From the Groupe de Resonance Magnétique
Nucléaire, Laboratoire Département de Chimie,
Synthèse Organique, Ecole Polytechnique, 91128 Palaiseau,
France and § Institut Jaques Monod, 4 place Jussieu,
75005 Paris, France
Received for publication, November 27, 2000
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ABSTRACT |
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The T4 endoribonuclease RegB is involved in the
inactivation of the phage early messengers. It cuts specifically in the
middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in coding sequences or in late messengers. In vitro RegB activity is very low but is
enhanced by a factor up to 100 by the ribosomal protein S1. In the
absence of clear sequence motif distinguishing substrate and
non-substrate GGAG-containing RNAs, we postulated the existence of a
structural determinant. To test this hypothesis, we correlated the
structure, probed by NMR spectroscopy, with the cleavage propensity of
short RNA molecules derived from an artificial substrate. A kinetic analysis of the cleavage was performed in the presence and absence of
S1. In the absence of S1, RegB efficiently hydrolyses substrates in
which the last G of the GGAG motif is located in a short stem between
two loops. Both strengthening and weakening of this structure strongly
decrease the cleavage rate, indicating that this structure constitutes
a positive cleavage determinant. Based on our results and those of
others, we speculate that S1 favors the formation of the structure
recognized by RegB and can thus be considered a "presentation protein."
The control of the pattern of gene expression is one of the main
means by which a prokaryotic or eukaryotic cell reacts to a variation
of its environment. The expression of a gene depends strongly on its
messenger concentration, which in turn is determined by the
transcription efficiency, but also by the mRNA decay rate. Messengers, indeed, may have very different degradation
susceptibilities. The half-lives of Escherichia coli
mRNAs, for example, range between 0.5 and 20 min and may change in
response to various factors (1). This could be very important for
short-lived messengers, in particular, since small variations of their
half-lives will temporarily induce very strong modifications in their
abundance (2).
The factors controlling mRNA half-lives are far from being
completely understood and are certainly different in prokaryotic and
eukaryotic organisms. In the former, in particular, the coupling of
transcription and translation events together with the absence of 5'
In this context, the S1-RegB system appears to constitute a very
interesting model. RegB, encoded in the bacteriophage T4 genome, is
involved in the specific degradation of phage early messengers,
including its own. Indeed, after infection by a
regB In this study, we have undertaken the identification of the molecular
basis of RegB selectivity and the definition of the nature of the role
of S1. In the absence of any clear sequence motif outside the GGAG, we
speculate that RegB activity might be dependent on an RNA
structural determinant. To test this hypothesis, we tried to correlate
the structure of a series of RNA molecules, probed by NMR spectroscopy,
with their cleavage susceptibility in the presence and absence of S1.
Several simple substrates of RegB were obtained by the SELEX method
(11). We have chosen one of them, possessing two GGAG sites, one
reported to be cleaved and the other not. From a comparison of the
properties of these two sites in several variants of this molecule, we
propose a model of the interactions between the RNA, S1, and RegB.
RegB and S1 Production--
The S1 protein was purified from an
E. coli MRE600 strain by affinity chromatography on a
poly(U)-Sepharose 4B (Amersham Pharmacia Biotech) column as described
(12). It was stored at 4 °C in 10 mmol.liter
The RegB gene has been introduced in a pET-7b vector (6). Its toxicity
prevents the transformation of any E. coli strain possessing
the T7 polymerase gene. A JM101 strain was therefore used, and RegB
production was induced by infecting the culture (at around 0.7 A650) with a RNA Production--
DNA oligonucleotide templates were purchased
from Eurogentec. Phenoxyacetyl
Most RNAs were produced using in vitro transcription
according to the method developed by Uhlenbeck and co-workers (14). In
addition, three (the S22cug, S22stem, and S22linear molecules, see
under "Results") were produced by chemical synthesis
(15).
Transcription--
Transcription reactions were performed in
40 mmol.liter RNA Purification--
To separate the full-length RNA molecule
from the shorter species, the chemically synthesized fragments were
purified using a Q-Sepharose high performance liquid chromatography
column running on a Beckman system. Before RNA loading, the column was
equilibrated with 80% buffer A and 20% buffer B (A: 5 mmol.liter
For of the transcription product, the RNA molecules could be separated
from the DNA matrices, abortive fragments, and free nucleotides using a
similar protocol. However, with this technique, it was impossible to
separate the correct RNA fragment from the high molecular weight
species. According to the interpretation above, the two products would
have complementary sequences and would thus be able to form very stable
double helices. Their separation could only be achieved by
polyacrylamide (18%) gel electrophoresis using denaturing conditions
(8 mol.liter RNA Cleavage Reactions--
Cleavage reactions were performed in
50 mmol.liter
The kinetics were generally followed for 2 h. Aliquots (5 µl)
were typically taken at 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, and
120 min. They were mixed with 3 µl of loading blue (8 mol.liter
Most reactions were repeated two or three times. In the case of the
S22linear fragment, the results were so surprising (see under
"Results") that we repeated the whole process (synthesis, purification, and kinetic study twice).
Kinetic Analysis--
The percentages of residual substrate and
product formed at a given time were evaluated from the ratio of the
corresponding band to the sum of all band intensities. Simple
phenomenological models were considered to represent the reactions: S
NMR Spectroscopy--
All experiments were recorded on a Bruker
DRX600 spectrometer equipped with a gradient TXI probe. The spectra
were processed off line using the GIFA software (17), and the data were
analyzed using either GIFC (18) or XEASY (19) programs.
H2O samples were prepared by dissolving the RNA in 400 µl
of 90% H2O, 10% D2O. The pH was adjusted to
about 6.5 by adding a small amount of NaOH. NaN3 (0.05%)
was added to prevent bacterial growth. D2O samples were
obtained by lyophilizing the corresponding H2O samples and
dissolving them in D2O. Two further lyophilizations were
generally performed to remove as much H2O as possible.
