(Received for publication, April 22, 1996, and in revised form, September 13, 1996)
From the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
CspA, the major cold-shock protein of
Escherichia coli, is dramatically induced during the
cold-shock response. The amino acid sequence of CspA shows 43%
identity to the "cold-shock domain" of the eukaryotic Y-box protein
family, which interacts with RNA and DNA to regulate their functions.
Here, we demonstrate that CspA binds to RNA as a chaperone. First, CspA
cooperatively binds to heat-denatured single-stranded RNA if it is
larger than 74 bases, causing a supershift in gel electrophoresis. A
minimal concentration of CspA at 2.7 × 105
M is absolutely required for this cooperative binding,
which is sufficiently lower than the estimated cellular concentration of CspA (10
4 M) in cold-shocked cells. No
specific RNA sequences for CspA binding were identified, indicating
that it has a broad sequence specificity for its binding. When the
142-base 5
-untranslated region of the cspA mRNA was
used as a substrate for ribonucleases A and T1, the addition of CspA
significantly stimulated RNA hydrolysis by preventing the formation of
RNase-resistant bands due to stable secondary structures in the
5
-untranslated region. These results indicate that binding of CspA to
RNA destabilizes RNA secondary structures to make them susceptible to
ribonucleases. We propose that CspA functions as an RNA chaperone to
prevent the formation of secondary structures in RNA molecules at low
temperature. Such a function may be crucial for efficient translation
of mRNAs at low temperatures and may also have an effect on
transcription.
The cold-shock response is a physiological response of living cells to temperature downshift, which is considered to be essential for cells to survive at low temperatures (for review, see Ref. 1). When an exponentially growing culture of Escherichia coli is shifted from 37 to 10 °C, there is a lag period of cell growth for approximately 4 h before cell growth is resumed (2). Cold-shock proteins are induced during this lag period, and a total of 17 cold-shock proteins have been identified by two-dimensional gel electrophoresis (2, 3, 4, 5).
CspA is the major cold-shock protein in Escherichia coli,
and its production reaches more than 10% of total cellular protein synthesized during the cold shock response (6). It has been suggested
that the expression of cspA is regulated at the level of
transcription (6, 7, 8, 9) as well as at the level of post-transcription (10,
11). CspA consists of 70 amino acid residues with a molecular mass of
7.4 kDa (6). The three-dimensional structure of CspA has been
determined both by NMR (12) and x-ray crystallography (13). CspA
consists of five anti-parallel sheets, which form a
-barrel
structure. RNA binding motifs RNP1 (Phe18-Gly19-Phe20) and RNP2
(Phe31-Val32-His33) are located on
-2- and
-3-sheet, respectively (12). Seven of eight aromatic
residues are distributed on the same surface of the CspA molecule,
suggesting potential interactions between CspA and nucleic acids
through hydrophobic interactions. Indeed, it has been shown that CspA
interacts with a 24-base ssDNA1 (12).
Interestingly, the amino acid sequence of CspA shows 43% identity to the "cold-shock domain" (CSD) of the eukaryotic Y-box protein family. All Y-box proteins characterized to date show varied nucleic acid binding activities (for review, see Refs. 14, 15, 16). Some Y-box proteins bind to DNA to affect gene transcription, DNA replication, and DNA repair, while other Y-box proteins interact with mRNA to affect translational efficiency. Furthermore, it has been demonstrated that the CSD is directly responsible for nucleic acid binding activities (17, 18).
In this paper, we demonstrate that CspA cooperatively binds to ssRNA. A
certain length of ssRNA (more than 74 bases) is required for the
cooperative binding. CspA binding to ssRNA displays a low sequence
specificity. A minimal concentration of CspA required for RNA binding
is 2.7 × 105 M, which is sufficiently
lower than the cellular concentration of CspA in cold-shocked cells
(10
4 M). In the presence of CspA, ssRNA
became much more sensitive to ribonucleases, and furthermore, secondary
structures formed in the RNA molecule that are resistant to
ribonucleases became highly susceptible to ribonuclease digestion.
These results indicate that CspA binding to RNA results in unfolding
RNA secondary structures. We propose that CspA functions at low
temperatures as an RNA chaperone, which binds to mRNAs with a low
sequence specificity to prevent the formation of secondary structures
in mRNAs. Such a function is considered to be important for
efficient translation of mRNAs at low temperatures.
All NTPs were purchased from
Boehringer Mannheim. [-32P]CTP (400 Ci/mmol) was
purchased from Amersham Corp. SP6 RNA polymerase was from New England
Biolabs. T7 RNA polymerase was from Promega. RNase inhibitor was
purchased from Boehringer Mannheim. All restriction enzymes were from
New England Biolabs.
CspA was purified as described previously (19).
Preparation of RNA FragmentsRNA fragments (fragments A-F;
see Figs. 1 and 2) were obtained as described previously (20).
Basically, RNA fragment A was prepared by transcribing the pGEMI
(Promega) plasmid cut with PvuII using SP6 RNA polymerase.
