From the Department of Biochemistry and Molecular
Biology, Penn State University, College of Medicine, Hershey,
Pennsylvania 17033 and the § Institute for Protein Research,
Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received for publication, November 20, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The cold shock protein CspB from
Bacillus subtilis binds T-based single-stranded DNA (ssDNA)
with high affinity (Lopez, M. M., Yutani, K., and Makhatadze,
G. I. (1999) J. Biol. Chem. 274, 33601-33608).
In this paper we report the results of CspB interactions with
non-homogeneous ssDNA templates containing continuous and non-continuous stretches of T bases. The analysis of CspB-ssDNA interactions was performed using fluorescence spectroscopy, analytical centrifugation and isothermal titration calorimetry. We show that (i)
there is a strong correlation between the CspB affinity and stoichiometry and the T content in the oligonucleotide that is independent of which other bases are incorporated into the sequence of
ssDNA; (ii) the binding properties of CspB to ssDNA templates with
continuous or non-continuous stretches of T bases with similar T
content is very similar, and (iii) the mechanism of interaction between
CspB and the T-based non-homogeneous ssDNA is mainly through the bases
(a stretch of three T bases located in the middle of the ssDNA
templates makes the binding independent of the ionic strength). The
biological relevance of these results to the role of CspB as an RNA
chaperone is discussed.
The detailed mechanism by which living cells survive a cold shock
remains controversial (1). It has been observed that in many bacteria
cold shock is accompanied by an increase in the expression levels of a
specific set of the so-called cold shock proteins
(CSPs)1 (2, 3). In
Bacillus subtilis the major CSP is a small acidic protein,
CspB, whose induction increases dramatically when the temperature is
down-shifted from 37°C to 15 °C (4). The first evidence of any
activity of CspB was demonstrated using gel retardation experiments,
and it was shown that CspB binds to single-stranded DNA (ssDNA)
containing the Y-box motif (ATTGG) (5, 6). Later, however, it was shown
that CspB binding is not limited only to this sequence (7, 8). Several
aromatic residues (Phe15, Phe17,
Phe27, Phe30, Phe38,
Trp8) and several basic amino acid residues (His29,
Lys7, Lys13, Lys39,
Arg56) have been proposed to be involved in the protein
interaction with ssDNA (9). The three-dimensional structure of the
protein shows that these amino acid residues are located in the same
side of the protein molecule (5, 6).
In our laboratory, using four different techniques (gel shift mobility
assays, isothermal titration calorimetry (ITC), analytical ultracentrifugation, and fluorescence spectroscopy), it was shown that
CspB binds preferentially to polypyrimidine ssDNA (8). CspB binding to
poly(dC) and poly(dT) has different properties showing higher affinity
for homogeneous T-based oligonucleotides with a binding site size of
6-7 T bases (8). The analysis of such binding according to the Epstein
model (10) showed that the CspB binding to T-based ssDNA template has
high affinity (Ka ~ 3 × 106
M Our results show that ssDNA binding properties of CspB are strongly
correlated with the T-base content in the ssDNA templates. Indeed,
ssDNA templates with similar T content have similar binding properties,
independent of whether the T bases form continuous or non-continuous
stretches. We also show that the forces involved in the CspB binding to
most of the non-homogeneous T-based ssDNA template are not via
electrostatic interactions of protein groups with the phosphate
backbone because no difference in the binding profiles is observed
under experimental conditions with different ionic strength. This
implies that the interactions of CspB with non-homogeneous T-based
ssDNA templates occur mainly through the bases. These observations
combined with the sequence analysis of 5'-UTR of cold shock-inducible
proteins suggests possible function of CspB as RNA chaperone.
CspB and ssDNA Purification--
CspB was overexpressed in
Escherichia coli and purified as described previously (8,
11). The protein concentration was measured spectrophotometrically
using the extinction coefficient Analytical Centrifugation Experiments--
The analytical
centrifugation experiments were performed in a Beckman XLA
ultracentrifuge. The runs were performed at 4 °C under two different
speeds, 20,000 and 25,000 rpm, until equilibrium was achieved (usually
>10 h). Absorbencies at 260 and 280 nm were recorded simultaneously
for three cells. Each cell contained only ssDNA, CspB, or CspB-ssDNA
complex. The ssDNA concentrations were 2 µM for 23ApT3
and 23ApT11 and 2.5 µM for 23CpT3, 23CpT7, and 23CpT11.
