4428–4434 Nucleic Acids Research, 2000, Vol. 28, No. 22
© 2000 Oxford University Press
Multiple conformational states of the hammerhead
ribozyme, broad time range of relaxation and
topology of dynamics
Marcus Menger, Fritz Eckstein1 and Dietmar Porschke*
Max Planck Institut für biophysikalische Chemie, D-37077 Göttingen, Germany and 1Max Planck Institut für
experimentelle Medizin, D-37075 Göttingen, Germany
Received August 11, 2000; Revised and Accepted September 29, 2000
ABSTRACT
The dynamics of a hammerhead ribozyme was
analyzed by measurements of fluorescence-detected
temperature jump relaxation. The ribozyme was
substituted at different positions by 2-aminopurine
(2-AP) as fluorescence indicator; these substitutions
do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the
ribozyme is very similar: a fast decrease of
fluorescence, mainly due to physical quenching, is
followed by a slower increase of fluorescence due to
conformational relaxation. In most cases at least
three relaxation time constants in the time range
from a few microseconds to ~200 ms are required for
fitting. Although the relaxation at different positions of
the ribozyme is similar in general, suggesting a global
type of ribozyme dynamics, a close examination
reveals differences, indicating an individual local
response. For example, 2-AP in a tetraloop reports
mainly the local loop dynamics known from isolated
loops, whereas 2-AP located at the core, e.g. at the
cleavage site or its vicinity, also reports relatively
large amplitudes of slower components of the ribozyme
dynamics. A variant with an A→G substitution in
domain II, resulting in an inactive form, leads to the
appearance of a particularly slow relaxation process
(τ ≈200 ms). Addition of Mg2+ ions induces a reduction
of amplitudes and in most cases a general increase
of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction
of relaxation curves recorded under corresponding
conditions. The changes induced in the relaxation
response by Mg2+ are very similar to those induced
by Ca2+. The relaxation data do not provide any
evidence for formation of Mg2+-inner sphere
complexes in hammerhead ribozymes, because a
Mg2+-specific relaxation effect was not visible.
However, a Mg2+-specific effect was found for a
dodeca-riboadenylate substituted with 2-AP, showing
that the fluorescence of 2-AP is able to indicate inner
sphere complexation. Amplitudes and time constants
show that the equilibrium constant of inner sphere
complexation is 1.2, corresponding to 55% inner
sphere state of the Mg2+ complexes; the rate constant
6.6 × 103 s–1 for inner sphere complexation is relatively
low and shows the existence of some barrier(s) on
the way to inner sphere complexes.
INTRODUCTION
The hammerhead ribozyme is a small catalytic RNA with a
core region containing 10 invariant nucleotides and with two
substrate binding arms of varying length (1–3). It cleaves the
substrate with a certain specificity 3′ to a triplet of the general
formula NUH where H can be adenosine, cytidine or uridine
but not guanosine. Activity requires divalent metal ions such as
Mg2+ or high concentrations of monovalent ions (4). The reaction
catalyzed is a phosphoryl transfer where the 2′-OH group of
the nucleoside at the cleavage site attacks the phosphorus at the
internucleotidic linkage in an in-line mechanism to produce a
terminal nucleoside 2′,3′-cyclic phosphate. Several X-ray
structures of this ribozyme have been determined, including
some structures of intermediate conformations, which probably
represent an approach to the transition state (5–7). The general
structure is consistent with several solution studies determined
mainly by FRET analysis (8–10).
Despite this information the structure of the active
conformer and details of the reaction mechanism are still
elusive. In particular the precise role of the metal ion is yet
uncertain. The overall kinetics of the hammerhead ribozymes
is dominated by the steps of substrate binding and/or product
release (11), which are determined by the well known mechanism
of RNA double helix formation and dissociation (12,13). The
cleavage rates are Mg2+-dependent and there is good evidence
that one of the roles of Mg2+ is to support the formation of the
catalytically competent structure. Mg2+-dependent conformational changes have been observed by gel mobility assays and
by FRET analysis (9,10). We have previously determined
equilibrium constants for Mg2+-induced conformation changes
using the fluorescence of 2-aminopurine (2-AP) incorporated
at various positions in the ribozyme (14). We report now on the
time dependence obtained by fluorescence detected temperature
jump relaxation for the same ribozymes as used in the previous
study to obtain insight into the internal dynamics of the
*To whom correspondence should be addressed. Tel: +49 551 2011438; Fax: +49 551 2011168; Email: [email protected]
Nucleic Acids Research, 2000, Vol. 28, No. 22 4429
ribozyme. We have demonstrated that 2-AP can be introduced
without loss of catalytic function (14). Substitutions at
different positions of the ribozyme facilitate an analysis of the
topology of the ribozyme dynamics. The data show that there
are both global and local modes of the dynamics, distributed
over a broad time range.
