From the ICSN-RMN, Ecole Polytechnique, 91128 Palaiseau, France and the § Institut Jacques Monod, 1 Place
Jussieu, 75005 Paris, France
Received for publication, December 13, 2002
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ABSTRACT |
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The RegB protein, encoded by the T4
bacteriophage genome, is a ribonuclease involved in the inactivation of
the phage early messenger RNAs. Its in vitro
activity is very low but can be enhanced up to 100-fold in the presence
of the ribosomal protein S1. The latter is made of six repeats of a
conserved module found in many other proteins of RNA metabolism.
Considering the difference between its size (556 amino acids) and that
of several RegB substrates (10 nucleotides), we wondered whether all
six modules are necessary for RegB activation. We studied the influence
of twelve S1 fragments on the cleavage efficiency of three short
substrates. RegB activation requires the cooperation of different sets
of modules depending on the substrates. Two RNAs are quite well cleaved
in the presence of the fragment formed by the fourth and fifth modules,
whereas the third requires the presence of the four C-terminal domains. However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or
tetra-modules are similar, suggesting a common interaction surface. In the case of the tetra-module the interactions
involve all four domains, raising the question of the spatial
organization of this region.
The bacteriophage T4, like many others, takes advantage of host
ribonucleases to mature and degrade its transfer and messenger RNAs (1,
2). However, T4 early transcripts are also inactivated by the
phage-encoded RegB protein synthesized shortly after infection (3-6).
The RegB protein is a ribonuclease that specifically cleaves in the
middle of the tetra-nucleotide GGAG (3), with a strong bias toward the
GGAG found in the Shine-Dalgarno sequence of the early messengers (6).
In addition, RegB probably also cleaves essential host messengers.
Indeed, it exerts a strong bacteriostatic/lethal effect on
Escherichia coli cells
(7).1 However, the in
vitro activity of RegB is very low compared with its in
vivo efficiency. In this context, of particular interest is the
observation that the enzyme activity can be enhanced by a factor of up
to 100 by the E. coli 30 S ribosomal subunit (9). In fact,
the S1 protein alone is sufficient to achieve the same level of
stimulation (9).
Working on the properties of RegB, we wished to analyze how S1 promotes
RegB reaction. S1 is the largest E. coli ribosomal protein
(for review, see Ref. 10). It is found in all Gram-negative and in
several Gram-positive bacteria (11). A smaller form has also been
described in plant chloroplasts (12). The protein plays an essential
role in the initiation of translation (13). It is, in particular,
required for the translation of messengers possessing weak (14) or no
(15) Shine-Dalgarno consensus sequence. It is thus likely that it
facilitates or even supplements the interactions between the
Shine-Dalgarno region of the messenger and the 16 S ribosomal RNA. In
addition, S1 is used by several phages. Besides its role in RegB
activation, it is one of the four subunits of the replicase of RNA
bacteriophages (for review, see Ref. 16) and it interacts with the
Considering the modular organization of S1, the discrepancy between the
size of its RNA-binding region and that of several RegB targets, and
the possible existence of two RNA-binding sites, we wondered whether
all six modules were necessary for RegB activation. To approach this
question, we cloned the sequences coding for twelve fragments of the S1
protein: the N-terminal bi-module, the C-terminal tetra-module,
and the two three-modules, the three bi-modules, and the four
mono-modules contained in the C-terminal region. The ability of all
these fragments to enhance RegB cleavage was tested, and the
interactions of two of them (the C-terminal tetra-module and the
bi-module containing the fourth and fifth domains) with two RNAs were
probed by NMR spectroscopy. The results indicate that RegB activation
requires the cooperation of different sets of modules depending on the
substrate but that all substrates tested interact similarly with either
the bi- or the tetra-module, suggesting a common interaction surface.
In the case of the tetra-module, the interactions involve the four
domains, reopening the question of the spatial organization of this region.
Cloning, Expression, and Purification of the S1 Protein and
Modules--
The S1 protein was purified from E. coli K12
strain ED8689 (M. Uzan, personal collection) by affinity
chromatography on a poly(U) Sepharose 4B (Amersham Biosciences)
column as described previously (10). It was stored at 4 °C
in 10 mmol·liter
The DNA polynucleotides corresponding to the S1 fragments were PCR
amplified from the strain used for S1 production and cloned in the
vector pET-15b between the NdeI (5') and
BamHI (3') sites, in phase with the 21-residue
poly-histidine tag. The different S1 fragments were obtained by
combining five 5'-oligonucleotides, corresponding to the beginning of
the first (5'-F1), third (5'-F3), fourth (5'-F4), fifth (5'-F5), and
sixth (5'-F6) module, and six 3'-oligonucleotides, corresponding to the
end of the second (3'-F2), third (3'-F3), fourth (3'-F4), fifth
(3'-F5), and sixth without the protein tail (3'-F6s) and with the
protein tail (3'-F6l) modules. A NdeI restriction site was
added to all 5'-oligonucleotides, an ochre (UAA) stop codon and a
BamHI restriction site to all 3'-oligonucleotides: 5'-F1,
GCGCATATGACTGAATCTTTTGC; 5'-F3,
AAACATATGAACAGCGCAGAC-GATCAG; 5'-F4,
GAAATTCCATATGTGGGTAGCTATCGCT; 5'-F5,
GCGCATATGTG-GCAGCAGTTCGCGG; 5'-F6,
GAAATTCCATATGTTCAACAAC-TGGGTTGC; 3'-F3,
AAGGATCCTTAATAACGTTTAGCGATA; 3'-F4,
TTGGATCC-TTAGTGGGTTTC-CGCGAA; 3'-F5, CGGGATCCTTAGTTCAGAGCAACCCAG; 3'-F6c, CGGGATCC-TTATTTCTCGTCAGCTCG; 3'-F6l, TTG- GATCCTTACTCGCCTTTAGCTG.