The protein concentration was 20-fold higher than the ssDNA
concentration. The CspB-ssDNA complex concentration was the same as in
the other cells. To allow for the complex formation, the protein was
incubated with the ssDNA at room temperature for 25 min prior to each
run. The partial specific volume for the protein, ssDNA, and complex
were 0.74, 0.55, and 0.65 cm3/g, respectively and
calculated as described previously (8).
ITC--
ITC experiments were performed using a VP-ITC (Microcal
Inc. Northampton, MA) instrument. The protein and ssDNA were dialyzed simultaneously in 50 mM Tris, pH 7.5, 100 mM
NaCl buffer. The protein concentration was usually between 10 and 15 µM. Typical concentrations of the ssDNA were between 85 and 135 µM. Series of several injections of 5 or 10 µl
each of solution containing ssDNA template into ITC cells containing
CspB were made until no further heat effect was observed. The heat of
dilution was measured by injecting identical amounts of ssDNA into the
ITC cell containing only buffer. The heat of the reaction was obtained by integration of the peak area after each injection of ssDNA solution,
using ORIGIN software provided by the manufacturer. Fluorescence Measurements--
The CspB fluorescence intensity
was measured using a FluoroMax spectrofluorometer with DM3000F software
and thermostated cell holder connected to a circulating water bath. The
equilibrium titrations were performed at low (0.3 µM)
protein concentration in 50 mM Tris-HCl, pH 7.5, with 100 mM NaCl (low ionic strength) or 1 M NaCl (high
ionic strength). Tryptophan fluorescence was excited at 287 nm, and the
emission was recorded at 349 nm. Stoichiometric titrations were
performed at high protein concentration (14 µM) in low
ionic strength buffer. The excitation and emission wavelengths were 300 and 349 nm, respectively. All experiments were performed at 25 °C,
and the initial volume in the sealed quartz cell was 1.1 ml. Small
aliquots of concentrated solutions of ssDNA were added into the cell
containing solution of CspB. The solution in the cell was gently
stirred during the titration and the intensity values were corrected by
dilution; inner filter effect and blanks were subtracted.
The equilibrium titration profiles, under conditions when only one
molecule of CspB was bound to the ssDNA template, were fitted to
Equation 1 (8).
Previously, using a combination of four different techniques, we
showed that CspB seems to bind T-containing homogeneous ssDNA template
with much higher affinity (Kd = 42 nM at
25 °C) than C-, G-, or A-containing 23-mer homogeneous ssDNA
templates (13). In this study we used hetero-ssDNA templates to
investigate how the change in length of a T stretch flanked by other
nucleotides (A, C, and G), the relative position of the T stretches
within the template and continuous versus non-continuous
stretches of T bases affect the thermodynamics of the CspB-ssDNA
interactions. With few exceptions the ssDNA templates were 23 nucleotides long (Table I). CspB-ssDNA
interactions were monitored using three different techniques:
fluorescence spectroscopy, isothermal titration calorimetry, and
analytical centrifugation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1) and moderate cooperativity
(
~ 16). To further characterize the CspB binding to T-based
ssDNA templates, two major questions must be answered. 1) How is CspB
binding to continuous stretches of T bases affected by the presence of
other bases in the oligonucleotide? 2) How is such a binding affected
when the T bases form non-continuous stretches? To address these
questions, we used fluorescence spectroscopy, analytical
centrifugation, and isothermal titration calorimetry and measured the
CspB binding properties to series of ssDNA (23-mers) in which the T
bases were placed in the middle of the oligonucleotide (23CpTi, 23ApTi,
and 23GpTi, where i = 3, 5, 7, 11, 15, and 17), forming a
continuous stretch but varying its length. The results of these
experiments were compared with the effect of non-continuous stretches
of T bases, the effect of location of the T bases within short ssDNA
template and the effect of the total length of the oligonucleotide on
the CspB binding. In addition, the nature of the mechanism responsible
for the CspB binding to non-homogeneous ssDNA template was investigated
by comparing the binding profiles at high (1 M) and low
(0.1 M) NaCl concentrations.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
280 = 5,690 M
1 cm
1
(8). The ssDNA oligonucleotides were obtained from Biosynthesis Inc.
and purified as described previously (8). The ssDNA concentration was
calculated considering the following extinction coefficients at 260 nm:
8400 M
1
cm
1 (for T), 12010 M
1 cm
1
(for G), 7050 M
1
cm
1 (for C), and 15,200 M
1 cm
1
(for A) (12).
Hcal was calculated by summing the individual
heats, corrected for the heats of dilution, and dividing by the total
number of moles of CspB present in the ITC cell during the experiment.
Detailed description of the ITC experiment has been reported previously (8).