Exchangeable proton assignments were obtained from the analysis of
one-dimensional and two-dimensional
NOE1 experiments recorded in
H2O. Typically, acquisition parameters were 2048 data
points over a 12-kHz spectral range, 64-128 scans, and 2-s relaxation
time. Generally, 512 increments were recorded in the two-dimensional
experiments. The water was always suppressed using a jump and return
reading pulse (20). The carrier was positioned at the water frequency,
and the delay between the two pulses was adjusted so that the
excitation maximum covered the region of the imino protons.
TOCSY (21), DQF-COSY ((22), and NOE spectroscopy (23) experiments were
also recorded in D2O. A MLEV17 sequence with a spin-lock
time of 60 ms was used for the TOCSY. NOE spectroscopy was realized
with mixing times between 70 and 500 ms. Acquisition parameters were
similar to those used in H2O with the exception of the
spectral width, reduced to 4.8 kHz. The residual HDO signal was
presaturated during the relaxation delay.
Choice of the Molecules--
Considering the lack of any clear
sequence motif distinguishing cleaved and uncleaved GGAG-containing
oligonucleotides, we hypothesized that RegB might recognize a
structural determinant. To test this, we decided to compare the
three-dimensional structure of good and poor substrates. One
possibility would have been to study two natural sequences, such as
motA (cleaved) and denV (uncleaved). However,
this presented two major difficulties. First, we have no information
about the relative position and distance between the GGAG and the
putative signal. Thus, it would have been difficult to define the
limits of the fragment to study. Second, the physiological role of the
sequence might require the presence of structural features on its own.
Differences between the cleaved and uncleaved sequences would thus have
been difficult to interpret.
We therefore took advantage of a published study carried out in the aim
of selecting good RegB substrates by the SELEX method (11). Two of the
reported molecules (the 22nd and 40th) looked particularly attractive
since both possess two GGAG sites, one cleaved, the other not. Using a
secondary structure predicted by mfold software (24), a
short fragment (hereafter designed S22cug) compatible with NMR
structural analysis and containing both sites, was isolated from the
22nd molecule. In addition, during the course of the study, we were led
to design several variants (Fig. 1). The
ends of the molecule were modified to obtain a better transcription
yield (S22gg). The influence of the structural environment on the
cleavage rate of the first site was studied with the series S26so (site
one, in which the second site has been eliminated and the terminal loop
shortened), S22turn (in which the terminal loop has been replaced by a
GNRA turn), and S22stem (in which the internal loop has also been
suppressed). Similarly, the second site alone was conserved in a
simplified molecule S22two, placed in the middle of the terminal loop
(S22twoloop) or in a linear fragment (S22linear).
Structural Analysis--
The RNA structures of five of these
molecules, the S22linear, S22cug, S22gg, S26so, and S22stem, (Fig. 1)
were probed by NMR spectroscopy. The secondary structure predictions
were not sufficient for our purpose; in particular, they are unable to
give information concerning possible structures adopted by the loop regions.
The resonances of the imino protons (found between 9.5 and 14 ppm in
spectra recorded in H2O) constitute a good probe of the oligonucleotide secondary structure. Their observation indicates, indeed, that the corresponding protons are involved in hydrogen bonds,
generally due to the formation of base pairs. In case of S22linear, a
resonance at 12 ppm is observed at 5 °C showing that at least one
base pair is formed at this temperature (Fig.
2). However, at 25 °C the spectrum is
exempt of sharp peaks, and the only remaining signal present between
10.5 and 11 ppm is characteristic of unpaired imino protons,
demonstrating the absence of structure in the conditions of the
cleavage reaction (37 °C). In all other cases, resonances are
observed in one-dimensional and two-dimensional spectra at both low and
high temperature. In the latter case, the lines are broader due to the
acceleration of the proton exchange rate. However, as shown in Fig. 2,
even if some become very weak, the number of peaks remains constant
throughout the explored temperature range. This is in agreement with
the determination of fusion temperatures, which are always above
37 °C (from 45 °C for the S22cug to 85 °C for the S22stem),
and indicates that the secondary structure observed at low temperature
is probably conserved in the condition of the enzymatic study.
The nucleotides involved in the secondary structure can be identified
by means of one-dimensional and two-dimensional NOE spectroscopy
experiments recorded in H2O. It is, in particular, easy to
discriminate between the GC and AU base pairs, the latter being
characterized by the presence of a strong NOE correlation between the
U-imino resonance and the very narrow peak (around 7 ppm) of the
A-H2 proton. In all cases analyzed, the observed number of
GC and AU base pairs was in agreement with the predicted structures.
The same experiments were also used when possible to assign the
resonances by analysis of sequential connectivity. The S22stem molecule
is predicted to form a long helical stem disrupted at the A position of
the GGAG (Fig. 1). The two strands of the helix are joined
by a short GUAA loop, believed to form a GNRA turn. The NOE pattern
allows the identification of a segment that matches the predicted stem.
In addition, one peak possesses a chemical shift (10 ppm) indicative of
a hydrogen bond with an oxygen atom. Such a hydrogen bond is expected
in the GNRA turn between the U-imino proton and an oxygen of the
backbone. In case of S22cug and S22gg, a stem-loop-stem-loop
organization is expected. The first stem has a predicted length of 5 (S22cug) or 6 (S22gg) residues, whereas the second is shorter (2 residues). The various spectra of the two molecules are nearly
identical, indicating similar structure. Five (of seven) of the S22cug
imino resonances were assigned. Their chaining matches that of the
first predicted stem. The two remaining peaks present the
characteristic features of GC base pairs but are so broad that it was
impossible to observe any sequential connectivity. They probably
correspond to the second short stem, its size and location between the
two loops resulting in low stability and consequently in the observed
peak broadening. The predicted structure of S26so is similar to that of
S22cug, as are the results. Seven imino resonances (six C, one U) are observed. Their assignment was complicated by the small dispersion of
the lines; however, by combining the results of different experiments performed at different temperatures, a chaining was identified corresponding to the first stem. The two remaining lines are very broad
and present no sequential connectivity. As in the case of S22cug, we
assumed that they correspond to the small GC/CG stem between the
internal and terminal loops.