Fragment B was prepared by transcribing the plasmid pSP65 cut with
XbaI using SP6 RNA polymerase. Fragment C was made by
transcription with SP6 RNA polymerase of pGEM3cs plasmid cut with
HaeII (21). Fragment D was made by transcription with SP6
RNA polymerase of pGEM3 plasmid cut with SacI. Fragment E
was prepared by transcribing pGEM3cs
cut with RsaI using
T7 RNA polymerase. Fragment F was synthesized by transcribing pGEM3 cut
with AccI using T7 RNA polymerase.
The RNA fragments, from A1 to A6, were synthesized in the way shown in
Fig. 3A. Briefly, fragment A1 was made by transcribing pGEMI
cut with HindIII by SP6 RNA polymerase. Fragment A2 was made
by transcribing pGEMI cut with EcoRI by SP6 RNA polymerase. Fragment A3 was made as follows. pGEMI was digested by
HindIII and EcoRI, followed by Klenow fill-in and
self-ligation. The resulting plasmid was cut with PvuII and
transcribed by SP6 RNA polymerase. Fragment A4 was prepared by cutting
pGEMI with XbaI and EcoRI, followed by
self-ligation. The resulting plasmid was cut with PvuII and
transcribed by SP6 RNA polymerase. Fragment A5 was made as follows. The
60-base pair fragment from pTH1 digested with SacI (22) was
inserted into the SacI site of pGEMI. The resulting plasmid
was digested by HindIII and transcribed by SP6 RNA
polymerase. Fragment A6 was made by transcribing the same plasmid used
for A5 cut with PvuII using SP6 RNA polymerase. All labeled
RNA fragments were labeled with [-32P]CTP. In
vitro transcription was carried out using the protocols provided
by the manufacturer.
Preparation of DNA Fragments
dsDNA fragment A was made as
follows. pGEMI was cut with HindIII and end-labeled by
Klenow filled-in with [
-32P]dATP. The linear DNA
fragment was then cut with PvuII followed by gel
purification of the resulting 88-base pair fragment A
. As a result,
only the minus strand DNA was labeled. The labeled minus strand DNA was
then obtained by heat-denaturing the labeled dsDNA fragment A
. The
plus strand DNA was labeled with [
-32P]ATP by T4
kinase after heat-denaturing dsDNA fragment A
. This procedure also
labeled the 3
-end-labeled minus strand DNA at its 5
-end as well.
RNA binding assay was carried out in a 15-µl reaction containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM KCl, 7.4% glycerol, 1 × 106 cpm RNA template, and purified protein. Mixtures were incubated on ice for 20 min and then loaded on a 8% polyacrylamide gel. The running buffer was 1 × TBE (50 mM Tris-Cl, 1 mM EDTA, 50 mM boric acid). Electrophoresis was carried out at 130 V for 3.5 h at 4 °C. The gel was vacuum-dried and visualized by autoradiography. The DNA binding assay was carried out in exactly the same manner.
Ribonuclease Assay32P-Labeled RNA substrates
were incubated with 3 µg of purified CspA in the binding buffer for
15 min on ice. The volume of the final reaction mixture was 15 µl.
RNase A or RNase T1 was added to the RNA-substrate solution, and the
mixture was kept on ice for 15 min in the case of fragment B or at
15 °C for 5 min in the case of the 5-UTR of the cspA
mRNA. Half of the mixture was loaded onto a 8% acrylamide gel. In
the case of fragment B, the remaining half was heat-denatured for 5 min
and subjected to 7 M urea, 10% acrylamide gel
electrophoresis. The region corresponding to the 5
-UTR from +1 to +142
of the cspA mRNA was placed under T7 promoter using
polymerase chain reaction, and in vitro transcription of the
polymerase chain reaction-amplified template was carried out in the
presence of [
-32P]CTP according to the protocol
provided by the manufacturer.
To examine whether CspA can bind to RNA, we
first utilized a partially double-stranded RNA as shown in Fig.
1. RNA fragments A and B were synthesized by in
vitro transcription, and fragment B was labeled with
[-32P]CTP. When fragments A and B were annealed, the
product had a 29-base pair double-stranded region with single-stranded
regions at the 5
-end (28 bases) and the 3
-end (41 bases) on fragment A and at the 5
-end of fragment B (9 bases) (Fig. 1). Using the annealed complex, gel retardation experiments were carried out with
different amounts of the purified CspA as shown in Fig. 1. When the
annealed complex that migrated at position b (lane
1) was heat-denatured and quickly cooled on ice before loading on the gel, 32P-labeled fragment B was separated from
nonradioactive fragment A and migrated at position a
(lane 2). When increasing amounts of CspA were added to the
annealed complex, its migration became retarded shifting from position
b to c (lanes 3-6). As a control, the
same amounts of bovine serum albumin (BSA) were added, where no shifted
band was observed (lanes 7 and 8). It is
interesting to note that the amounts of CspA used in this assay were
rather high; 0.5, 1, 2, and 3 µg of CspA was added in a final 15-µl
reaction mixture in lanes 3, 4, 5, and
6 in Fig. 1, respectively. In addition, the shift caused by
CspA was discontinuous as the band was suddenly shifted from position
b to c at 2 µg of CspA (lane 5) and
more notably at 3 µg (lane 6). The CspA concentration in
the reaction mixture at 3 µg was calculated to be 2.7 × 10
5 M. At 1 µg (lane 4), no
shift was observed. This result suggests a cooperative binding of CspA
to RNA.