MATERIALS AND METHODS
The hammerhead ribozymes (Fig. 1) and the dodeca-riboadenylate
substituted with 2-AP, A(pA)6p(2-AP)(pA)4 [2-AP-oligo(A)], were
synthesized by the solid phase method and purified as previously described (14,15). A standard buffer B containing 0.1 M
NaClO4, 50 mM cacodylic acid/Tris pH 7.2 was used for most
of the measurements.
Fluorescence intensities were measured using an SLM 8000
spectrofluorimeter, interfaced to a PC for data collection and
averaging. The fluorescence was excited at 320 nm with a
bandwidth of 1 nm; the emission was measured at 380 nm with
a bandwidth of 16 nm. Before measurements, all solutions
were centrifuged within the cuvette in a low-speed desk-top
centrifuge for 2 min, in order to clear the solution from dust
particles. After thermostating in the cuvette holder for 5 min,
the fluorescence intensity was measured for 400 s. There was
no indication of a slow process under these conditions. After
correction by reference measurements, the data were fitted by
least-squares fitting routines using the facilities of the Gesellschaft
für wissenschaftliche Datenverarbeitung mbH (Göttingen,
Germany). The model used for fitting of the titration data has
been described previously (14).
Fluorescence detected temperature jump relaxation was
measured with an instrument of the type described by Rigler et al.
(16) with improvements by C. R. Rabl (unpublished results). A
200 W Xe/Hg arc lamp together with a Schoeffel GM250
monochromator was used as the light source. The fluorescence
was excited at 313 nm and collected behind cutoff filters
GG385 (2 mm) from Schott (Mainz, Germany). The transients
were initially stored on a Biomation 1010 waveform recorder
and finally transmitted to the facilities of the Gesellschaft für
wissenschaftliche Datenverarbeitung mbH, for exponential
fitting by deconvolution procedures either according to
Porschke and Jung (17) or Diekmann et al. (18).
Stopped flow data were measured with fluorescence detection
(excitation/emission wavelengths and cutoff filters as in T-jump
experiments) using an instrument developed at the Max Planck
Institut für biophysikalische Chemie. The stopped flow
construction of this instrument is equivalent to that used in a
recently described stopped flow field jump instrument (19).
RESULTS
Design of hammerhead constructs
The hammerhead ribozymes used in the present investigation
were cis-cleaving constructs with 49 nt in which cleavage was
inhibited by the presence either of 2′-deoxycytidine at position
17 for hammerheads HH1, HH3, HH4 and HH5 or of 2′-deoxy-2′aminocytidine for HH2 (Fig. 1). The constructs were designed to
analyze various parts of the molecule for any characteristic contribution to its dynamics and to metal ion binding. Thus, 2-AP
reporter groups were substituted next to the 3′-side of the cleavage
Figure 1. (A) Sequence and secondary structure of hammerhead ribozyme
variants; numbering according to Hertel et al. (20). (B) Model of the hammerhead
ribozyme structure for the sequence used in the present investigation with the
positions of substitutions; based on the crystal structure of Pley et al. (21),
assuming that the essential parts of the structure remain unchanged.
site (position 1.1 in HH1, HH2 and HH5), at the catalytic core
close to the cleavage site (position 7 in HH3) and at the periphery
in the stem III tetraloop (position L3.3 in HH4). Ribozyme HH5
was also modified at position 14, where an adenosine was
exchanged by a guanosine, abolishing cleavage activity (22).
4430 Nucleic Acids Research, 2000, Vol. 28, No. 22
Figure 2. Fluorescence detected relaxation curves (change of fluorescence
intensity ∆I as a function of time t) for hammerhead ribozymes HH1–HH5
induced by temperature jumps from 2.0 to 10.6°C in buffer B. The data are
shown with a pretrigger recording period of 40 µs, i.e. the time before application
of joule heating. The time constant of the initial decrease of the fluorescence
is determined by the heating time of 8.25 µs and the detector risetime of 5.4 µs
(adjusted in these experiments to a relatively slow detection mode).