The study of the S22cug molecule was carried further by looking at the
nonexchangeable protons (Fig. 3). The
analysis of the aromatic-H1' correlations on the one hand
and of the imino- and amino-aromatic correlations on the other hand
confirmed the previously obtained results but could not provide any
further assignment. In fact, a close examination of the spectra
recorded in D2O reveals that all nontrivial signals involve
the helix residues. This strongly suggests that the two loop regions
are not structured. Interestingly, we also observed that the line width
of the cytidine and uridine H5-H6 scalar
correlations (as determined in a TOCSY experiment) varies with respect
to the location of the residue (Fig. 4).
These protons form a rigid system. Thus, all variations of their line
shape reflect a variation of the residue dynamics (25). The five
correlations corresponding to the residues of the first helix were
assigned. As shown in Fig. 4, their line width increases regularly from the free end of the helix (C1) to the other end (C26). The four other
correlations are located in the short helix (C8 and C21) and in the
terminal and internal loops (C16 and C23, respectively). These peaks
could not be assigned. However, two behave like the C26. The others are
very broad, and their observation depends strongly on the temperature
and on the spectrometer field (data not shown). The narrow lines
correspond to residues in a single environment or, more likely here, to
residues with a fast exchange rate (with respect to the chemical shift
difference) between two conformations or environments. On the contrary,
the widening or disappearance of the peaks reflects a slower
(fast-intermediate or intermediate) exchange rate. The behavior of the
various peaks thus confirms that the loop regions are in equilibrium
between several conformations. The peak widening from the free end of the helix to the other end in particular could be interpreted as the
result of an increasing perturbation of the corresponding residue
environment due to the loops conformational exchange.
Kinetic Analysis of Cleavage--
A kinetic analysis of the
cleavage reaction has been carried out on all substrates in the
presence and absence of S1. To be able to compare the cleavage rates,
all experiments were done rigorously in the same conditions.
As an illustration of the quality of the data, the gel autoradiographs
of the experiments with the S22, S26so, S22turn, and S22linear
molecules are presented in Fig. 5. The
different bands are well defined, and their sizes can be unambiguously
determined by comparison with the ladder. In a few cases, a smear was
observed in the middle region of the gel. Generally, it was very slight and did not interfere with the analysis. However, in case of the S22twoloop, the smear prevented the quantification of the bands. The
control lanes always showed a unique spot corresponding to the
full-length, indicating that there is neither degradation nor cleavage
in absence of RegB.
S22cug (Fig. 5A) and S22gg correspond to a fragment of the
initial SELEX molecule (11). They possess two GGAG sequences, but the
first was thought to be resistant. Given that the molecules are labeled
in 5', two bands (corresponding to the full-length and one product) are
thus expected. In fact, three are observed whose lengths are in
agreement with cutting at both sites. The unexpected cleavage at the
first site was studied with the S26so (Fig. 5B), S22turn
(Fig. 5C), and S22stem molecules, which differ by the number
of loops. The S26so (having an internal and a terminal loop) and
S22turn (in which the terminal loop is replaced by a GNRA tetra loop)
are cleaved as expected, whereas the S22stem (further lacking the
internal loop) appears to be resistant, even in the presence of S1.
Similarly, we tried to analyze the susceptibility of the second site
with the S22two, S22twoloop and S22linear series. This led to the most
surprising results. S22two, in which the region comprising the
first site and the internal loop has been removed, is cleaved by RegB
alone but not in the presence of S1. When the site is displaced from
the edge to the middle of the loop in the S22twoloop, it becomes
fully resistant to cleavage. Finally, the S22linear (Fig.
5D), which corresponds to the loop region of the initial
molecule, gives rise to the very low appearance of two products.
Strikingly, neither of them has the expected length (8 and 9 nucleotides instead of 10). It seems that the cleavages occur
immediately upstream and between the two Gs of the GGAG
sequence. All these experiments were repeated two or three times to
eliminate any doubt. In the case of S22linear, in particular, the whole
process (RNA synthesis and purification, cleavage reaction, and
analysis) was done twice.
To analyze the reaction kinetics, we chose to use simple
phenomenological models compatible with the amount of data we disposed. We only considered the forward (hydrolysis) reaction because the backward (synthesis) reaction is very unlikely in the presence of
water. We also neglected product inhibition. As shown later, this
hypothesis is not always justified, although it has no impact on
the initial rate determination. For the S26so, S22turn, and S22two
molecules, a simple S
All kinetic rate constants are reported in Fig.
7. They have been grouped in two series
corresponding to the analysis of the two sites. Strikingly, we observe
that the second site (in the terminal loop region) is cleaved much more
efficiently than the first (at the junction of the two helices) when we
look at the two molecules S22cug and S22gg. In the absence of S1, the
rate constant ratio is around 15. It is lower in its presence (about 4)
but remains significant. The first site is cleaved with the same
efficiency in the S22cug, S22gg and S26so molecules, suggesting that
the cleavage of the second site in S22cug and S22gg has only a weak
influence on the properties of the first. The replacement of the
terminal loop by a GNRA tetra-loop induces a drastic reduction in the
rates (25-fold in the absence of S1, 7-fold in its presence). The
cleavage seems completely abolished by the further elimination of the
internal loop. All this suggests at first sight a strong correlation
between the stability of the RNA at or near the GGAG site and the
cleavage efficiency. However, the results are more puzzling when
considering the second site. The simultaneous elimination of the first
site and the internal loop in the S22two molecule induces a reduction
in the cleavage rate (by a factor of 4) in the absence of S1 but, more
surprisingly, abolishes cleavage in the presence of S1. Furthermore,
the cleavage efficiency becomes null or very low when the GGAG site is
in the middle of the loop (S22twoloop) or when the fragment is
linearized (S22linear). In this latter case, in addition, the enzyme
specificity is lost, as mentioned above.