The fragments were overproduced in the E. coli strains
BL21(DE3)pLysS (24). Cultures were induced around A
0.6 using 1 mmol·liter Ribonucleotide Synthesis and Purification--
All
oligoribonucleotides were synthesized chemically on a Amersham
Biosciences LKB Gene Assembler Plus apparatus using Amersham Biosciences polystyrene beads for primer support and phenoxyacetyl Enzyme Assays and Kinetic Analysis--
Cleavage reactions were
performed in 50 mmol·liter NMR Experiments--
All NMR experiments were performed
on a Bruker DRX600 spectrometer equipped with a gradient triple
resonance TXI probe. The spectra were processed off-line using
the GIFA software (26) running under LINUX on a personal
computer and analyzed using XESAY (27). The
fast-HSQC2 sequence (28) was
used for all experiments.
A first series of HSQC was realized on all fragments in a standard NMR
buffer (20 mmol·liter
Four series of titration experiments were carried out to analyze the
interactions between the F45 and F3-6 bi- and tetra-modules and the
JR10 and Selex26 substrates. The F45/Selex26 and F3-6/Selex26 series
were realized with two different RNA batches, and in the case of the
F3-6/JR10, the HSQC were recorded at two temperatures (30 and
35 °C); all others were recorded at 30 °C. The protein was
dialyzed against Tris (pH 8.0) buffer and used at a concentration of
250 µmol·liter Choice and Production of the S1 Fragments and of the
Substrates--
The S1 protein is the largest E. coli
ribosomal protein (556 amino acids in K12). It is composed of
six repeats of a conserved domain, called the S1 domain, and is found
in many other proteins involved in the RNA metabolism. Considering
this, we wondered whether the six modules were necessary for RegB
activation. The fragment consisting of the two N-terminal modules (F12)
has been reported to be involved in the S1 interaction with the
ribosome, whereas that encompassing the four C-terminal domains (F3-6)
is responsible for RNA binding (10). We thus decided to isolate these
two fragments together with all F3-6 sub-fragments: the two
three-modules (F3-5 and F4-6), the three bi-modules (F34, F45, and
F56) and the four mono-modules (F3, F4, F5, and F6) (Fig. 1). The module extremities were chosen
according to the alignment published by Bycroft et al. (29).
The linkers preceding and following each fragment were always included,
and all fragments containing the sixth module but one were constructed
with the C-terminal 30-amino acid tail. Indeed, the F6 domain was
cloned twice, once with the C-terminal tail (F6long) and once without (F6short). All fragments were easily cloned in a pET-15b vector and
expressed. All but the F5 were purified from the soluble fraction. The
F5 fragment was produced as inclusion bodies but could be recovered by
an unfolding-refolding procedure (see "Material and Methods").
To compare the activity of all these fragments on RegB kinetics, we
needed substrates whose cleavage was markedly accelerated by the whole
protein. In addition, because one of our objectives was also to study
the interactions between the substrates and the protein fragments by
NMR, we wanted to use RNA molecules as short as possible. In their
initial study of the role of S1 in RegB activation, Ruckman
et al. (9) used a decanucleotide whose cleavage is
very low in the absence of S1 but is accelerated by a factor of about
80 in its presence. This molecule (CUUUGGAGGG, hereafter designed JR10)
clearly met our criteria. In addition, in a previous study (23), we
looked at the influence of the substrate conformation on RegB cleavage
efficiency. For this, we took advantage of a published study (30)
carried out in the aim of selecting good RegB substrates, in the
presence of S1, by the SELEX method, and we used several short
fragments derived from one of the reported clones. Two of the molecules
we analyzed at this occasion (Selex12 and Selex26) also suit our
present requirements. As shown in Fig. 1, these two Selex molecules
present strong similarities to each other and are very different from
JR10. They are longer (20 nucleotides for Selex12 and 23 for Selex26).
Their composition is strongly biased against U (only one each), whereas
JR10 possesses five G, three U and only one A and one C. They present
similar secondary structure as predicted by the mfold
software (31), whereas JR10 seems to have no stable conformation.
Enzymatic Analysis of S1 Fragment Influence on RegB
Activity--
We first tested the effect of all S1 fragments on the
Selex12 and Selex26 oligoribonucleotides by following the reaction
kinetics for 20 min. The electrophoresis gels and kinetic curves
corresponding to the cleavage by RegB alone or in the presence of all
but the mono-modules are shown in Fig. 2.
The data first show that there is, as expected, a marked difference
between the cleavage ratio in the presence of S1 (nearly 100% after 20 min) and in its absence (less than 5%). They also demonstrate that the
different fragments have variable effects on the reaction rate
enhancement but have no influence on the position of the cutting event.
Finally, they indicate that all fragments have similar effects on the
Selex12 and Selex26 oligonucleotides.