Broad spectrum of relaxation indicates existence of many
conformational substates
The general form of the relaxation curves induced by temperature
jumps in solutions and reported by the fluorescence of 2-AP is
very similar for all the hammerhead ribozyme variants: in a
first part of the relaxation curves the fluorescence decreases
with time constant(s) below the time resolution of the instrument
(τ ≤1 µs); in a second part the fluorescence increases with time
constants in the range from a few microseconds up to ~200 ms
(Fig. 2). The first part is mainly due to the physical processes
of thermal quenching, whereas the second part reflects conformational relaxation of the ribozymes. Exponential fitting of
these relaxation curves shows the existence of at least three
conformational relaxation processes in most cases. Approximate
values of relaxation time constants are compiled in Table 1. An
example with at least four conformational relaxation processes
required for a satisfactory fit is shown in Figure 3 for HH3.
Because of the limited amplitudes and the close spacing of the
processes on the time scale, it is virtually impossible to deduce
exact values for the time constants. Thus, the existence of a
continuous spectrum of time constants cannot be excluded.
In spite of this problem in the determination of time
constants and the similarity of the general relaxation response,
a more detailed comparison of the relaxation curves obtained
for the hammerhead variants reveals clear differences (Fig. 2).
One extreme case is that of HH4 with 2-AP in the tetraloop,
which mainly exhibits a relatively fast relaxation response,
which has been observed and characterized previously for
isolated hairpin loops (23). The other extreme is HH5, which is
not active in catalysis because of the A→G substitution at
position 14. In this case the relaxation extends to relatively
long times with a rather high amplitude. Thus, it is possible to
identify characteristic features of given ribozyme variants.
Figure 3. Relaxation curve for HH3 induced by a temperature jump from 2.0
to 10.6°C in buffer B with 5 mM Mg2+, shown in two different time scales (a)
and (b). The line marked with circles represents the reference curve measured
with a solution of tryptophan under identical experimental conditions; this
reference is used for deconvolution (17). The line without noise represents a
least squares fit with the time constants 0.56, 5.0, 54, 98 and 8800 µs; the
corresponding relative amplitudes are –215.2, 59.7, 18.6, 20.7 and 16.3%. The
two lower panels show the residuals for the fast and the slow time scale. The insert
shows the autocorrelation function of the residuals.
Table 1. Relaxation time constants τi of hammerhead ribozymes
HH1–HH5 from fluorescence detected temperature jump relaxation
measurements
The arrows indicate the change of the respective time constants observed
with increasing concentration of metal ions: Ý = increase of τ (≡ slower
relaxation), ß = decrease of τ (≡ faster relaxation); ô = direction not
clearly defined.
Search for Mg2+-inner sphere complexes
Measurements of chemical relaxation can be used for an
analysis of metal ion binding with respect to inner sphere
Nucleic Acids Research, 2000, Vol. 28, No. 22 4431
Figure 4. Fluorescence detected relaxation curves for hammerhead ribozymes
HH1–HH5 induced by temperature jumps from 2.0 to 10.6°C in buffer B with
5 mM Mg2+. The data are shown with a pretrigger recording period of 40 µs,
i.e. the time before application of joule heating. The time constant of the initial
decrease of the fluorescence is determined by the heating time of 7.25 µs and
the detector risetime of 5.4 µs (adjusted in these experiments to a relatively
slow detection mode). The conditions are identical to those in Figure 2 except
for the addition of 5 mM Mg2+.
complexation. Previous studies (24,25) demonstrated that
inner sphere complexation of Mg2+ ions is associated with a
characteristic relaxation process in the microsecond time
range, which is not observed for Ca2+-ions. Thus, the hammerhead relaxation was monitored over a broad range of Mg2+- and
Ca2+-ion concentrations from 0.5 to 100 mM. A sample set of
relaxation curves obtained at 5 mM Mg2+ for the various
hammerhead species is shown in Figure 4. Addition of Mg2+
ions decreased the relaxation amplitudes and in most cases
increased the relaxation time constants. These changes reflect
the increased electrostatic shielding resulting from ion binding
to the ribozyme. Any additional and specific effect associated
with Mg2+-binding, indicating inner sphere binding, has not
been detected. However, detection would only be possible if
this inner sphere effect would be associated with an amplitude
at least as high as the amplitude of the conformational relaxation
occurring in the same time range. Because the conformational
relaxation extends over the whole time range from a few
microseconds to ∼200 ms, it is difficult to get unequivocal
evidence for any additional relaxation process in the same time
range unless the additional process is associated with a sufficiently
high amplitude.