Since its discovery in 1988 (7), one of the most intriguing and
exciting question concerning the T4-encoded endoribonuclease RegB has
been the study of its specificity. First, it has been established that
the enzyme cut in the middle of a very precise sequence (GGAG). In
addition, it has been shown that not all GGAG sequences are cleaved
in vivo. The enzyme efficiently processes most of the
GGAGs found in the intergenic (in particular Shine-Dalgarno) regions of
the early T4 messengers (6, 7, 26). On the contrary, it has no activity
on most of those located in the Shine-Dalgarno regions of late
messengers or in coding sequences (6). By transforming an uninfected
cell with two plasmids, one coding for RegB, the other for a T4 natural
messenger, it was demonstrated that the pattern of RegB sensitivity
observed in vivo can be reproduced in the absence of other
T4 factors (6). However, this does not imply that RegB alone is able to
distinguish between the two classes of GGAG sequences. It was, indeed,
also shown that RegB possesses by itself a very low activity that can
be accelerated by a factor up to 100 in the presence of the ribosomal
protein S1 (8). In this condition it is not clear which of the two proteins carries the specificity. On the one hand, since S1 is involved
in the recognition of the Shine-Dalgarno regions by the ribosome, it
has been proposed that it could act as a nonspecific mediator between
the RNA and RegB by increasing the enzyme concentration in the vicinity
its targets through protein-protein interactions (6, 11, 26). In this
way, S1 in this system would play a role similar to that of the K RNA
binding domain of the RNase E (27). On the other hand, in the absence
of evidence of any direct interaction between S1 and RegB, it is also
possible that S1 acts as a presentation protein and modulates the
efficiency of the system through specific interactions with the RNA, as
recently described in the case of the Hfq1 protein for example (5). This raises two questions. What are the respective contributions of
RegB and S1 in the modulation of cleavage efficiency, and what are the
signals recognized by RegB, by S1 or by both proteins on target (or
non-target) RNAs?
To address these questions, few results are available in the literature
concerning the enzymatic properties of the RegB-S1 system. The
efficiency of RegB in the presence or absence of S1 has been evaluated
in conditions similar to those used here (1 µmol.liter Compared with these data, our results raise a series of questions.
First, both GGAGs of the S22cug and S22gg molecules are cleaved in our
experiments, whereas only the second was found to be processed by the
authors of the SELEX study. However, it must be noticed that our work
uses neither the same molecules nor the same experimental conditions.
The original clone 22 is circular and longer than our fragments (75 residues instead of 32). The RNA/RegB and S1/RegB ratios are higher in
the SELEX procedure than in our experiments. More puzzling are the
differences in cleavage efficiency.
Looking at the effect of RegB alone (in the absence of S1), the results
obtained on the first site indicate a strong correlation between the
secondary structure of the molecule and cleavage susceptibility. It has
been suggested that the presence of stable secondary structure at or
near the scissile bond would constitute a major negative determinant of
RegB efficiency, which would be maximal on unstructured fragments (28).
At first sight, our results seem to support this idea. Indeed, the
differences between the S22cug, S22gg and S26so (which have similar
rate constants) are not thought to deeply perturb the stability of the
two stems flanking the first GGAG. In contrast, the replacement of the
terminal loop by a GNRA turn (in the S22turn) is likely to stabilize
the short stem, as most certainly will the suppression of the
internal loop in the S22stem molecule. This hypothesis would also
explain why the second site of the S22gg and S22cug molecules, located
in a flexible loop, is cleaved more efficiently than the first.
However, the same site placed in the center of the loop (S22twoloop) or
in a linear fragment (S22linear) is no longer processed. Moreover, the
comparison of our rate constants with those reported by Ruckman
et al. (8) indicates that the second site is cleaved in the
absence of S1 much more efficiently than "good substrates" in the
presence of S1. In these conditions, even if we cannot exclude the
possibility that the presence of stable secondary structure impedes
cleavage, our results demonstrate the existence of a positive
determinant favoring interactions between RegB and S22 molecules. The
particular properties of the S22linear fragment, which was designed to
conserve the sequence but not the structure surrounding the second
site, indicate that this structure contributes strongly to the
determinant. In addition, they also suggest that the determinant may
play a role not only in the efficiency but also in the specificity of the enzyme. The S22linear, indeed, is not only cleaved with low efficiency but also with wrong specificity, whereas all other molecules
including the linear JR decanucleotide are processed at the correct
position. A striking difference between the two linear fragments is
that ours (GAGAAACGGAGCACA) is rich in A and G, whereas the
JR (CUUUGGAGGG) has a single A and several CUs. It is thus
tempting to suppose that the enzyme has more difficulty recognizing the
correct site in the AG-rich context of the S22linear fragment but is
guided either by the sequence, in the JR fragment, or by the structure,
in all other S22-derived molecules. The precise nature of the
structural determinant is difficult to assess with the elements we
dispose of at this stage of our study, but several guesses can be made.