As expected, the F3-6 (C-terminal) fragment induces the same
enhancement as the whole S1 protein, whereas the F12 (N-terminal) has
no influence on the cleavage rate. All other fragments can be
classified in three groups. The F3-5 three-module has the same effect
as S1 and F3-6. The F45 and F4-6 fragments also enhance the cleavage
rate but to a lesser extent, with cleavage ratios about half that
obtained using S1, F3-5, and F3-6 proteins. Finally, the F34 and F56
bi-modules and all mono-modules have no effect. Looking in detail at
the kinetic curves, a difference appears between the two substrates.
The cleavage is higher for the Selex12 substrate than for Selex26 in
the presence of S1 and the F3-6, F3-5, and F4-6 fragments but is
lower in the presence of the F45 bi-module. We previously observed (32)
that Selex12 was cleaved better than Selex26 in the presence of S1.
Interestingly, the ratio between the cleavage values of the two
substrates is conserved (Selex12 cleavage/Selex26 cleavage ~1.2) for
the four S1, F3-6, F3-5, and F4-6 proteins. The second observation
is more puzzling, but it is difficult to determine whether it is really
significant, considering that the data on the S1, F3-6, F3-5, and
F4-6 kinetics came from one gel and that on F45 from another. In fact,
the values obtained for RegB alone (Fig. 2, both gels) and
in the presence of F12 (first gel) or of F34 and F56
(second gel) are also lower for the second gel than for the first.
In a second series of experiments, we looked at JR10 cleavage. We first
notice that although S1 clearly enhances the cleavage of this
substrate, the effect is less pronounced than for Selex26 and Selex12.
We thus decided to follow the kinetics
for 1 h in the presence of S1 and
the F3-6, F3-5, and F4-6 fragments (Fig. 3, left
gel). Interestingly, this experiment indicates that the C-terminal region (F3-6), as expected, is able to promote cleavage, but to a lesser extent than the whole S1 protein. It also shows that
the F3-5 and F4-6 three-modules have little, if any, effect on
cleavage efficiency. A second set of reactions was then performed using
the bi-modules (F34, F45, and F56) and repeating the three-modules. Considering the projected slow rate of the reaction, only two points
were recorded at 30 and 60 min. The cleavage was indeed very low, but
we remark that after 30 min, the product ratio obtained in the presence
of the F3-5 and F4-6 three-modules (around 5%) is actually twice
that obtained in the presence of the F34, F45, and F56 bi-module
(around 2.4%), suggesting a slight effect of the former.
Choice of NMR Conditions--
Two fragments of the S1 protein were
of particular interest: the F3-6 C-terminal region and the F45
bi-module, which is the smallest fragment able to promote cleavage by
RegB and is part of all active fragments. To try to understand the
system better, we decided to analyze the interactions between these
fragments and the substrates by nuclear magnetic resonance. More
precisely, we decided to follow the effect of increasing amounts of RNA
on the HSQC spectra of both fragments. The HSQC experiment correlates the chemical shifts of the backbone and side-chain 15N
nucleus with those of their attached protons and can be considered a
fingerprint of the backbone structure, because any structural perturbation will modify the spectrum.
Before doing the experiments, we needed to determine the best
conditions. We first recorded an HSQC on all fragments using a standard
NMR buffer (20 mmol·liter
However, the NMR and SAXS experiments were recorded, as explained
above, at pH 6.0, whereas the cleavage experiments were performed at pH
8.0. It was thus necessary to see whether the equilibrium had the same
properties in both conditions. Accordingly, we recorded the NMR spectra
of the F3-6 fragments and reran the SASX experiment in the cleavage
buffer at pH 8.0. As expected, the HSQC spectra are less well defined
in these conditions due to the acceleration of the proton exchange
rate. However, we observed no differences between 30° and 35 °C.
In addition, the SAXS experiment gives an apparent mass equal to the
theoretical mass (47 kDa) at both temperatures. The protein is always
monomeric at pH 8.0. Therefore, although the spectra are cleaner at pH
6.0, we decided to perform the interaction experiment at pH 8.0 to be
able to correlate the biological and spectroscopic results. A high
concentration of Tris (50 mmol·liter NMR Analysis of the Interactions between F3-6 and F45 Fragments
with JR10 and S26--
The perturbations
induced by JR10 and Selex26 on the F45 and F3-6 fragments are
presented in Figs. 5 and 6, respectively. In each case, we show the HSQC of the protein in the absence or presence of a stoichiometric amount of RNA and the corresponding difference map. As previously indicated, the quality of the spectra is
poorer at pH 8.0 than at pH 6.0. This is not too damageable in the case
of the F45 HSQC, where almost all peaks seen at pH 6.0 are still
identifiable at pH 8.0. The problem is more serious in the case of the
F3-6 fragment. Observation of the periphery of the spectra suggests
that almost all peaks are actually present, but they are very difficult
to identify and to analyze in the center because of a strong
overlapping. Fortunately, even in these conditions, striking
differences are observable between the spectra recorded with and
without RNA. In addition, it is possible to rely on the difference map.
The interaction between the F3-6 fragment and the JR10 RNA (F3-6/JR10
experiment) was analyzed at two temperatures (30° and 35 °C). The
corresponding maps are nearly identical. In addition, we checked many
of the effects seen on the maps by visual comparison of the HSQC or of
one-dimensional extracts. In all cases we found noticeable differences
between the reference and stoichiometric spectra. It is worth noticing
that the reverse is not true. Several HSQC differences, readily
observable, have no correspondence in the difference maps, suggesting
that the sensitivity of the latter is low. In this way we may lose
several effects, but, on the contrary, there are probably very
few false positive hits.