Q represents the quenching effect on the CspB Trp
fluorescence after each addition of ssDNA, Qmax
is the maximal quenching obtained upon complete saturation of the
protein with ssDNA, [CspB]tot and
[ssDNA]tot are the total concentration of protein and
ssDNA in solution, respectively, and Ka is the
equilibrium association constant for the CspB-ssDNA interaction.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Sequence of the oligonucleotides used throughout this study
Effect of 23-mer Non-homogeneous ssDNA Templates with Different Number of T Bases on the CspB Tryptophan Fluorescence-- The changes in the protein intrinsic tryptophan fluorescence is a widely used to monitor protein-DNA interactions (e.g. Refs. 14 and 15). The cold shock protein CspB has only one Trp residue that is located in the putative nucleic acid binding face of the protein (9). The fluorescence intensity of this Trp is dramatically quenched upon interaction with T-based ssDNA templates (8). This quenching of CspB Trp fluorescence correlates with the degree of binding upon the CspB-ssDNA interactions (8).
Fig. 1 (A-C) shows the
quenching effects caused by non-homogeneous ssDNA templates 23CpTi,
23GpTi, and 23ApTi (i = 3, 5, 7, 11, 15, and 17; see Table I for
sequences) on the CspB intrinsic Trp fluorescence. In all cases there
is a shift in the titration profiles toward lower ssDNA concentrations
as the number of T bases in ssDNA template increases. The difference in
the binding competences, defined as [ssDNA]0.5, between
XT3 and XT17, where X represents C, G,
or A bases, is about 1 order of magnitude (from ~4 × 107 M to ~6 × 10
8 M, respectively). Comparing
the three series of ssDNA templates (23CpTi, 23GpTi, and 23ApTi), it is
clear that ssDNA templates with the same T content behave in very
similar fashion in the 23CpTi and 23GpTi series (Fig. 1, A
and B). In fact, there is a good correspondence between the
binding competences for the ssDNA templates with the same number of T
bases in both series (Fig. 2). The
correlation coefficient is 0.98, indicating that the interaction is
independent of the presence of C or G bases surrounding the stretches
of T. This result strongly indicates that the CspB interactions with
these ssDNA templates are probably independent of three-dimensional
structure of ssDNA in agreement with previous results (13).
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The titration profiles of CspB with the 23ApTi template series are different than the ones obtained with the 23CpTi or 23GpTi series (Fig. 1, compare C with A and B). The titration profiles for the ssDNA templates 23ApT3, 23ApT5, 23ApT7, and 23ApT11 overlap. This result can be explained considering that, in the 23ApTi series, some of the ssDNA templates have the ability to form double-stranded DNA due to the classical Watson-Crick base pairing, as will be further supported by the analytical centrifugation data (see below). However, ssDNA templates with larger number of T bases (23ApT15 and 23ApT17) do show an enhancement in binding, as seen by the decrease in the [ssDNA]0.5 as the stretch of T bases becomes longer (Fig. 1C).
Taken together, these results indicate that there is a strong correlation between the [ssDNA]0.5 and the T content in the template when the T bases are located in the middle of the ssDNA template and form a continuous stretch. Observed differences in [ssDNA]0.5 for different templates can arise from the changes in the affinity of binding and/or in the stoichiometry of the interaction.
It must be noted that the ssDNA templates used in this study are not homogeneous and relatively short (most of them are 23-mer). Unfortunately, no rigorous mathematical model has been developed for the analysis of the titration profiles in which more than one molecule of protein binds to the short and heterogeneous matrix (14, 16). To the best of our knowledge, the mathematical models available to date have been developed either for homogeneous and infinitely long linear templates (17), or for homogeneous and short lattices (10) or even homogeneous templates for which linearity between the signal change and the fractional saturation does not necessarily hold during the binding reaction, but not for short non-homogeneous templates (for reviews, see Refs. 14 and 16). We thus are not able to estimate the CspB/ssDNA binding affinities. We can, however, experimentally estimate the stoichiometry of the CspB binding to non-homogeneous ssDNA templates using analytical centrifugation and isothermal titration calorimetry.
Stoichiometry of Non-homogeneous ssDNA-CspB Complexes-- For homogeneous ssDNA templates, it was shown that three CspB molecules bind to 23pT and only one CspB molecule binds to 23pC (8). However, the templates used in present study are not homogeneous, and thus different stoichiometry among templates with different T-content is expected.