In particular, it will be noticed that the first site of S26so and both
sites of S22gg and S22cug are better cleaved than the linear fragments and share the same global structural organization. In both, the A of
the GGAG is free, the last G is involved in a short (two
residue) helix, and this helix is followed by a nonstructured loop
region. The main difference is that the two first Gs of the GGAG are also engaged in base pairs in the first site but belong to a loop in the second. The most important parameter seems to
be the presence of the short helix involving the last G. The cleavage
rate is, indeed, very weak or null in all molecules lacking this
element (JR, S22twoloop, S22linear). The elimination of the loop
reduces the rate by a factor 2 (second site in S22two versus S22cug) to 10 (first site in S22turn versus S22gg). Its
length and precise sequence seem to be of little importance, as
indicated by a comparison of the first site cleavage in S22gg or S22cug and S26so. Finally, the comparison of the two sites in the S22gg and
S22cug molecules suggests that the involvement of the first two Gs in
base pairs induces a diminution of the rate by a factor about 15 (13 for S22cug, 16 for S22gg). It thus seems that a good RegB substrate can
be formed by a short helix involving the last G of the GGAG
site flanked by two nonstructured regions. However, we cannot exclude
the possibility of other motifs.
The second aspect of our study concerns the role of the S1 protein. It
is more difficult to analyze because we have only indirect information
(the consequence of the presence of S1 on the RegB cleavage rate).
Nevertheless, several remarks seem pertinent. A first puzzling point is
the very low value of the rate enhancement observed at both sites of
the S22cug and S22gg molecules (about 4 and 1.1 for the first and
second site, respectively). However, in the presence of S1, even the
first site is cleaved as efficiently as the JR molecule. The low
enhancement value thus appears as a consequence of the intrinsic high
cleavage rate obtained in the presence of RegB alone. Also remarkable
is the fact that the GGAGs not cleaved in the absence of S1 (those of
the S22stem, S22twoloop, S22linear) are not processed in its presence
either. Interestingly, this is also the case for one GGAG of the long T7 transcript studied by Ruckman et al. (8). Finally,
although S1 seems unable to convert a nonsubstrate GGAG into a
substrate, it can on the contrary completely inhibit RegB activity (S22two).
Given our first conclusion, that RegB efficiently cleaves a GGAG when
the RNA molecule possesses a particular structure, it seems plausible
to propose that S1 acts by promoting its formation. According to this
hypothesis, a substrate will be a molecule able to adopt the correct
conformation (even if it is not the stablest), whereas a nonsubstrate
will be unable to reach it. Accordingly, the second site of the S22cug
and S22gg molecules is very well processed, and the rate is only
marginally enhanced because the molecules already possess a near ideal
conformation. On the contrary we may suppose that the S22stem,
S22twoloop and S22linear are no longer able to adopt it either because
they are engaged in very stable alternative structures (likely the case
of S22stem) or because the sequence is no longer suitable (likely the
case of S22linear).
In conclusion, our results indicate that the discrimination between a
substrate and a nonsubstrate molecule depends on two positive
determinants: the presence of GGAG and the possibility to adopt a
particular conformation, a short stem between two loops in the present
case. The efficiency of RegB alone is controlled by the structure of
the molecule and can be much higher than previously thought (the S22gg
second site is cleaved 400 times faster than the JR oligonucleotide).
S1 probably acts by promoting the appearance of the correct RNA
conformation and thus can be considered a presentation protein. In
addition, it is not excluded that it could participate in the
regulation of the system by discriminating between presentable and
nonpresentable substrates or by inhibiting the RegB activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3' exonucleases impose specific constraints on the process (3).
However, in all cases, mRNA stability seems under the control not
only of cis-acting but also of trans-acting factors (4). Indeed, the
importance of RNA-binding proteins in the control of mRNA decay
rates is becoming more and more evident. It has recently been shown,
for example, that the stability of the E. coli ompA mRNA
is modulated by its interactions with the host factor I (HfqI) protein,
whose concentration depends, in turn, on the growth rate of the
bacteria (5). Similarly, several families of proteins, such as the
poly(A) or AU-rich sequence binding proteins, have been implicated in
the control of eukaryotic messenger degradation (2).
strain, the half-life of the early
mRNAs is increased by a factor of three, whereas that of the middle
and late mRNAs remains unaffected. Several studies have established
that this effect is due to differences in the enzyme activity toward
the three classes of messengers and not to its inactivation during the
middle phase (6). More precisely, it has been shown that RegB
specifically cleaves between the G and A of GGAG sequences in the
Shine-Dalgarno regions of the early messengers. Surprisingly, a few
GGAG motifs carried by early and middle mRNA and all motifs carried
by late mRNA escape RegB processing, as also do most tested motifs
within coding sequences. This indicates that RegB recognizes not only
the GGAG sequence but also another signal, which remains unknown to
date. Another striking point is that the RegB activity determined
in vitro is very low in comparison with what can be inferred
from the in vivo observations. Considering the specificity
of the enzyme, it had been postulated that the ribosome might play a
role (7). This has been verified. In vitro it was
demonstrated that RegB activity can be accelerate by a factor of up to
100 (depending on the substrate) by the addition of the ribosomal
protein S1 to the reaction mix (8). S1 is the largest ribosomal protein
(556 amino acids). It is composed of six repetitions of a conserved
domain, called the S1 motif, known to be an RNA/DNA binding motif also
found in many prokaryotic and eukaryotic proteins involved in RNA
metabolism. Its presence is strictly required to initiate the
translation of most mRNAs (9), but its precise mechanism of action
remains unknown. In our system, it seemed reasonable to postulate an
interaction between S1 and the RNA, but this has not been demonstrated.
In addition, the nature of S1 action has not been elucidated. It was
possible to imagine a nonspecific role. S1, indeed, has been reported
to be an unwinding protein (10), but this model was not completely
satisfying. In particular, it did not explain the rate enhancement
observed on small unstructured RNAs (8). On the contrary, we could
imagine that S1 participates in enzyme specificity by selecting several targets.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 Tris-Cl (pH 7.4), 6 mmol.liter
1 magnesium acetate, 0.1 mmol.liter
1 EDTA, 1 mol.liter
1 NH4Cl, and 7.2 mmol.liter
1
mercaptoethanol.