Binding of metal ions to the hammerhead ribozyme HH1
was also analyzed by stopped flow experiments with fluorescence
detection. Under the conditions of these experiments the
fluorescence was affected by a photoreaction and, thus, the
stopped flow reaction curves obtained after mixing of
ribozyme with Mg2+ had to be corrected by subtraction of the
change due to the photoreaction (measured after equilibration
of ion binding). Addition of 10 mM Mg2+ to 1.6 µM ribozyme
in the standard buffer at 10°C showed the existence of reaction
effects with time constants ranging from a few milliseconds
over ∼40 s up to ∼20 min (data not shown). These data
demonstrate that the spectrum of reaction effects in hammerhead ribozymes extends over a wide time scale.
Figure 5. Difference relaxation curves obtained by subtraction of a relaxation
curve measured for HH1 from a corresponding curve measured for HH2.
(a) Buffer B; (b) buffer B + 5 mM Mg2+. Experimental conditions as described
in the legends to Figures 2 and 4.
The effects observed upon addition of Ca2+-ions to the
hammerhead ribozyme HH1 are very similar to those observed
upon addition of Mg2+, both in temperature jump and stopped
flow experiments (data not shown). Any clear difference in the
data obtained for Mg2+ and for Ca2+ could not be identified.
Thus, the Mg2+/Ca2+ comparison, which clearly indicated
Mg2+-inner sphere complexation in other cases (cf. below), did
not indicate inner sphere complexes in the case of hammerhead
ribozymes.
Difference relaxation curves illustrate topology of
relaxation response
The changes of the conformational relaxation observed upon
addition of Mg2+ ions provide some information on the nature
of the interactions in the hammerhead ribozyme. These changes
are more apparent in difference relaxation plots than in the
original data. An example is given for HH2 (Fig. 5) using the
data for HH1 as a reference: a difference is clearly visible in the
absence of Mg2+, but disappears at 5 mM Mg2+. The ribozyme
HH2 is distinguished from HH1 by a 2′-NH2-substitution of the
nucleotide at the 5′-side of the cleavage site. This substitution
leads to a small perturbation of the structure, which is reflected
in its relaxation in the absence of Mg2+, but is not visible any
more at 5 mM Mg2+. Because of electrostatic repulsion,
intramolecular interactions are expected to be more labile in
the absence of Mg2+. Addition of 5 mM Mg2+ reduces repulsion,
increases the stability of interactions and, thus, masks the
perturbation introduced by the 2′-NH2-group.
An effect in the opposite direction is found for HH4, where
again HH1 is used as a reference. These ribozymes differ in the
position of the reporter groups: in HH4 the 2-AP is at the
periphery in the stem III tetraloop, whereas in HH1 it is in the
core next to the cleavage site. The difference between these
two ribozymes is clearly visible in the absence of Mg2+ and
increases upon Mg2+ addition (Fig. 6). As mentioned above,
the relaxation amplitudes decrease upon Mg2+ addition in all
cases. However, the result shown in Figure 6 demonstrates that
this decrease is much stronger for HH1 than for HH4. Thus,
4432 Nucleic Acids Research, 2000, Vol. 28, No. 22
Figure 6. Difference relaxation curves obtained by subtraction of a relaxation
curve measured for HH1 from a corresponding curve measured for HH4.
(a) Buffer B; (b) buffer B + 5 mM Mg2+. Experimental conditions as described
in the legends to Figures 2 and 4.
Figure 7. Fluorescence detected relaxation curve for 2-AP-oligo(A) in buffer
B + 20 mM Mg2+ induced by a temperature jump from 2.0 to 10.6°C. The line
marked with circles is the tryptophan reference curve used for deconvolution;
least squares fitting provides a value of 95 µs for the chemical relaxation
effect.
Mg2+ addition stabilizes the interactions in the core more
strongly than those at the periphery.
Mg2+-inner sphere complex reported by 2-AP-fluorescence
in a substituted dodeca-riboadenylate
The relaxation effects obtained for hammerhead ribozymes in
the presence of Mg2+ ions and the absence of evidence for
formation of inner sphere complexes raises the question
whether the fluorescence of 2-AP can be used to detect inner
sphere complexes. Because oligoriboadenylic acids are known
to form Mg2+-inner sphere complexes (24,25), an oligoriboadenylic acid with 2-AP-substitution was analyzed by the same
approach used for the hammerhead ribozymes. Fluorescence
detected temperature jump relaxation using 2-AP-oligo(A)
revealed a clear relaxation effect with a time constant of ~100 µs
in the presence of mM-concentrations of Mg2+ (Fig. 7), which
was not observed in the presence of Ca2+ under the same
conditions (data not shown). This result demonstrates
formation of Mg2+-inner sphere complexes and shows that the
fluorescence of 2-AP can be used to detect these complexes.