The stoichiometry for several of the 23CpTi-CspB and 23ApTi-CspB complexes was determined by analytical centrifugation. The apparent molecular masses of the ssDNA, CspB, and the ssDNA-CspB complex were measured simultaneously in the same experiment. The results are summarized in Table II. Under our experimental conditions, the average molecular masses for the protein and the 23CpTi ssDNA (8.0 ± 0.4 and 7.1 ± 0.2 kDa, respectively) are in good correspondence with the expected values for monomeric species (7.5 and 6.9 kDa, respectively) (Fig. 3A). Considering the apparent molecular masses of the CspB:23CpTi complexes and individual species (Table II), we find that two molecules of CspB bind to 23CpT3, 23CpT7, and 23CpT11. Although this result is clear for the 23CpT7 and 23CpT11 templates, a lower value was obtained for 23CpT3 (the ratio CspB:23CpT3 is 1.5). Nevertheless, isothermal titration calorimetry experiments (see below) support our conclusion that more than one molecule of CspB binds to 23CpT3. We can therefore conclude that the difference in binding competences observed in the equilibrium titration profiles from 23CpT3 to 23CpT11 (Fig. 1A) is due to an increase in the protein effective binding affinity and not due to a change in the stoichiometry of the interaction. It is notable that the increase in effective binding affinity directly correlates with the T-content in the ssDNA.
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The calculated stoichiometries for 23ApT3 and 23ApT11 show that these
oligonucleotides bind only one CspB molecule (Table II). This is not
surprising for 23ApT3 because CspB does not bind to homogeneous A-based
templates (8). So, if one molecule of CspB binds to the three T bases
in 23ApT3, this will represent an "imperfect" binding because we
have shown previously using 23pT template that the CspB binding site
size is 6-7 bases long (8). An imperfect binding should translate into
a lower affinity of the protein for this ssDNA, as is observed
experimentally (Fig. 1C). When only one molecule of protein
binds to the ssDNA template, the interaction is accurately described by
the classical binding equation (see Equation 1, under "Materials and
Methods"), and it has been used to describe the thermodynamics of
binding in different protein-DNA systems (8, 18). From the fit of the titration profile of CspB with ssDNA templates to Equation 1, the
calculated equilibrium association constant (Ka) for
CspB interaction with 23ApT3 is (4.3 ± 0.1) × 106 M1, which is
indeed lower than the effective affinity for 23pT ~5 × 107 M
1 (8). In
contrast, 23ApT11 has enough T bases to bind one molecule of CspB with
the same affinity as 23pT. Experimentally, however, we observe that the
normalized titration curve for 23ApT11 overlaps with that for 23ApT3
(Fig. 1C), indicating that 23ApT3 and 23AT11 have comparable
affinities. The fit of the CspB:23ApT11 titration curve to Equation 1
gives an equilibrium constant Ka of (4.4 ± 0.1) × 106 M
1,
identical to that of 23ApT3 but an order of magnitude lower than the
effective affinity for 23pT. The reason why the CspB molecule bound to
23ApT11 presents lower affinity than expected seems to be related to
the fact that this non-homogeneous template has the ability to form
double-stranded DNA. Indeed, the measured molecular mass for the
23ApT11 template is higher than that predicted for the ssDNA (10.1 ± 0.1 kDa versus 6.8 kDa, respectively, Table II),
indicating probably the presence of a significant population of
double-stranded DNA under our experimental conditions. CspB does not
have detectable affinity to double-stranded DNA (5, 6). Thus, if at
least part of the 23ApT11 forms double-stranded DNA via classical
Watson-Crick A-T base pairing, the effective concentration of ssDNA
will be overestimated and that would result in a lower apparent
affinity, as is observed experimentally (Fig. 1C).
The stoichiometry of the CspB interaction with some of the
oligonucleotides within the 23CpTi series was also studied using ITC.
The protein solution (in the calorimetric cell) was titrated with the
ssDNA (in the syringe) until no heat effects were observed. Under
experimental conditions with high protein concentration (11-15
µM CspB in solution), we observe that the titration
profiles are very steep and rapidly level off (Fig. 3B),
allowing determination of the stoichiometry of the interaction from the
breaking point in the titration profile. The breaking point for the
23CpT15 template is ~ 0.3, indicating that 3 molecules of CspB
bind to 23CpT15. The titrations with 23CpT11 and 23CpT5 are shifted
toward lower stoichiometry. The breaking point for the titrations of
these two templates is at a ratio ssDNA/CspB ~ 0.5, indicating
that 2 molecules of CspB bind to 23CpT11 and 23CpT5 templates. Finally, the titration with 23CpT3 is not as sharp as the other ones; however, the curve could not be fitted according to Equation 1 using 1:1 stoichiometry of CspB/ssDNA complex (Fig. 3B). This result
suggests that more than one molecule of protein binds to the 23CpT3
template and supports our conclusion based on the analytical
centrifugation experiments that probably two CspB molecules bind to
23CpT3 template. The enthalpies of the interaction per binding site for
these oligonucleotides were 105 ± 9 kJ/mol, comparable to the
values obtained for 23pT,
119 ± 6 kJ/mol (8). Similarity of the
enthalpies of interactions with different T-based ssDNA templates
suggests similar structural features for the CspB-ssDNA complexes.