CE6 phage (multiplicity
of 10) carrying the T7 polymerase gene (13). The protein was purified
on a nickel nitrilotriacetic acid (Qiagen) column. Strikingly, its
complete elution required a very high (500 mmol.liter
1) imidazol concentration. After
dialysis, the protein was stored at
20 °C in 20 mmol.liter
1 Tris-HCl (pH 8.0), 200 mmol.liter
1 NaCl, 0.2 mmol.liter
1 dithiothreitol, and 50% glycerol.
-RNA phosphoramidite were
bought from Amersham Pharmacia Biotech. RNA chemical synthesis was
performed on a Amersham Pharmacia Biotech Gene Assembler Plus apparatus.
1 Tris-HCl (pH 8.0), 1 mmol.liter
1 spermidine, 5 mmol.liter
1 dithiothreitol, 0.01% Triton
X-100, 80 mg.ml
1 40% polyethylene glycol,
4-16 mmol.liter
1 MgCl2, 2 mmol.liter
1 each nucleotide, 400 nmol.liter
1 template, and 0.01 mg.ml
1 T7 RNA polymerase. Before large scale
production, all transcriptions were tested on small volumes, and the
products were analyzed by gel autoradiography to check the quality of
the matrices and to optimize the conditions (the MgCl2
concentration, in particular). In all cases, two major species were
observed, one corresponding to the awaited product, the other having a
much higher molecular weight. Similar observations have been reported
by several authors and interpreted as the result of an
RNA-dependent RNA polymerase activity of the T7 polymerase
(16). Different matrices and conditions were tested to try to eliminate
this species, without success.
1 potassium phosphate (pH 6.5), 4 mol.liter
1 urea; B: same as A with 1 mol.liter
1 NaCl). All species could be
separated using a linear gradient: 20% B
80% B in 80 min.
1 urea) and high temperature
migration (60-80 °C). Typically, 2 mg of RNA could be loaded
on a 3-mm gel. After migration, the bands were detected by UV-shadowing
and cut from the gel, and the RNA was recovered using a Schleicher & Shuell electroelution apparatus. The elution buffer was 50 mmol.liter
1 Tris borate (pH 8.0), 1 mmol.liter
1 EDTA. The oligonucleotides were
concentrated by lyophilization or ethanol precipitation and dialyzed
twice, first against 20 mmol.liter
1 phosphate
buffer (pH 6.4), 5 mmol.liter
1 EDTA, 100 mmol.liter
1 NaCl and then against pure
H2O.
1 Tris-HCl (pH 8.0), 1 mmol.liter
1 dithiothreitol, and 0.1 mmol.liter
1 EDTA at 37 °C. To compare the
reaction rates, we always used the same conditions: 1 µmol.liter
1 32P-labeled RNA, 0.2 µmol.liter
1 RegB, and 0 or 0.4 µmol.liter
1 S1 in a final volume of 80 µl. 60 units of RNasin were added to prevent the eventuality of
parasite cleavage by RNase A-like enzymes. In parallel with each
reaction, a control was performed by incubating the RNA in the same
conditions (buffer, S1, RNasin) without RegB. In addition, a ladder,
obtained by a partial digestion of the substrate, was always added to
obtain a measurement of the product lengths.
1 urea, 20 mmol.liter
1 EDTA, 10% glycerol, and 0.05%
bromophenol blue), heated 2 min at 98 °C, and put on ice. The
substrates and products were separated by gel electrophoresis (18%
polyacrylamide, 8 mol.liter
1 urea) and
detected by autoradiography. The band intensities were quantified using
a Molecular Dynamics PhosphorImager.
P or S
P1
P2, according to the
number (one or two) of cleavage sites. The corresponding equations were
fitted to the experimental curves. It was not always possible to obtain
a correct adjustment between the model and all data points. In these
cases, only the first points were taken into account.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
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Fig. 1.
Sequence and secondary structure of
the RNA molecules used in the study. A, the S22cug
molecule corresponds to a fragment of the original SELEX 22nd clone
(11). Its ends were modified in S22gg to allow a better transcription
yield. Both have two GGAGs, designated in the text as the first
(red) and the second (blue). The S26so, S22turn,
and S22stem molecules (B) were designed to study the effect
of structural perturbations on the first cleavage site; S22two,
S22twoloop, and S22linear (C) were designed to study the
effect of perturbations on the second site. The proposed secondary
structures were calculated with the mfold software (24). They
have been confirmed by NMR spectroscopy for S22cug, S22gg, S26so,
S22stem, and S22linear.
View larger version (26K):
[in a new window]
Fig. 2.
Temperature-dependant variations of the
one-dimensional NMR spectra of the S22linear (A),
S26so (B) S22stem (C), and S22cug
(D) imino protons. The spectra were recorded in
H2O by using a jump and return water suppression sequence
(20). A vertical expansion or compression of the display (indicated on
the right of the spectra) was applied in several cases. The spectrum
recorded on the S22linear molecule at 300 K indicates that this
molecule is not structured at this temperature. In contrast, all other
spectra are characteristic of the involvement of imino protons in base
pairs. The comparison of the spectra at low and high temperatures shows
that the secondary structure is conserved at high temperature, even if
several lines become very broad due to the increased exchange rate
between the imino and solvent protons.
View larger version (27K):
[in a new window]
Fig. 3.
H6/8-H6/8 and
H6/8-H5/1' regions of a NOE spectroscopy
spectrum recorded on the S22cug molecule. The spectrum was
recorded in D2O at 290 K. The
H6/8-H6/8, H6-H5, and
H6/8-H1' intra-residual and sequential
correlations used to confirm the long stem assignment are indicated by
solid lines. Several others are designated by dashed
lines and are labeled above the figure. Only a small number of
peaks are present on this spectrum. Most of them correspond to trivial
intra-residual correlations (such as H5-H6) or
concern the residues of the long stem. This strongly indicates that the
loop regions of the S22cug molecule do not possess a single structure
but are rather in equilibrium between several conformations.