We first looked at the interactions between the F45 bi-module and
either the Selex26 or the JR10 molecule (Fig. 5). Strikingly, many
effects are visible in both maps, indicating that the F45 fragment
actually interacts not only with the Selex26 molecule but also with
JR10. Furthermore, all effects observed in the presence of JR10 are
also found with Selex26. At first sight, the reverse does not seem
true. The difference map seems more complicated with Selex26. Two
series of effects are highlighted by small blue and
red circles in the F45/Selex26 difference map
(Fig. 5). They correspond, respectively, to the appearance, in the
presence of the RNA, of two positive (blue circles) and four
negative (red circles) peaks that are absent in the
reference spectrum. These effects are not visible in the F45/JR10
difference map. However, direct observation of the corresponding HSQC
indicates that the two positive (blue) peaks and, at least,
two of the negative (red) peaks do actually appear in the
presence of JR10 also. They are simply much fainter. This seems to be a
general observation. In the presence of RNA many peaks shift
(juxtaposition, on the difference map, of a blue and a
red dot of similar intensity). As exemplified by the two
effects indicated by arrows, the shifts are smaller in the
presence of JR10 than in the presence of Selex26. Altogether, these
observations strongly suggest that the JR10 and Selex26 molecules both
interact with the same area of the F45 fragment, but with different affinities.
The same analysis was carried out on the F3-6 tetra-module (Fig. 6).
As previously indicated, the spectra are very crowded, and it is more
difficult to observe directly the effects on the HSQC. However, the
difference maps reveal that there are many, actually more than in case
of the F45 bi-module. As previously, the two maps seem very similar at
first sight, the main difference being again the intensity of the
effects. However, the processes seem more complicated. A peak is
indicated by an arrow on the reference HSQC. Strikingly, as
shown in Fig. 6, its intensity increases in the presence of the Selex26
RNA, whereas it decreases slightly in the presence of JR10. Similarly,
we also indicated another effect on the difference maps, very strong
for the interaction with Selex26 but absent with JR10. This suggests
that although many induced effects are identical (with different
intensities) for the two RNA molecules, others are specific to Selex26.
We also compared the two F3-6 difference maps with the HSQC spectra of
the F45, F3-5, and F6short fragments recorded in the same conditions (Fig. 7). It is clear that many effects
are not superimposed with the F45 peaks, indicating that they
correspond to residues not belonging to this fragment. Indeed, most of
these peaks are readily superimposed with peaks of either the F3-5
HSQC (in red on Fig. 7) or the F6short HSQC (in
blue). Some remain unassigned. They may correspond to the
C-terminal tail of the protein, or to peaks involved in an interface
(whose chemical shifts would be different in the whole protein and in
the fragments analyzed).
The use of HSQC to detect the effect of RNA addition on the F45 and
F3-6 fragments focuses the analysis on the backbone of the proteins.
Nevertheless, it also shows that several side-chains are involved in
the complexes. The four peaks highlighted by red circles in
Fig. 5 actually correspond to correlations between the N As indicated in the introduction, the S1 protein is composed of
six repeats of a conserved module of about 80 amino acids organized in
a five-stranded The cleavage experiments first confirm that the ability of S1 to
enhance the RegB rate is due to the C-terminal region of the protein
(F3-6 fragment), but they also suggest that the removal of the
N-terminal part alters its properties (as indicated by the comparison
of the F3-6 and S1 efficiencies with JR10 as substrate). They also
demonstrate that the different C-terminal modules are not equivalent.
The F45 fragment is the only bi-module able to accelerate the cleavage
reaction (using the Selex molecules), but to a lesser extent than the
entire protein. The third module is always required and is sufficient
in the case of the Selex substrates to compensate for the difference.
The sixth module has no influence on the cleavage of the Selex
molecules but is strictly required for JR10. These experiments also
show that there is a strong cooperativity between the modules. The
fourth and fifth modules are only active when associated in the F45
fragment, as shown by the absence of activity of the F4 and F5
mono-modules and of the F34 and F56 bi-modules. The third module is
only active when associated with the fourth and fifth in the F3-5
fragment, as shown by the absence of activity of the F3 and F34
fragments. Finally, the sixth is only active when associated with the
three others in the F3-6 fragment, as shown by the absence of
activity of the F6 and F56 fragments and by the very weak effect of the F4-6 three-module.
A second observation is that the efficiency of the fragments depends on
the substrates. The F45 fragment is sufficient to partially activate
the cleavage of the Selex molecules but not of JR10. The F3-5
tri-module is sufficient to reproduce the effect of the entire S1
protein on the two Selex molecules, but the complete C-terminal region
(F3-6) is required to accelerate the cleavage of JR10. Interestingly,
a similar property of the sixth module (indispensable for one activity,
dispensable for another) has been recently reported in the initiation
of translation (34). The authors showed that the S1 ssyF
mutant codes for a protein lacking the sixth module (the last 92 residues of the protein, more precisely). They also showed that this
mutation hardly affects the initiation of translation of the E. coli messenger RNA but abolishes the autorepression of S1
translation. This strongly suggests that the sixth module is
indispensable for the down-regulation of the translation of S1
but dispensable for the initiation of translation. A related
observation has also been made with the nucleolin protein (35).
Nucleolin consists of four RNA binding domains of about 80 amino acids.