In summary, the stoichiometries for the CspB interaction with the 23CpTi oligonucleotides determined by analytical centrifugation and ITC are in good agreement. The results show that 23CpT3, 23CpT5, 23CpT7, and 23CpT11 bind two CspB molecules. Knowing that the CspB binding site size is 6-7 T bases (8), our results suggest that not only the T bases but also the C bases are somehow involved in the CspB binding to those ssDNA templates. In addition, the ITC experiments show that there are changes in the total number of protein molecules bound to 23-mer ssDNA templates (two or three CspB molecules bind to 23-mer ssDNA templates depending on the number of T bases in the oligonucleotide).
Properties of the CspB Binding to 23-mer ssDNA with Non-continuous
Stretch of T Bases--
The experiments described in the previous
sections have shown that CspB interactions with ssDNA templates vary
with the number of T bases forming a continuous stretch (Table II,
Figs. 1 (A-C) and 3B). To evaluate how the
different content of T bases forming non-continuous stretches of T
might affect the CspB-ssDNA interaction, the ATTGG sequence was
incorporated in four 23-mer oligonucleotides: 23CT2,
23CT4, 23CT8, and 23CT12
(see Table I for sequence). The reason we chose this sequence to
be incorporated into the ssDNA was that initially it was considered
that CspB binds specifically to ATTGG sequence (19). Later, however, it
has been shown that the CspB binding to ssDNA is not limited to this
sequence (7, 8). Nevertheless, it constitutes a good model to study the CspB interaction with 23-mer ssDNA with non-continuous stretches of T
bases. It is important to note that the T bases in these non-continuous
stretches are only one or two bases apart. The normalized equilibrium
titrations with these oligonucleotides are shown in Fig.
4. The binding curves for the CspB
titration with 23CT12 (with a total of 12 T) and 23CpT11
(with a total 11 T) overlap. The same is observed for the CspB
titrations with the other pairs of templates: 23CT8 (8 T
bases) and 23CpT7 (7 T bases), 23CT4 (4 T bases), and
23CpT5 (5 T bases) and 23CT2 (2 T bases) and 23CpT3 (3 T
bases). These results suggest that the [ssDNA]0.5 for
these oligonucleotides containing non-continuous stretches of T bases
separated by one or two bases is very comparable to the values obtained
for ssDNA with similar T content forming continuous stretches of T. The
importance of the potential implications of these findings is obvious.
First, it is not the local structure of the ssDNA that is important for
CspB binding. Second, not all bases in the CspB binding site are
equally important for the interaction. The latter is particularly
significant because non-continuous stretches of T bases are more often
found in DNA, as compared with long and continuous stretches of T
bases.
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CspB Binding to ssDNA Templates Containing Two Continuous Stretches
of T Bases--
The next step was to study the CspB binding to
non-homogeneous oligonucleotides of different length with stretches of
T in which the T bases were more than two bases apart and not in the middle of the template but situated at the ends. A constant stretch of
seven C bases was always in the middle of these templates, which are
flanked by different number of T bases (Table I). The reason C bases,
as opposed to A or G, were selected to be in the middle of the
oligonucleotides was to avoid possible double-stranded DNA formation
(in the presence of A bases), and we chose seven because it appears
that the size of the CspB binding site on T-based ssDNA template is
6-7 nucleotides long (8). Fig.
5A shows the titration
profiles of CspB with T18C7T18, T14C7T14, and T9C7T9 under
stoichiometric conditions. Since titrations were performed under
stoichiometric conditions, we observe, from the breaking point in the
titrations, that about 6 nucleotides are covered by a molecule of CspB
in agreement with our previous observations (8). These results indicate
that, when the T content in the ssDNA template is high (72% or more)
and the continuous stretches of T bases are on both ends of the ssDNA,
these templates behaved as if they were homogeneous. Fig. 5B
shows the normalized equilibrium titrations of CspB with T18C7T18,
T14C7T14, T9C7T9, T7C7T7, and T5C7T5. It is clear that the
[ssDNA]0.5 for these ssDNA templates increases as the
length of the ssDNA decreases, being more dramatic for T5C7T5. A
similar effect was observed by Kowalczykowski et al. (20).
