappearance of the
signals due to imino protons. Fig. 6shows that the imino proton
signals of G, G, G, and
G (stem II) did not disappear completely even at 70
°C though all other signals were lost below 50 °C.
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.
(dotted line). The transition
temperature was calculated to be 58 or 66 °C, respectively
.
Effects of the Order of Addition to the Reaction Mixture
of R32, Substrate, and Mg
Because the NMR data indicated that Mg Ions on Rates of
Reaction ions are required for establishment of the active
ribozyme-substrate complex in our R32 system, we examined the effects
of the order of addition of each component by measuring cleavage rates
under single-turnover conditions. When Mg ions were
added last to the preincubated solution of ribozyme and substrate at 0
°C, the rate of reaction was lower than when either ribozyme or
substrate was preincubated with Mg ions (Fig. 8). These data indicate that in the absence of
Mg ions at 0 °C, the ribozyme was trapped, at
least in part, in an inactive conformation. We should add, however,
that at the measurement temperature of 37 °C, the order of addition
of each component did not affect the kinetic behavior (data not shown).
Therefore, at the relatively high temperature of 37 °C, inactive
conformers can undergo rapid conformational changes to establish the
active ribozyme-substrate complex once Mg ions are
added. This phenomenon explains why we did not have to preheat R32 to
90 °C prior to our kinetic measurements (preheating did not change
the kinetic
behavior(13, 15, 19, 20, 22) ).
In other words, Mg induces a fairly rapid transition
between inactive and active folds, even without significant heating of
the sample.
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.
The Thermostable GAAA Loop in
R32
Tetranucleotide loops of GNRA and UNCG
(where N can be any nucleotide and R can be either A
or G) are known as unusually thermostable
loops(30, 31) , and several GNRA (32, 33) and UNCG (34, 35, 36) tetraloop hairpins have been
investigated by NMR spectroscopy. The GAAA loop is a member of the
GNRA family, and Heus and Pardi have reported the solution
structure of the GAAA tetraloop(32) . In this structure, the
first residue (G) and the last residue (A) in the loop form a
sheared-type G:A base pair, and the phosphodiester backbone has a turn
between the first residue (G) and the second residue (A). In the imino
proton spectrum of R32 without both RdC11 and Mg ions, the signal from the imino proton of G was
observed at 10.61 ppm. In the study by Heus and Pardi of the GAAA loop,
the signal from the imino proton of guanosine in the loop was also
observed in the low field region, where signals from nonhydrogen-bonded
imino protons are located(32) . The rates of exchange of these
imino protons with water protons should be slow, because these imino
protons are thought to be buried deep inside the loop. Our NMR data
confirm that a thermostable GAAA loop exists and base pairing of stem
II also occurs in R32, in accord with previous
studies(4, 5) . These structural units were also
observed in the crystal structure(8) .The Non-Watson-Crick, 3-Base Pair Duplex in
R32
The x-ray studies indicated the existence of a
non-Watson-Crick, 3-base pair duplex adjacent to stem II in the crystal
structure(8, 9) . The duplex consists of
A:G, G:A, and
U:A. A:G and
G:A form sheared G:A base pairs (hydrogen
bonds were formed between 3N of guanosine and 6NH of
adenosine and between 2NH of guanosine and 7N of adenosine; (37) and (38) ), and U:A form
a non-Watson-Crick base pair (hydrogen bonds form between 6NH of adenosine and 2CO of uridine, and between 1N of adenosine and
2` OH of uridine).. 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. 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. , 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.4-Base Pair Duplex Including a G:U Base Pair in
R32
One-dimensional NOE experiments suggested that
G-C-U-G and
C-G-G-C in
R32 formed a base pair duplex that included a wobble G:U base pair (Fig. 3). Previously, Heus and Pardi studied the substrate-free
ribozyme by NMR spectroscopy(4) , but neither a 4-base pair
duplex that included a wobble G:U base pair nor a 3-base pair
non-Watson-Crick duplex that followed stem II were observed. Because
the sequence of their ribozyme in the region that corresponded to
4-base pair duplex in R32 was not complementary, in the case that they
examined, the 4-base pair duplex did not form. Moreover, because this
4-base pair duplex would be expected to stabilize the 3-base pair
non-Watson-Crick duplex, this region was probably more flexible in the
ribozyme studied by Heus and Pardi(4) . This difference
probably explains why a 4-base pair duplex and a 3-base pair
non-Watson-Crick duplex were observed only in the R32 ribozyme. 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).The Effect of Mg
The
specific cleavage reaction of the hammerhead ribozyme requires a
divalent cation and ribozymes are recognized as
metalloenzymes(11, 12, 13, 14, 15, 16, 17) .
X-ray studies by McKay's group showed that a strong coordination
site for a metal ion exists in the non-Watson-Crick 3-base pair duplex
of the central core (the N7 atom of G Ions on R32 and the pro-Rp oxygen of the phosphate of A)(8) ,
and Klug's group also identified this metal binding
site(9) . Our experiments showed that the signals from the
imino protons of G, G, and U disappeared upon the addition of Mg ions (see Fig. 4, b and h). Although the imino proton
signal of G of R32 in the absence of RdC11 did not
disappear upon the addition of Mg ions (see Fig. 4, b and h), the imino proton signals of
G and those of the neighboring residue, U, were
lost upon the addition of Mg ions in the presence of
the uncleavable substrate, RdC11 (see Fig. 5). These results may
support the existence of the strong binding site for a divalent ion
indicated by the crystallographic studies(8, 9) .
Alternatively, Mg ions may function to destabilize
the duplex formed by the residues of the central core. Unfortunately,
our analysis could not identify the exact binding site of the catalytic
Mg ion in the R32 ribozyme system, as previous NMR
studies could
not(4, 5, 6, 7, 39) . A
further examination is required toward a detailed structural
characterization or toward locating the site of metal binding, for
instance by using Mn (if it does fold the RNA in a
similar way to Mg) as a paramagnetic probe. 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) . 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. 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. 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.
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.
Conclusion
Our data can be summarized as follows:
(i) the secondary structure of R32 in the absence of Mg ions is as shown in Fig. 3; (ii) the R32-RdC11 complex
cannot form without Mg ions because the recognition
arms of R32 form intramolecular base pairs (the recognition arms are
closed); and (iii) the addition of Mg ions (but not
Na ions) causes the recognition arms to be opened (a
prerequisite for the ribozyme-substrate interaction because
Mg ions induce binding of the substrate RNA to the
R32 ribozyme). Mg ions were thought previously to
play an integral role in catalytic function (13, 15) and not in structural stability, and the
folding of the ribozyme that is dependent on Mg ions
has not been observed in previous NMR studies(4, 7) .
Recently, electrophoretic studies demonstrated that the ribozyme folds
in response to the concentration and type of ion present(18) .
Mg ions induced co-linear alignment of stems II and
III with a variable angle subtended by stem I that depended on the
concentration of Mg ions. Moreover, the proposed
Mg-binding site (18) may explain the
disappearance of our signals from the 3-base pair
U-G-A:G-A-A duplex. In this report, we showed that Mg ions
function to induce structural changes that are favorable for
recognition of the substrate RNA. This is the first NMR study to
indicate that the structural change in a ribozyme that is required for
substrate recognition can be induced by Mg ions.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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