Measurements of the relaxation effect at different Mg2+
concentrations show a dependence of time constants and
amplitudes (Fig. 8), which is consistent with the following
mechanism
k 12
1
Mg2+ + oligo(A) ⇔ OSC
k 21
k 23
OSC ⇔ ISC
k 32
2
In a first reaction step Mg2+ combines with oligo(A) to an outer
sphere complex OSC, which is converted in a second, relatively
slow reaction to an inner sphere complex ISC. Because the first
reaction step is known to proceed without activation barriers as
a diffusion controlled reaction, we use k12 = 1 × 1010 M–1 s–1.
The overall binding constant
Figure 8. Reciprocal relaxation time constants 1/τ (filled circles) and relative
fluorescence amplitudes ∆F/F (open circles) obtained by temperature jump
measurements for 2-AP-oligo(A) at different Mg2+ concentrations in buffer B at
10.6°C. The dotted and the continuous lines represent a combined fit of these
data according the reaction model specified in equations 1 and 2. The parameters
are: k12 = 1 × 1010 M–1s–1; k21 = 6.3 × 107 s–1; k23 = 6.6 × 103 s–1; k32 = 5.5 × 103 s–1;
α = 0.01; β = 2.38; ∆ln K1 = 0; ∆ln K2 = –0.0222.
K = K1(1 + K2) = (k12/k21)/[1 + (k23/k32)]
3
was determined by fluorescence titrations. The values obtained
for Mg2+ and Ca2+ (350 and 320 M–1, respectively) are identical
within the limits of accuracy (±100 M–1). Furthermore, the
relative change of the fluorescence observed in titration experiments ∆ [≡ (fluorescence intensity in the limit of saturation
with Me2+)/(fluorescence intensity before addition of Me2+)] is
related to the individual quantum yields by
∆ = [1/(K2 +1)]α + [K2/(K2 +1)]β
4
where α and β are the quantum yields of OSC and ISC relative
to that of the free oligonucleotide, respectively. Again the ∆-values
Nucleic Acids Research, 2000, Vol. 28, No. 22 4433
obtained for Mg2+ and Ca2+ (1.30 and 1.26, respectively) are
identical within the limits of accuracy (±0.02).
Fitting of the data in Figure 8 provided the parameters k23 =
6.6 × 103 s–1 and k32 = 5.5 × 103 s–1. Thus, the inner sphere
binding constant K2 = k23/k32 is 1.2 and the outer sphere binding
constant K1 is 160 M–1. Due to mutual coupling of the enthalpy
changes and the fluorescence quantum yields, these parameters
cannot be determined as individual independent values. The
combination of these parameters, which has been used to
describe the experimental data in Figure 8, is presented in the
legend to this figure.
DISCUSSION
The fluorescence of 2-AP provides a unique approach to the
analysis of reaction states and their dynamics in nucleic acids
at specific sites. 2-AP may be introduced at various positions
within nucleic acid sequences without any serious perturbation
of the structure and its fluorescence can be used to report on
any process occurring in the environment. It has been shown
previously that 2-AP can be introduced into hammerhead
ribozymes without loss of catalytic function (14).
In the present investigation 2-AP was used to analyze the
dynamics and topology of hammerhead ribozymes. Previously
the fluorescence of 2-AP has been demonstrated (23) to indicate
changes of base stacking interactions in its environment in an
oligoriboadenylate with a time constant of <1 µs. Thus, the
relaxation effects described here with time constants in the
range from a few microseconds to 200 ms must be attributed to
internal conformational relaxation of the hammerhead
ribozyme. Most of our constructs show at least three relaxation
processes which according to theory (26,27) demonstrate the
existence of at least four conformational states of the ribozyme. In
some cases even more relaxation effects have been observed.
Because the relaxation effects are closely spaced on the time scale,
it has not been possible to obtain satisfactory evidence for the
existence of discrete processes and, thus, a continuous spectrum of
relaxation effects reflecting a corresponding spectrum of
conformational states cannot be excluded.
Although a structural assignment of individual relaxation
effects is not possible, the experimental data clearly demonstrate the existence of local modes of conformational relaxation.
Hammerhead ribozymes are relatively small RNA molecules
and, thus, a general global mode of relaxation may have been
expected. However, the experiments show particularly large
amplitudes of slow relaxation processes in the catalytic core,
whereas fast processes are preferentially reflected from the
loop region at the periphery.