They studied the binding of gene 32 protein to poly(rA) as a function
of the oligonucleotide length (>400, 120, and 56 nucleotides). A
rigorous binding analysis of the titration profiles showed that the
affinity and cooperativity was the same for all three oligonucleotides,
although more protein was needed to saturate the 56-mer poly(rA) than
to saturate the 120-mer or >400 mer. This effect has been also
observed for other proteins interacting with shorter oligonucleotides,
i.e. the mitochondrial Y-box protein RBP16 interacting with
guide RNAs. RBP16 has a cold shock domain and interacts with guide RNAs
through an oligo(U) tail (21). This interaction required higher
[RNA]0.5 (defined as the molar excess required to achieve
50% inhibition of the RBP16 binding to 32P-labeled guide
RNA gA6) as the length of the oligo(U) decreased from 40 to 4 nucleotides (22).
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Importance of the Relative Position of the T Bases in 23-mer ssDNA
Templates--
The effect of relative position of the T bases within
ssDNA template was investigated by comparing the CspB binding to the 23GpTi series (the T bases are in the middle) and the 23pTiG series (in
which the T bases are on both ends; see Table I for sequence) keeping
constant the total length of the ssDNA template at 23 bases. Fig.
6 shows the normalized profiles for the
titrations of CspB with the 23pTiG oligonucleotides. The binding curves
for the 23pT20G, 23pT18G, and 23pT16G overlap. The CspB binding
competence for these ssDNA templates is about 5.8 × 108 M, which is very close to the
CspB binding competence for 23pT, 6.1 × 10
8 M (8). This result again
indicates that when the T content in non-homogeneous ssDNA templates is
high (70% in 23pT16G or more) and the T bases are on both ends of the
ssDNA templates they behave as if they were homogeneous. We can also
conclude, from this and previous results with the TiC7Ti series, that
as long as the stretch in the middle is short enough (30% or less of
the total length of the template) the oligonucleotide will behave as
homogeneous 23pT independent of which bases are in the middle, C or G
(Figs. 5B and 6A, respectively). As the T content in the ssDNA templates decreases (e.g. 23pT12G, 23pT8G, and
23pT6G series), there is a corresponding increase in the
[ssDNA]0.5 (Fig. 6A). To understand whether
this effect is due to the difference in the number of T bases or due to
the different position of these bases within the oligonucleotides, we
plotted the [ssDNA]0.5 for the 23pTiG and 23GpTi series
as a function of the T content (percentage of T bases) in the ssDNA
(Fig. 6B). The dependence of the CspB binding competences on
the T content in the 23pTiG and 23GpTi templates is very comparable for
both series of ssDNA, except for the 23pT6G template in which "end
effects" or possible aggregation of the G bases may play a role. This
similar behavior suggests that it is the T content in the ssDNA
templates, and not its relative position, which defines the properties
of CspB binding to T-based ssDNA, although some "end effects"
cannot be completely ruled out. The importance of the "end effects"
was clearly demonstrated for the interaction of the Y-box protein RBP16
from Trypanosoma brucei, which contains a cold shock domain
motif, with 34-mer oligonucleotides (22). The 34-mer oligonucleotides
had a patch of four adjacent uridylates, forming the binding site, and
the rest of the oligonucleotide was oligo(dC). The tetraU patch was moved from 5' to 3' at certain intervals and the binding of RPB16 to
these different templates was compared. It was shown that binding is
weaker when tetraU patch was located close to the 5' or 3' ends of the
template. Furthermore, Pelletier et al. (22) showed that the
affinity also depends on whether tetraU patch is located at 5' or 3'
end.