It is able to bind two kinds of substrate: RNA fragments containing a
short hairpin (NRE for nucleolin recognition elements) or an
unstructured stretch of 11 conserved nucleotides (ECM for evolutionary
conserved motif). Interestingly, the first two domains of nucleolin are
necessary and sufficient for its specific interaction with the NRE
hairpin, whereas all four domains are required for the binding of the
unstructured ECM.
Several hypotheses can account for the above results. First, it was not
excluded that some of them result from structural problems induced by
the truncation of the protein in fragments (some mono- or bi-modules,
for example, may lose their structure). Alternatively, we may speculate
that S1 possesses different RNA-binding sub-sites, one recognized by
the Selex molecules across the fourth and fifth modules, another
binding JR10 and spanning the third and sixth modules. This hypothesis
is supported by several studies suggesting the existence of two S1
polynucleotide-binding sites (19-22). However, we may also imagine
that both molecules interacts with the F45 fragment but that JR10 needs
more contacts (with the third and sixth modules) because its affinity
is too low or because its interactions with the F45 fragment alone are
insufficient to allow the proper orientation or conformation.
The NMR experiments first demonstrated that all fragments are
structured and stable and that the modules have conserved their integrity in all of them. In addition, together with the SAXS experiments, they strongly support the existence of a monomer-dimer equilibrium of the F3-6 fragment at pH 6.0. At first sight, this provided an elegant model to explain our results, in particular the
strong cooperativity between the third and sixth modules observed in
JR10 cleavage. Indeed, even with the hypothesis of a completely extended organization of the fragment, it is possible to bring the
third module of one molecule in close vicinity to the sixth of a second
in a symmetric dimer. It thus seemed possible to propose that the Selex
molecules interact with the fourth and fifth modules either only in the
monomer or in both the monomer and the dimer states, whereas the JR10
would bind the third and sixth modules in the dimeric form only.
However, the subsequent controls at pH 8.0 indicated that the F36
fragment is always monomeric in cleavage buffer and thus ruled out this model.
Actually, the NMR interaction experiments carried out at pH 8.0 show
that the two substrates bind both the F45 and F3-6 fragments. In the
case of the F45 fragment, the effects induced by the addition of both
RNA seem similar. Their intensities, however, are different, suggesting
that the ability of the F45 fragment to accelerate the cleavage of
Selex26 but not of JR10 could be due to a stronger affinity of the
protein for the first substrate. As for the F3-6 protein, most of the
effects were observable in the two experiments, with the same bias in
their intensities. However, we found at least two effects specific to
interactions with Selex26. According to these results, the cleavage
activation of the Selex and JR10 substrates obtained with different
sets of modules would not result from interactions of the two RNAs with
different sub-sites of the protein, but are more likely due to
different use of the same interaction area. Interestingly, at the end
of their study of nucleolin (35), the authors note that they "were
unable to detect interactions of [the NRE and EMC] RNA with the same
nucleolin molecule." This suggests that the situation we encountered
here (different action of the fragments toward the two families of substrate, but strong similarities between the effects induced by the
interaction, suggesting a common interaction surface) may be shared by
the two systems.
The comparison of the F36 difference map with the HSQC spectra of the
F45, F3-5 and F6short proteins clearly shows that the binding of both
RNAs on the F3-6 fragment induces modifications not only on the fourth
and fifth modules but also on the third and sixth. This was not
unexpected in the case of the JR10 substrate but it is more surprising
with the Selex26 RNA. Again, this observation raises the problem of S1
spatial organization. First, we have to point out that the technique we
used cannot distinguish between direct contact and indirect
modification of the protein structure. And, considering the very large
number of effects observed, it is unlikely that they all correspond to
direct contacts. It is thus very likely that the binding of the
substrates to the F3-6 fragment induces structural perturbations of
the protein. Under these conditions, it is not impossible that there is
no direct interaction between the RNA and the third and/or sixth
modules, but merely coupling between the substrate binding and a
perturbation of the structure and/or positioning of the two modules.
Obviously, with this hypothesis, the fact that the third and sixth
modules are necessary for complete activation of the reaction implies that the coupling acts in both directions: i.e.
that it is necessary to allow the correct substrates binding. However,
this is not the only possibility. A comparison was made between the
cryo-electron microscope map of a S1 containing E. coli
ribosome and an x-ray crystallographic structure of the 30 S sub-unit
lacking S1 (36). The deduced shape of the S1 protein is complex, with a
central globular portion and two stretched arms, very different from
the tri-axial body proposed in view of the SAXS experiments. In
addition, the S1 protein possesses two cysteines, both in the fourth
module. One (Cys-349) is exposed in the native protein but is protected in the presence of RNA (8). The other (Cys-292) is protected in the
entire protein but is accessible in the M1S1 truncated form of S1
lacking the sixth module (10). This suggests a structural relationship between the fourth and the sixth modules. It thus seems
possible to imagine that the four C-terminal modules of S1 are actually
able to interact simultaneously with short RNA substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-protein of phage
(17). The fact that both its physiological
function and its role in the RegB system involve the Shine-Dalgarno
region suggests that S1 recognizes a similar signal in both cases.