This investigation complements the earlier study (14) where
fluorescence changes of the same 2-AP-containing hammerhead ribozymes were monitored as a function of Mg2+ concentration. The data indicated the presence of multiple binding
sites with varying degrees of affinity towards this metal ion.
The present results indicate at least three conformational
changes at a constant Mg2+ concentration on the pre-steady
state time scale. Thus a picture emerges of a rather flexible
molecule which undergoes numerous conformational changes
to reach its steady state structure. At present it is not possible to
assign these changes to a particular structural change. The
ribozyme constructs described here are not cleaved and thus
additional changes are expected for the cleavage reaction to
occur. This is evident from the X-ray structures in the ground state
which all require at least one additional local conformational
change to reach the transition state to be compatible with an in-line
cleavage step (5–7).
RNA folding has been the subject of considerable interest
(28). In particular, the folding of the domains of the Tetrahymena
ribozyme, which is one of the large ribozymes, has also been
studied by pre-steady state kinetics. One study employed timeresolved hydroxyl radical footprinting to follow the folding of
the entire molecule (29). Another looked at the folding of the
P4–P6 subdomains by stopped flow changes of covalently
attached pyrene (30). There is a hierarchy of folding events
which range from rates of 2 to 0.02 s–1. Thus, the rates in this
system are considerably slower than those detected by
temperature jump relaxation for the much smaller hammerhead
ribozyme. However, except for the fastest folding of the P5a–P5c
domain the processes monitored for the Tetrahymena
ribozyme are associated with the formation of domain–domain
interactions. The resolution of the methods used for the
Tetrahymena ribozyme would not have detected the fast
internal changes found for the hammerhead ribozyme.
In addition to the time range and topology of conformational
relaxation, temperature measurements have been used in the
present investigation to analyze the type of RNA–Mg2+ interactions. On the basis of phosphorothioate interference studies
it is generally accepted that one Mg2+ should be coordinated to
the pro-Rp phosphate oxygen at the cleavage site (1,31).
Although this has not been shown by X-ray analysis of the
ground state structures, a Mg2+ in the vicinity of this position
could be identified in the X-ray structure approaching the
transition state (6). Using the Rp-diastereomer of a phosphorothioate group at the cleavage site, an inner sphere complex
with Hg2+ has been observed by UV–Vis spectroscopy (32).
There are a number of other metal ion binding sites detected by
uranyl-induced photocleavage (10), NMR spectroscopy (33),
X-ray structural analysis (5) and phosphorothioate interference
(22,34–36). Molecular dynamics simulations have provided
evidence for two Mg2+ ions near the active site (37). A site at
G5 previously identified as inhibitory in the presence of
Tb(III), by displacing an essential Mg2+, has been further
characterized by luminescence spectroscopy (38). These
experiments indicate that Tb(III) at this position makes two to
three inner sphere contacts with the RNA.
Thus, there is evidence for the formation of inner sphere
complexation with metal ions other than Mg2+. It would of
course be desirable to demonstrate such type of complexation
also with this metal ion. Fluorescence detected temperature
jump relaxation measurements are suitable to identify the
presence of Mg2+-inner sphere complexation on the basis of the
characteristic rate constant for inner sphere substitution
(25,39,40). This approach has been successful in the analysis
of a Mg2+ binding site in the anticodon loop (41) of tRNAPhe
and of Mg2+-inner sphere complexes with oligoriboadenylates
(24,25). The example described here for the interaction of 2-APoligo(A) with Mg2+ as a model system demonstrates that
temperature jump relaxation detected by 2-AP fluorescence
can distinguish between inner and outer sphere complexation.
However, the relaxation spectra of the hammerhead ribozymes
were too broad to identify the presence of Mg2+-inner sphere
complexation. This does not exclude the existence of such
4434 Nucleic Acids Research, 2000, Vol. 28, No. 22
complexes, because their characteristic signal may be hidden
in the broad range of conformational relaxation processes.
In summary, application of the fluorescence temperature
jump technique to hammerhead ribozymes containing 2-AP
demonstrates the existence of many different conformational
states. The time range of relaxation processes extends from a
few microseconds up to 0.2 s. Evidence for a further extension
of this time range up to 20 min is provided by stopped flow
experiments. Thus, the time scale of conformation changes in
hammerhead ribozymes is more extensive than expected. The
present investigation also provides clear evidence for the
existence of local relaxation modes. This approach should also
be useful for the future assignment of relaxation effects to
given structure changes.
ACKNOWLEDGEMENTS
We thank U. Kutzke for her expert preparation of RNA samples.