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Effect of High Ionic Strength on the CspB Binding to Non-homogeneous ssDNA-- It has been shown that CspB binding to 23pC is strongly dependent on the salt concentration and could be practically abolished in 1 M NaCl, whereas the CspB binding to 23pT is independent of salt concentration (8). These results were interpreted to mean that there are two different modes of CspB interactions with polypyrimidine ssDNA templates. In mode I, the interactions of CspB with poly(dC) occurs predominantly through the phosphate backbone and thus is largely electrostatic and strongly dependent on the ionic strength. In mode II, the interactions of CspB with poly(dT) occurs predominantly through van der Waals and stacking interactions with the bases and are thus independent of the ionic strength. We thus investigated which of these two modes of interactions was involved in the CspB binding to non-homogeneous ssDNA templates. This was done by comparing the CspB binding to different oligonucleotides (with continuous and non-continuous stretches of T bases) measured in low (0.1 M) and high (1 M) NaCl concentrations. It was anticipated that if the CspB binding to C bases is independent of the presence of T bases, then under 1 M NaCl concentration the CspB binding to the C bases in the ssDNA would be dramatically impaired, which would translate in a lower quenching effect on the CspB Trp fluorescence upon binding. Qualitative results of the analyses presented in Table I show that the presence of 1 M NaCl did not affect the titration profiles of CspB with 23CpTi (i = 3-23 forming a continuous stretch of T bases). These results suggest that CspB interacts directly with the T bases, mainly through hydrogen bonding, van der Waals interactions, and hydrophobic effects, but not through electrostatic interactions of protein groups with the phosphate backbone. How many T bases are required in order to have a salt-independent binding? To answer this question, the interactions of CspB with two other ssDNA, 23CpT1 and 23CpT2, with just one or two T bases (Table I) at low and high salt concentrations were studied. It was found that for these two oligonucleotides there is an effect of 1 M NaCl on the titration profiles. The decrease, however, is not as pronounced as in the case of 23pC, where 1 M NaCl completely abolishes CspB binding (8). We thus can conclude that the switch from mode I to mode II of CspB-ssDNA interactions requires presence of at least 3 T bases in the template.
The only other templates, which also exhibit the dependence of the binding on the ionic strength, are 23CT2 and 23pT6G (Table I). In the case of 23CT2, such a behavior is understandable because this template contains only two T bases. The 23pT6G template contains six T bases arranged into two triplets. According to the observations made on the other ssDNA templates, the presence of three T bases should render salt-independent quenching effect on CspB-ssDNA interactions. However, these T bases are located at the 5' and 3' ends of the 23pT6G template, and the end effects might be important. Indeed, the effect on 1 M NaCl on CspB titration is significantly reduced in 23pT8G and completely abolished in 23pT12G (Table I). We thus propose that the switch from mode I to mode II binding for CspB ssDNA requires at least 3 T bases located within the ssDNA template or 4 T bases if located at the 5' or 3' ends.
These results suggest that the mechanism of the CspB interaction with
most of the studied non-homogeneous T-based ssDNA templates is through
the bases and does not involve electrostatic interactions of protein
groups with the phosphate backbone. The large enthalpies of binding to
the non-homogeneous T-based ssDNA templates (105 ± 9 kJ/mol)
also support this conclusion because binding to the 23pC (which is
strongly salt-dependent) is accompanied by 4 times lower
enthalpy,
25 ± 2 kJ/mol (8). Moreover, in terms of the binding
enthalpy, the non-homogeneous T-based oligonucleotides behaved as the
homogeneous 23pT, indicating that hydrogen bonding, van der Waals
interactions, and hydrophobic effects are the major forces responsible
for CspB binding to ssDNA templates (8).
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DISCUSSION |
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In this study it has been shown that the ability of the cold shock protein CspB from B. subtilis to bind ssDNA templates correlates with the content of T bases present in the template. Moreover, the binding competences of CspB for continuous stretches of T are independent of the surrounding bases (Fig. 2), suggesting that the local structure of the ssDNA does not play significant role in the interactions. Comparable binding properties are observed when CspB binds continuous or non-continuous stretches enriched in T bases (Figs. 4, 5, and Fig. 6B). This result suggests that the presence of T is not equally important at all position of the binding site consisting of 6-7 bases. Our findings also strongly suggest that, in the CspB interaction with non-homogeneous ssDNA templates, it is the protein binding to the T bases which drives the interaction. Just three T bases are enough to switch the mechanism of interaction from CspB interacting with ssDNA phosphate backbone (like in 23pC) to CspB interacting directly with the bases (like in 23pT and most of the other studied ssDNA series) as the CspB binding becomes independent of the ionic strength (Table I). Our recent studies have shown that binding of CspB to ssDNA-template based on another pyrimidine nucleotide U is much more similar to the binding characteristics of 23pT than 23pC (13). In particular, binding of CspB to 23pU under the conditions of high and low ionic strengths are comparable (13).