However, the nature of this signal is still an open question. The
protein is able to bind poly(A), poly(U), and poly(C) oligonucleotides
(10). Using a procedure based on UV cross-linking, the S1-binding site on Q
phage mRNA was shown to contain a poly(U) stretch located upstream of the Shine-Dalgarno GGAG sequence (18), but other results
indicate that S1 can also act as a poly(A)-binding protein (19). In
agreement with these results, it has been proposed that S1 possesses
two RNA-binding sites, one with better affinity for poly-pyrimidines,
the others for poly-purines (19-22). The existence of two different
RNA-binding sites may result from the fact that S1 consists of six
repetitions of a conserved module found in many other proteins involved
in RNA metabolism. The first two modules are responsible for the
interactions of the protein with the ribosome, whereas the four
C-terminal modules interact with mRNA (10). This latter region has
been proposed to be elongated in shape, with a length of about 200 Å (10) but it fixes short RNA molecules of about 10 nucleotides.
Similarly, S1 is able to accelerate the RegB-mediated cleavage of an
unstructured decanucleotide (9) and of several structured molecules of
about 20 nucleotides (23).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 Tris-Cl (pH 7.4), 0.1 mmol·liter
1 EDTA, 50 mmol·liter
1 NaCl,
and 7 mmol·liter
1
-mercaptoethanol.
1
isopropyl-1-thio-
-D-galactopyranoside and
harvested after 4 h. The proteins were purified on a Co-agarose
TALON resin, using the manufacturer's (Clontech)
recommended protocol and conserved in 20 mmol·liter
1
Tris (pH 8.0), 200 mmol·liter
1 NaCl, and 5 mmol·liter
1
-mercaptoethanol. In the case of the F5
fragment (corresponding to the fifth module, see Fig. 1), the protein
forms inclusion bodies. After harvesting, the cells were resuspended
and sonicated in 20 mmol·liter
1 Tris (pH 7.4), 300 mmol·liter
1 NaCl, 6 mol·liter
1
guanidium chloride, and 10% glycerol. The insoluble material was
removed by centrifugation, and the supernatant was incubated in the
presence of the resin. After several wash cycles, the extraction buffer
was first exchanged against 20 mmol·liter
1 Tris (pH
7.4), 300 mmol·liter
1 NaCl, and 8 mol·liter
1 urea and then against 20 mmol·liter
1 Tris (pH 7.4), 300 mmol·liter
1 NaCl (renaturation buffer). The protein was
then recovered as the others.
-RNA phosphoramidites. The protocol was adapted from that used for
DNA synthesis, the main differences being the increase of the coupling
time (20 min) and the use of
5-ethylthio-1H-tetrazol as activator (25). The
terminal 5'-O-dimethoxytrityl group was removed on the
synthesizer at the end of the synthesis. The oligoribonucleotides were
cleaved from the support and unprotected accordingly to Leroy and
co-workers (25). The fragments were purified using a Q-Sepharose
high pressure liquid chromatography column running on a Beckman system.
Prior to RNA loading, the column was equilibrated with 80% buffer A
and 20% buffer B (buffer A, 5 mmol·liter
1 potassium
phosphate (pH 7.0), 4 mol·liter
1 urea; buffer B, same
as A with 1 mol·liter
1 NaCl). All species could be
separated using a linear gradient: 20%B to 80%B in 80 min.
1 Tris-HCl (pH 8.0),
20 mmol·liter
1 NaCl, 1 mmol·liter
1
dithiothreitol, and 0.1 mmol·liter
1 EDTA at 37 °C.
In case of the JR10 substrate (Fig. 1), 1 µmol·liter
1
32P-labeled RNA was incubated in the presence of 0.2 µmol·liter
1 RegB and 0.4 µmol·liter
1 S1 fragment in a final volume of 35 µl.
60 units of RNasin were added to prevent the eventuality of parasite
cleavage by RNase A-like enzymes. In these conditions, the cleavage of
the Selex12 and Selex26 molecules was too fast to be correctly
analyzed. The amount of enzyme and of S1 fragment was, thus, reduced by
a factor of 2. In parallel with each reaction, a control was performed by incubating the RNA in the same conditions (buffer, S1 fragment, and
RNasin) without RegB. In addition, a ladder, obtained by partial digestion of the substrate, was always added to obtain a measurement of
the product lengths. Aliquots (5 µl) were typically taken at 5, 10, 20, 30, 45, and 60 min for JR10 and 1, 3, 5, 10, and 20 min for the
Selex molecules. They were mixed with 3 µl of loading blue (8 mol·liter
1 urea, 20 mmol·liter
1 EDTA,
10% glycerol, and 0.05% bromphenol buffer), 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 by phosphorimaging (Molecular Dynamics). Most
reactions were carried two or three times.
1 phosphate (pH 6.3), 200 mmol·liter
1 NaCl) at 30 °C. The three- and
tetra-modules were also probed at 25 and 35 °C. To prepare
the interaction experiments, a second series was also recorded in 50 mmol·liter
1 Tris (pH 8.0), 20 mmol·liter
1 NaCl, 4 mmol·liter
1
-mercaptoethanol at 30 and 35 °C for the F6short, F45,
F3-5, and F3-6 fragments. In all cases, the concentration varied
between 250 µmol·liter
1 and 1 mmol·liter
1.