View larger version (15K):
[in a new window]
Fig. 4.
H6-H5 region of a
TOCSY spectrum recorded on the S22cug molecule. The spectrum was
recorded in D2O at 290 K. The contour plot of the
two-dimensional TOCSY spectrum and the one-dimensional slices taken at
the peak frequencies are reported. They readily indicate a variation of
the peak width with the localization of the corresponding residues. The
C1 and U2 residues at the free end of the long stem display the
sharpest correlations. In contrast, the C26 residue (at the other end
of the long stem) and the C8, C16, C21, and C23 residues (in the two
loops and in the short stem) correspond to very broad peaks. This
confirms that the two loops are in equilibrium between several
conformations and indicates that the exchange rate between the
conformations belongs to the fast-intermediate NMR domain.
View larger version (49K):
[in a new window]
Fig. 5.
Activity of RegB alone or in the presence of
S1 on the S22cug (A), S26so (B),
S22turn (C), and S22linear (D)
molecules. The substrates were incubated with RegB in the absence
(RegB) or presence (RegB + S1) of the ribosomal
protein S1 as described. Aliquots were removed at 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, and 120 min. The first lane of each
gel corresponds to the labeled RNA, and the last corresponds to a
degradation control performed by incubating the RNA in the reaction mix
for 2 h in the presence of S1 but in the absence of RegB. The
length of the band obtained in the course of the reaction is indicated
on the right of the gel autoradiogram together with the corresponding
cleavage position. nt, nucleotides.
P scheme, associated to a single rate
constant (k1) was adopted. Two parallel
reactions, S
P1 (k1) and S
P2
(k1'), were considered for the S22linear
substrate. Finally, for the S22cug and S22gg, we had to make an
additional assumption. We assumed that the difference between the two
rate constants is sufficient to allow us to consider the unique pathway P
S1
S2 (i.e. to neglect the P
S2 direct
reaction). In all cases, the model was over-determined, i.e.
the number of experimental curves was larger than the number of
parameters to be fitted. When considering the kinetic cleavage of
S22cug (with and without S1) and S22gg (without S1), the experimental
and fitted curves remain in good agreement even after 2 h (Fig.
6 and data not shown), indicating that
our assumption concerning the rate differences is correct. However, in
all other cases, the time course of the reaction is not correctly
described by the model. We were always able to obtain a good fit during
the first 15 (S26so without S1) to 40 min (S22turn) but observed after
that a slowdown of the kinetics. This suggests that the enzyme is
inhibited by at least one of the reaction products. In fact, in all
cases the beginning of the divergence corresponds to the formation of
about 20% product.
View larger version (31K):
[in a new window]
Fig. 6.
Kinetic analysis of the S22cug
(A) and S26so (B) cleavage
reactions. The intensities of all bands were quantified and
expressed in percentage as the ratio of the band intensity to the sum
of all bands. The data were fitted by a simple S P for S26so
(B) or S
P1
P2 for S22cug. The experimental
curves are represented by solid lines, and the fitted curves
are represented by dashed lines. The good agreement obtained
with the S22cug (A) molecule indicates that the simple model
we have used is sufficient to analyze the result. In the case of S26so
(B), the fit is only possible for the first 15 min, strongly
suggesting that the enzyme is inhibited by the reaction product.
nt, nucleotides.
View larger version (14K):
[in a new window]
Fig. 7.
Representation of the kinetic constants
determined in the presence and absence of S1 protein on each
substrate. The constant rate in the presence (pale
blue) or absence (orange) of S1 were determined by
fitting the experimental curves as explained under "Materials and
Methods." The values are indicated above the bar in
min 1.103 (i.e. 90 represents 0.090 min
1). The first five
couples of values correspond to the cleavage of first site (red
GGAG); the five following correspond to the cleavage of second
site (GGAG in blue). JR designates the short
decaoligonucleotide studied by Ruckman et al. (8).
S22twoloop has two sites. The first is cleaved, but we could not
determine the rate constant values.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 RNA, 0.1 µmol.liter
1 RegB, 0.2 µmol.liter
1 S1) on two model substrates: a
112-nucleotide T7 transcript and a small decanucleotide CUUUGGAGGG (8)
hereafter designated JR. Interestingly, the long transcript possessed
two GGAGs, one of which (located in the Shine-Dalgarno region) is
cleaved. In the absence of S1, very low turnover was measured (1 fmol·min
1 for the decanucleotide and 1.8 fmol·min
1 for the long transcript,
corresponding to less than 1% of the molecules cut in 60 min). This
turnover is accelerated in the presence of S1 by a factor of 70-80 (80 fmol·min
1 for the JR decanucleotide and 120 fmol·min
1 for the long transcript,
i.e. about 25% cleaved in 60 min). It must be pointed out
that the original paper (8) is lacking one experimental parameter (the
total amount of enzyme used or, equivalently, the reaction volume)
preventing the conversion of turnover values to rate constants.
Fortunately, these parameters could be found in the Ph.D. thesis of J. Ruckman (28), leading to the rate estimation reported in Fig. 7.
Subsequently, the SELEX method was used to obtain RNA molecules that
are specifically processed by RegB in the presence of S1 (11).
Forty-eight sequences were reported. All but one possess a GGAG motif.