If the biological function of CspB is indeed related to its high
affinity for stretches of ssDNA enriched in T bases, there are two
T-rich regions identified in DNA. One occurs at factor-independent transcription termination signals (see Refs. 23-27 for reviews). The
other is frequently located downstream from the promoter sequences, as
part of the sequences contained within 5'-UTR of cold shock proteins
(9). Recently, we have shown that the CspB binding parameters for 23pU
are quite comparable to the ones for 23pT (13) (although the former has
somewhat lower affinity, it might be related to the fact that we used
d(pU) instead of r(pU)). There are two cold shock boxes enriched in U
bases within the cspB 5'-UTR (Fig.
7), which are highly conserved in another
member of the cold shock family of proteins from B. subtilis, the cspC 5'-UTR (7). We have performed CspB
binding studies with each of these motifs using ssDNA templates with U
bases substituted by T and found that
CspB binds with high affinity, as it is expected from their high T
content.2 Thus, our results support the idea that indeed
these motifs could have a regulatory role in vivo as
suggested by Graumann et al. (7). Since the CspB binding
affinity increases as the temperature decreases (8), the protein
binding to the cold shock Box1 and Box2 is more favored at low
temperatures. Protein binding to such regions would prevent formation
of secondary structure of the 5'-UTR (Fig. 7); hence, CspB would
facilitate translation at low temperatures. This 5'-UTR region has been
shown to be important in stabilizing the mRNA for the major cold
shock protein CspA from E. coli (28). Comparison of the
5'-UTRs among the cold shock proteins in E. coli clearly
shows that the proteins that are cold-inducible (CspA, CspB, CspG, and
CspI) have very long 5'-UTR (>100 bases) and have tendency to form
similar secondary structures at low temperatures (Fig. 7). The same
appears to be true for 5'-UTR of CspB and CspC from B. subtilis. The average Gibbs energy of secondary structure
formation for the cold shock-inducible 5'-UTR calculated according to
SantaLucia (29) is (260 ± 50) kJ/mol. However, those proteins
that are not cold-inducible (CspC, CspD, CspE) have short 5'-UTRs
(40-90 bases), have relatively low secondary structure stability,
(120 ± 50) kJ/mol, and thus low tendency to fold at low
temperatures. This correlation suggests that, upon cold shock, the
increase in CspB induction might be essential for keeping its 5'-UTR
region unstructured. Comparison of the 5'-UTR regions among cold shock
proteins in E. coli has showed that there is a well
conserved 11-base sequence (cold shock box) (28). However, the sequence
of the cold shock box in the 5'-UTR of cold-inducible proteins in
E. coli (UGACGUACAGA) is very different from the cold shock
boxes found in the same region in B. subtilis
(AUUAUUUUUGUUC) that is very rich in U bases (Fig. 7). It is
conceivable that cold shock proteins have diverse functions. The
ability to facilitate transcription antitermination has been suggested
for CspA from E. coli (30), whereas B. subtilis
CspB might be preventing mRNA folding and thus facilitating
translation at low temperatures. This possible functional difference is
supported by the observation that cold-inducible proteins from
different bacteria may recognize different sequences, as we have
already demonstrated for CspB from B. subtilis and CspA
from E. coli (8, 13).
|
Our results suggest a model for the role of the cold shock protein CspB
from B. subtilis. At low temperatures and low concentration of CspB, the nascent mRNA will fold, thus interfering with
translation. It is important to note that cold shock boxes are located
within two putative stem structures of the 5'-UTR (Fig. 7). In the
presence of CspB, protein binding to the cold shock boxes 1 and 2 can
be the driving force to prevent the mRNA to adopt stem structure. Thus, CspB binding will effectively prevent secondary structure formation and thus will facilitate translation. Probably, two CspB
molecules will bind with high affinity per each of two cold shock
boxes, as these boxes are about 14 nucleotides long and, although not
completely homogeneous, are highly enriched in U bases (Fig. 7). This
would suggest that cold shock proteins possess RNA-chaperone activity
(31), a possible function of cold shock proteins that is in accord with
the earlier observations. It has been shown that both CspA (32, 33) and
CspB (7) bind RNA and increase its susceptibility to the ribonuclease
digestion, thus indicating that CspA/CspB binding prevents the
formation of secondary structure of RNA, i.e. acting as an
RNA chaperone.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Fried for many helpful suggestions and Miyo Sakai for performing experiments on Beckman XL-A.
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FOOTNOTES |
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* This work was supported by Human Frontier in Science Program Grant RPG-0036/1997M.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: Dept. of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, PA 17033. Tel.: 717-531-0712; Fax: 717-531-7072; E-mail: makhatadze@psu.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010474200
2 M. M. Lopez and G. I. Makhatadze, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CSP, cold shock protein; UTR, untranslated region; ssDNA, single-stranded DNA; ITC, isothermal titration calorimetry.
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