1. The corresponding amount of RNA was
dispatched in four fractions and lyophilized after dialysis against
pure water and pH neutralization. Each titration point was done by
removing the NMR sample from the tube, mixing it with the lyophilized
RNA and putting it back in the NMR tube. Five HSQC were thus recorded
for each series, corresponding to RNA/protein ratios of 0, 0.25, 0.5, 0.75, and 1. All recording parameters were kept rigorously
constant; the only modification concerned probe tuning and field
shimming. The experiments were carried out with 200 rows of 512 points,
each row corresponding to 128 scans. The spectral width was set to 22 (15N) and 5 (1H) ppm and the frequency offset
to 7300 (15N) and 4800 (1H) Hz. The spectra
were processed identically, to obtain 512 × 512 points real
matrices. Small adjustments of the zero order phase in the
2
(1H) dimension (less than 5°) were necessary to phase the
matrices correctly. The difference maps were obtained by subtracting
the reference matrix from the one of interest.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence of the S1 protein, fragments of the
protein, and RNA substrates used in this study. The S1 domains are
indicated in red in the sequence, the linkers in
blue, and the residues conserved in at least three modules
in green. The domain limits and the alignment are
those proposed by Bycroft et al. (29). The same colors
(red for the domains, blue for the linkers) have
been used for the representation of the fragments. As indicated, all
but the F6short include the linkers preceding and following the first
and the last domain, respectively. The sixth module was produced twice:
once with the C-terminal extension of the protein (F6long)
and once without (F6short). The sequences of the three RNA
substrates are represented with their predicted secondary structure,
calculated with the mfold software (31). The GGAG cleavage site is
indicated in red.
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Fig. 2.
Cleavage experiments carried out on the
Selex12 (left) and Selex26 (right)
substrates. The upper gels correspond to cleavage in
the presence of the S1 protein, the C-terminal part (F3-6
tetra-module), the N-terminal part, and the two three-modules F3-5 and
F4-6. The lower gels correspond to cleavage in the presence
of the three bi-modules F34, F45, and F56. In both cases, the cleavage
by RegB alone has been added as a reference. In each experiment the
fives lanes correspond to the analysis of the reaction after
1, 2, 5, 10, and 20 min. The curves obtained after quantification are
represented at the bottom of the figure.
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Fig. 3.
Cleavage experiments carried out on the JR10
substrate. The gel on the left corresponds
to the RNA cleavage in the presence of S1 protein, the N-terminal
bi-module, the C-terminal F3-6 tetra-module, and the F3-5 and F4-6
three-modules. The reaction was analyzed at 5, 10, 20, 30, 45, and 60 min. The corresponding curves are represented below. The
gel on the right corresponds to RNA cleavage in
the presence of S1 protein, the two F3-5 and F4-6 three-modules, and
the three F34, F45, and F56 bi-modules. In each case, the analysis was
carried out at 30 and 60 min. The percentage of cleavage after one
hour, deduced from the two experiments, is represented in the
histogram. The value of cleavage in the presence of S1
protein was used to normalize the (small) differences between the two
gels.
1 phosphate, 200 mmol·liter
1 NaCl, 5 mmol·liter
1
-mercaptoethanol) at pH 6.0. This pH was chosen because of the better quality of the NMR spectra at acidic pH because the exchange rate of the amide proton is slower. In all cases, the quality of the
spectra is indeed very high (Fig. 4). The
number of peaks is close to that expected. Even in the case of the
F3-6 fragments, despite the crowding of the central region, more than
300 peaks, among 380, are readily identifiable. They are well defined,
with similar intensities and their 1H frequencies are
spread over nearly 4 ppm. This indicates that all fragments are
correctly folded, the large 1H spectral width being in
agreement with the module-expected high content in
-strands (29).
The behavior of the F3-6 fragment, however, was puzzling. The spectrum
previously discussed and reported in Fig. 4C was recorded at
35 °C but below 30 °C only a few peaks remained (Fig.
4D). By comparison with the spectra of the F6long and
F6short fragments, the remaining peaks could be assigned to the
30-amino acid C-terminal tail of the protein, which was demonstrated, by the same, to be unstructured. The disappearance of the peaks below
30 °C could be due either to the formation of a stable (and reversible) high molecular weight multimer or to the existence of an
equilibrium in the intermediate exchange regime between two protein
states (e.g. between two conformations or between a monomer
and a dimer). To discriminate between these two possibilities, we
recorded a small angle x-ray scattering experiment (SAXS) on a 250 µmol·liter
1 F3-6 sample in NMR buffer at 30 and
35 °C. This experiment allows precise determination of the particle
mean mass. We measured an identical value of 57.1 kDa at the two
temperatures, compatible with a monomer-dimer (70/30%)
equilibrium.
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Fig. 4.
HSQC spectra recorded at pH 6.0 with F6short
(A), F6long (B), F45
(C), F4-6 (D), F3-6 at 35 °C
(E), and F3-6 at 25 °C (F).
All spectra but that presented in E were recorded at
25 °C. The peaks present in the F6long spectrum and absent in
F6short (and thus corresponding to the C-terminal extension) are
indicated by small boxes in the F6long (B)
spectrum. These peaks are more intense than the others, and the
dispersion of their 1H chemical shifts is small (less than
1 ppm), strongly indicating that the C-terminal tail is not structured.
The same peaks are found in F (F3-6 at 25 °C), showing
that the only part of the protein observable in these conditions is
also its C-terminal tail.
1) was also
conserved to ensure that the RNA addition (final concentration: 250 µmol·liter
1) would not modify the pH of the solution.
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Fig. 5.