In 33% of them, the GGAG is part of a GGAGGA strong Shine-Dalgarno
sequence, and most (but not all) are followed by a long stretch of A or
C nucleotides. Strikingly, nearly all GGAG sequences are found
at the 5' extremity of the variable region. Several of these clones
have been further tested for their cleavage susceptibility in the
presence or absence of S1 in conditions rather different from those
used in the first study or by ourselves (4 µmol.liter
1 RNA, 0.1 µmol.liter
1 RegB, 0.8 µmol.liter
1 S1). Two classes of molecules
could be identified. The members of the first are not appreciably
cleaved by RegB alone but are very well processed in the presence of S1
(around 80% in 10 min), whereas those of the second are cleaved at the
same low rate (less than 5% in 10 min) in the presence or absence of
S1. A partial correlation was found between the AC richness and the S1
dependence of the rate. However, none of the observations leads to a
clear signal identifying the substrates.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Drs. F. Dardel and D. Fourmy for help with the RNA transcription and Drs. M. Kochoyan, J.-L. Leroy, and K. Snoussi for help with the chemical preparation of some of the molecules. Drs. J.-B. Créchet and E. Jacquet are acknowledged for their advice on kinetic experiments. We thank Dr. R. d'Ari for very helpful comments on this manuscript.
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FOOTNOTES |
---|
* This work was supported by the French CNRS program Physique et Chimie du Vivant.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Institute of Molecular Biology, National Chung Hsing University, 250 Kuo Kuang Rd., Taichung 402, Taiwan, Republic of China.
To whom correspondence should be addressed. Tel.: 33 1 69 33 48 32; Fax: 33 1 69 33 30 10; E-mail:
francois.bontems@polytechnique.fr.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M010680200
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ABBREVIATIONS |
---|
The abbreviations used are: NOE, nuclear Overhauser enhancement; TOCSY, total correlation spectroscopy; DQF-COSY, double quantum filtered correlation spectroscopy.
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REFERENCES |
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1. | Coburn, G. A., and Mackie, G. A. (1999) Prog. Nucleic Acid Res. Mol. Biol. 62, 55-108[Medline] [Order article via Infotrieve] |
2. | Ross, J. (1996) Trends Genet. 12, 171-175[CrossRef][Medline] [Order article via Infotrieve] |
3. | Petersen, C. (1993) in Control of Messenger RNA Stability (Belasco, J. G. , and Brawerlan, G., eds) , pp. 117-145, Academic Press, Inc., New York |
4. | Derrigo, M., Cestelli, A., Savettier, G., and Di Liegro, I. (2000) Int. J. Mol. Med. 5, 111-123[Medline] [Order article via Infotrieve] |
5. |
Vytvytska, O.,
Jakobsen, J. S.,
Balcunaite, G.,
Andersen, J. S.,
Baccarini, M.,
and von Gabain, A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14118-14123 |
6. | Sanson, B., Hu, R., Troitskaya, E., Mathy, N., and Uzan, M. (2000) J. Mol. Biol. 297, 1063-1074[CrossRef][Medline] [Order article via Infotrieve] |
7. | Uzan, M., Favre, R., and Brody, E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8895-8899[Abstract] |
8. |
Ruckman, J.,
Ringquist, S.,
Brody, E.,
and Gold, L.
(1994)
J. Biol. Chem.
269,
26655-26662 |
9. | Sørensen, M. A., Fricke, J., and Pedersen, J. (1998) J. Mol. Biol. 280, 561-569[CrossRef][Medline] [Order article via Infotrieve] |
10. | Subramanian, A. R. (1983) Prog. Nucleic Acid Res. Mol. Biol. 28, 101-142[Medline] [Order article via Infotrieve] |
11. | Jayasena, V. K., Brown, D., Shtatland, T., and Gold, L. (1996) Biochemistry 35, 2349-2356[CrossRef][Medline] [Order article via Infotrieve] |
12. | Subramanian, A. R., Rienhardt, P., Kimura, M., and Suryanarayana, T. (1981) Eur. J. Biochem. 119, 245-249[Abstract] |
13. | Studier, F. W., and Moffat, B. A. (1986) J. Mol. Biol. 189, 113-130[Medline] [Order article via Infotrieve] |
14. | Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Nucleic Acids Res. 15, 8783-8798[Abstract] |
15. | Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workmann, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. (1995) Nucleic Acid Res. 23, 2677-2684[Abstract] |
16. | Cazenave, C., and Uhlenbeck, O. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6972-6976[Abstract] |
17. | Delsuc, M.-A. (1989) in Maximum Entropy and Bayesian Methods (Skilling, J., ed) , pp. 285-290, Kluwer Academic Publishers Group, Dordrecht, Netherlands |
18. | Rouh, A., Delsuc, M.-A., Bertrand, G., and Lallemand, J.-Y. (1993) J. Magn. Reson. 102, 357-359[CrossRef] |
19. | Bartels, C., Xia, T.-h., Billeter, M., Günter, P., and Wüthrich, K. (1995) J. Biomol. NMR 6, 1-10 |
20. | Plateau, P., and Guéron, M. (1982) J. Am. Chem. Soc. 104, 7310-7311 |
21. | Brauschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528 |
22. | Rance, M., Sørensen, O., Bodenhausen, G., Wagner, G., Ernst, R. R., and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun. 117, 479-485[Medline] [Order article via Infotrieve] |
23. | Kumar, A., Ernst, R. R., and Wüthrich, K. (1980) Biochem. Biophys. Res. Commun. 95, 1-6[Medline] [Order article via Infotrieve] |
24. | Zuker, M., Mathews, D. H., and Turner, D. H. (1999) in RNA Biochemistry and Biotechnology (Barciszewski, J. , and Clark, B. F. C., eds) , pp. 11-43, NATO ASI series, Kluwer Academic Publishers Group, Dordrecht, Netherlands |
25. | Lane, A. N., and Lefèvre, J.-F. (1994) Methods Enzymol 239, 596-619[Medline] [Order article via Infotrieve] |
26. | Sanson, B., and Uzan, M. (1993) J. Mol. Biol. 233, 429-446[CrossRef][Medline] [Order article via Infotrieve] |
27. | Kaberdin, V. R., Walsh, A. P., Jakobsen, T., McDowall, K. J., and von Gabein, A. (2000) J. Mol. Biol. 301, 257-264[CrossRef][Medline] [Order article via Infotrieve] |
28. | Ruckman, J. (1993) Ph.D. thesis , University of Colorado |