The facilities of the Gesellschaft für wissenschaftliche Datenverarbeitung mbH, Göttingen, were used for data processing. This
work was financially supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
REFERENCES
1. Birikh,K.R., Heaton,P.A. and Eckstein,F. (1997) The structure, function
and application of the hammerhead ribozyme. Eur. J. Biochem., 245, 1–16.
2. Lilley,D.M.J. (1999) Structure, folding and catalysis of the small
nucleolytic ribozymes. Curr. Opin. Struct. Biol., 9, 330–338.
3. Carola,C. and Eckstein,F. (1999) Nucleic acid enzymes. Curr. Opin.
Chem. Biol., 3, 274–283.
4. Murray,J.B., Seyhan,A.A., Walter,N.G., Burke,J.M. and Scott,W.G.
(1998) The hammerhead, hairpin and VS ribozymes are catalytically
proficient in monovalent cations alone. Chem. Biol., 5, 587–595.
5. Wedekind,J.E. and McKay,D.B. (1998) Crystallographic structures of the
hammerhead ribozyme – relationship to ribozyme folding and catalysis.
Ann. Rev. Biophys. Biomol. Struct., 27, 475–502.
6. Murray,J.B., Terwey,D.P., Maloney,L., Karpeisky,A., Usman,N.,
Beigelman,L. and Scott,W.G. (1998) The structural basis of hammerhead
ribozyme self-cleavage. Cell, 92, 665–673.
7. Scott,W.G. (1998) RNA catalysis. Curr. Opin. Struct. Biol., 8, 720–726.
8. Tuschl,T., Gohlke,C., Jovin,T.M., Westhof,E. and Eckstein,F. (1994)
A three-dimensional model for the hammerhead ribozyme based on
fluorescence measurements. Science, 266, 785–789.
9. Bassi,G.S., Murchie,A.I., Walter,F., Clegg,R.M. and Lilley,D.M.J. (1997)
Ion-induced folding of the hammerhead ribozyme – a fluorescence
resonance energy transfer study. EMBO J., 16, 7481–7489.
10. Bassi,G.S., Mollegaard,N.E., Murchie,A.I.H. and Lilley,D.M.J. (1999)
RNA folding and misfolding of the hammerhead ribozyme. Biochemistry,
38, 3345–3354.
11. Stage-Zimmermann,T.K. and Uhlenbeck,O.C. (1998) Hammerhead
ribozyme kinetics. RNA, 4, 875–889.
12. Porschke,D. and Eigen,M. (1971) Cooperative non-enzymic base
recognition III. Kinetics of the helix-coil transition of the
oligoribouridylic·oligoriboadenylic acid system and of oligoriboadenylic
acid alone at acid pH. J. Mol. Biol., 62, 361–381.
13. Craig,M.E., Crothers,D.M. and Doty,P. (1971) Relaxation kinetics of
dimer formation by self complementary oligonucleotides. J. Mol. Biol.,
62, 383–401.
14. Menger,M., Tuschl,T., Eckstein,F. and Porschke,D. (1996) Mg2+dependent conformational changes in the hammerhead ribozyme.
Biochemistry, 35, 14710–14716.
15. Tuschl,T., Ng,M.M., Pieken,W., Benseler,F. and Eckstein,F. (1993)
Importance of exocyclic base functional groups of central core guanosines
for hammerhead ribozyme activity. Biochemistry, 32, 11658–11668.
16. Rigler,R., Rabl,C.R. and Jovin,T.M. (1974) A temperature-jump apparatus
for fluorescence measurements. Rev. Sci. Instrum., 45, 580–588.
17. Porschke,D. and Jung,M. (1985) The conformation of single-stranded
oligonucleotides and of oligonucleotide-oligopeptide complexes from
their rotation relaxation in the nanosecond time range.
J. Biomol. Struct. Dyn., 2, 1173–1184.
18. Diekmann,S., Hillen,W., Morgeneyer,B., Wells,R.D. and Porschke,D.
(1982) Orientation relaxation of DNA restriction fragments and the
internal mobility of the double helix. Biophys. Chem., 15, 263–270.
19. Porschke,D. (1998) Time resolved analysis of macromolecular structures
during reactions by stopped-flow electrooptics. Biophys. J., 75, 528–537.
20. Hertel,K.J., Pardi,A., Uhlenbeck,O.C., Koizumi,M., Ohtsuka,E.,
Uesugi,S., Cedergren,R., Eckstein,F., Gerlach,W.L., Hodgson,R. and
Symons,R.H. (1992) Numbering system for the hammerhead.