Analysis of the NMR interaction experiments
between the F45 bi-module and either Selex26 (upper
spectra) or JR10 (lower spectra). In
both cases the reference HSQC (left), the HSQC in
the presence of a stoichiometric amount of RNA (middle), and
the difference map (right) are represented. The positive
peaks are in blue (or in black in the reference
spectra), the negative in red. The differences clearly
visible between the reference and the interaction spectra are indicated
by circles or square boxes. The red
circles correspond to the appearance, in the presence of the RNAs,
of correlations between the N and H
of
arginine side-chains. These peaks are negative because they are folded
back in the spectra, their actual N frequencies (about 85 ppm) being
outside of the spectral window analyzed. The green circles
correspond to the disappearance of asparagine and/or glutamine
NH2 side-chain correlations. Finally, the blue
circles show the appearance of two peaks in an empty region of the
reference spectra. The arrows indicate two peaks undergoing
a marked shift in the presence of the RNAs. As discussed under
"Results," the shifts are larger for the interaction with
the selex26 RNA molecule, suggesting a higher affinity of the protein
for Selex26 than for JR10.
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Fig. 6.
Analysis of the interactions between the
F3-6 teta-module and either Selex26 (upper spectra)
or JR10 (lower spectra). The disposition of the
spectra and the color code are identical to those used in Fig. 5. The
only difference is that the blue circles are used here to
indicate peaks whose intensities increase in the presence of the RNAs
and the arrows designate two effects that depend on the
substrate considered. The arrows on the HSQC spectra
indicate a correlation whose intensity increases in the presence of
Selex26 and decreases in the presence of JR10. The arrow on
the difference maps indicates a strong peak shift in the presence of
Selex26 that is not observed in the presence of JR10.
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Fig. 7.
Identification of the module involved in the
interactions between the F3-6 tetra-module and Selex26 or JR10.
The difference maps corresponding to the interactions of the F3-6 with
Selex26 or JR10 have been superimposed on the F45 HSQC in A
and B, respectively. The green circles
indicate effects seen on the difference map not corresponding to peaks
in the HSQC. These effects must concern residues not belonging to the
F45 bi-module. These circles have been reported on the HSQC of the
F3-5 three-module (C) and the F6short mono-module
(D). The red circles probably correspond to peaks
belonging to the third module and the blue circles to peaks
belonging to the sixth module. The green circles correspond
to effects whose localization is ambiguous.
and H
atoms of arginine side-chains. Their appearance in
the presence of RNA strongly suggests that they become involved in
hydrogen bonds (more precisely, it indicates that the exchange rate of the proton with the solvent is slowed down in the complex). Similarly, the strong effects indicated by green circles in Figs. 5 and
6 correspond to the disappearance of asparagine or gluatamine
NH2 side-chain correlations, indicating that at least two
of these side-chains participate in the formation of the complex.
Another striking observation is the number of effects seen on the
difference maps. The addition of either the JR10 or the Selex26 RNA
leads to modifications in the intensity or position of about 100 peaks of the F3-6 fragment for example. It seems very unlikely that all
these modifications correspond to amino acids directly involved in
contacts with the RNA. It is more likely that many of them have an
indirect origin such as an induced structural modification of the
protein or a change in the amide proton exchange rate. The latter
interpretation, in particular, is supported by the observation that the
intensity of many peaks increases (blue dots in the
difference maps) in the presence of RNA, suggesting a slower exchange rate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrel. The distance between the N and C termini of
each module is ~30 Å (29), a value compatible with that deduced for
the entire protein (between 230 and 280 Å, with an axial ratio of
about 10) from hydrodynamic or x-ray scattering experiments (10). In
contrast with this large size, the dimension of the RNA-binding site
seems rather small. It has been evaluated by different techniques to
correspond to unstructured fragments of about 10-12 nucleotides (10,
18, 19), with lengths of about 40 Å. The generation by the SELEX
method of high affinity RNA ligands to S1 provided molecules consisting
of a pseudoknot whose central helix was also formed by 10 nucleotides (33). The S1 protein is able to promote the cleavage of unstructured decanucleotides (e.g. JR10 used in this study). Considering
the apparent discrepancy between the size of S1 and that of its RNA targets, we wondered whether all modules were necessary for a given
biological activity or whether each has evolved to acquire specialized
functions. A first answer to this question is provided by the
observation that the first two modules are involved in ribosome
binding, whereas the four following are responsible for the mRNA
recognition, but the question remains open for the four C-terminal
modules. We thus decided to analyze the ability of several fragments of
the S1 protein, and in particular of all sub-fragments of the
C-terminal region, to promote the RegB cleavage of several short RNAs.
![]() |
ACKNOWLEDGEMENTS |
---|
The SAXS experiments were carried out at the Laboratoire pour l'Utilization du Rayonnement Electromagnétique (Universite d'Orsay). We are very grateful to Dr. J. Perez for performing and analyzing them. We thank Dr. J.-L. Leroy for help with the chemical preparation of the RNA molecules. We also thank Dr. R. d'Ari for helpful comments on this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the French Fondation pour la Recherche Médicale and by the Ecole Polytechnique.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.
¶ To whom correspondence should be addressed: Groupe ICSN-RMN, Ecole Polytechnique, F91128 Palaiseau, France. Tel.: 33-1-69-33-48-55; E-mail: Francois.Bontems@polytechnique.fr.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M212731200
1 F. Saida, M. Uzan, J.-Y. Lallemand, and F. Bontems, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HSQC, heteronuclear single-quantum coherence; SAXS, small angle x-ray scattering experiment; NRE, nucleolin recognition elements.
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REFERENCES |
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