Nucleic Acids Res., 20, 3252.
21. Pley,H.W., Flaherty,K.M. and McKay,D.B. (1994) Three-dimensional
structure of a hammerhead ribozyme. Nature, 372, 68–74.
22. Ruffner,D.E. and Uhlenbeck,O.C. (1990) Thiophosphate interference
experiments locate phosphates important for the hammerhead RNA selfcleavage reaction. Nucleic Acids Res., 18, 6025–6029.
23. Menger,M., Eckstein,F. and Porschke,D. (2000) Dynamics of the RNA
hairpin GNRA tetraloop. Biochemistry, 39, 4500–4507.
24. Porschke,D. (1979) The mode of Mg++ binding to oligonucleotides. Inner sphere
complexes as markers for recognition? Nucleic Acids Res., 6, 883–898.
25. Porschke,D. (1995) Modes and dynamics of Mg2+-polynucleotide
interactions. In Cowan,J.A. (ed.), The Biological Chemistry of
Magnesium. VCH Publishers, New York, NY, pp. 85–110.
26. Eigen,M. and De Maeyer,L. (1963) Relaxation methods.
In Weissberger,A. (ed.), Technique of Organic Chemistry. Wiley,
New York, NY, pp. 895–1054.
27. Bernasconi,C.F. (1976) Relaxation Kinetics. Academic Press, New York, NY.
28. Treiber,D.K. and Williamson,J.R. (1999) Exposing the kinetic traps in
RNA folding. Curr. Opin. Struct. Biol., 9, 339–345.
29. Sclavi,B., Sullivan,M., Chance,M.R., Brenowitz,M. and Woodson,S.A.
(1998) RNA folding at millisecond intervals by synchrotron hydroxyl
radical footprinting. Science, 279, 1940–1943.
30. Silverman,S.K. and Cech,T.R. (1999) RNA tertiary folding monitored by
fluorescence of covalently attached pyrene. Biochemistry, 38, 14224–14237.
31. Scott,E.C. and Uhlenbeck,O.C. (1999) A re-investigation of the thio effect
at the hammerhead cleavage site. Nucleic Acids Res., 27, 479–484.
32. Cunningham,L.A., Li,J. and Lu,Y. (1998) Spectroscopic evidence for
inner-sphere coordination of metal ions to the active site of a hammerhead
ribozyme. J. Am. Chem. Soc., 120, 4518–4519.
33. Hansen,M.R., Simorre,J.P., Hanson,P., Mokler,V., Bellon,L., Beigelman,L.
and Pardi,A. (1999) Identification and characterization of a novel high affinity
metal-binding site in the hammerhead ribozyme. RNA, 5, 1099–1104.
34. Knoll,R., Bald,R. and Fürste,J.P. (1997) Complete identification of nonbridging
phosphate oxygens involved in hammerhead cleavage. RNA, 3, 132–140.
35. Wang,S.L., Karbstein,K., Peracchi,A., Beigelman,L. and Herschlag,D.
(1999) Identification of the hammerhead ribozyme metal ion binding site
responsible for rescue of the deleterious effect of the cleavage site
phosphorothioate. Biochemistry, 38, 14363–14378.
36. Murray,J.B. and Scott,W.G. (2000) Does a single metal ion bridge the A-9
and scissile phosphate groups in the catalytically active hammerhead
ribozyme structure? J. Mol. Biol., 296, 33–41.
37. Hermann,T., Auffinger,P., Scott,W.G. and Westhof,E. (1997) Evidence
for a hydroxide ion bridging two magnesium ions at the active site of the
hammerhead ribozyme. Nucleic Acids Res., 25, 3421–3427.
38. Feig,A.L., Panek,M., Horrocks,W.D. and Uhlenbeck,O.C. (1999) Probing
the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using
luminescence spectroscopy. Chem. Biol., 6, 801–810.
39. Diebler,H., Eigen,M. and Hammes,G.G. (1960) Relaxations-spektrometrische
Untersuchungen schneller Reaktionen von ATP in wässriger Lösung.
Z. Naturf., 15b, 554–560.
40. Diebler,H., Eigen,M., Ilgenfritz,G., Maass,G. and Winkler,R. (1969)
Kinetics and mechanism of reactions of main group metal ions with
biological carriers. Pure Appl. Chem., 20, 93–115.
41. Labuda,D. and Porschke,D. (1982) Magnesium ion inner sphere complex
in the anticodon loop of phenylalanine transfer ribonucleic acid.
Biochemistry, 21, 49–53.