In order
to find out which strand in the annealed complex (A or B) is
responsible for CspA binding, fragments A and B were labeled with
[-32P]CTP separately. Both fragments were
heat-denatured before the addition of CspA. As shown in Fig.
2, the same amount of CspA used in Fig. 1 was able to
cause a mobility shift of fragment A (lanes 1 and
2) but not fragment B (lanes 3 and 4).
To investigate whether CspA binding to heat-denatured ssRNA has any
sequence specificity, four other RNA fragments (fragments C, D, E, and F) shown in Fig. 2 were synthesized by in vitro
transcription. They were also labeled with [
-32P]CTP,
and a gel retardation assay was carried out as described above. Among
the new RNA fragments, fragments C (lanes 5 and
6) and E (lanes 9 and 10) were found
to form complexes with CspA, as judged from their retarded migration in
the gel. In contrast, CspA did not bind to fragments D (lanes
7 and 8) and F (lanes 11 and
12).
Those fragments incapable of CspA binding (fragments B, D, and F) were
shorter in length (39, 52, and 46 bases, respectively) than those
fragments to which CspA was able to bind (fragments A, C, and F are 98, 107, and 103 bases, respectively). Such a size dependence of RNA
binding has also been observed with FRGY2, a Xenopus Y-box
protein (18). In order to examine the effect of the length of ssRNA on
CspA binding, a number of deletions or insertions were created based on
fragment A as shown in Fig. 3A. The results
of a gel retardation assay performed with these fragments are shown in
Fig. 3B. CspA could not bind to fragment A1 (32 nucleotides)
and A2 (56 nucleotides), which resulted from deleting 66 (fragment A1)
and 42 bases (fragment A2) from the 3-end of fragment A (lanes
1 and 2 and lanes 3 and 4,
respectively). When an internal 46-base region was removed, leaving the
42-base 3
-end sequence that was deleted in fragment A2, a minor
retardation was observed with the resulting fragment A3 (52 nucleotides; lanes 5 and 6). When the internal
deletion became shorter by 22 bases, CspA was able to bind well to the
resulting fragment A4 (74 nucleotides), causing a supershift
(lanes 12 and 13) as observed with fragment A
(lanes 7-9). These results demonstrate that CspA binding to ssRNA requires a certain length of the target RNA. Fragment A4 is the
shortest template that is capable of CspA binding.
Based on the fact that CspA caused a minor retardation of fragment A3,
it was postulated that CspA binding to heat-denatured ssRNA may also
have some sequence specificity that exists in the 3-end region of
fragment A. To test this possibility, a 60-base sequence from pTH1 (22)
digested with SacI was added to the 3
-end of fragment A2,
creating fragment A5 of 116 bases in length, which is longer than
fragment A (see Fig. 3A). CspA was found to hardly bind to
fragment A5 (Fig. 3B, lanes 14 and
15). This result indicates that there is a preferable CspA
binding sequence located at the 3
-end region of fragment A in addition
to the length required for CspA binding. The 56-base 5
-end sequence of
fragment A (fragment A2) does not contain such a sequence as required
for the CspA binding. Note that the 60-base sequence added to fragment
A2 did not have any negative effect on the CspA binding, since the
insertion of the sequence at the middle of fragment A resulting in
fragment A6 did not inhibit the supershift of fragment A6 (lanes
16 and 17). When the nucleotide sequences of fragments
A, C, and E, all of which showed specific interaction with CspA, were
compared with each other, a 15-base sequence common to all three
fragments was found: CUAUAGUGAGUCGUA, from base 66 to 80 in fragment A
and from base 25 to 39 in fragment C and CUAUAGUGucUCcUA in fragment E
from base 25 to 39 (Fig. 2; mismatched bases in lowercase type). This
region in fragment A (8 nucleotides at a time) was randomized, and the
SELEX method (23) was applied to screen the most preferable sequences
for CspA binding. However, this approach was unsuccessful, since CspA
was still able to bind to the fragments with the randomized
sequences.
We
further examined the cooperation of CspA binding to ssRNA using
fragment A. As shown in Fig. 4, when 1 µg of CspA was
added to 32P-labeled fragment A in a 15-µl reaction
mixture, a minor retardation was observed (compare lane 2 with lane 1). However, when 3 µg of CspA were added in the
same final volume (15 µl) as in the case of lane 2, the
band was supershifted (lane 3). This supershifted band did
not change its migration even when a 24-fold excess of nonradioactive
fragment A was added to the reaction mixture (lane 4),
suggesting that all RNA molecules added were shifted. A similar result
was obtained when a 5-fold (lane 6) or 24-fold (not shown) excess of 32P-labeled fragment A was added, indicating that
CspA binding to ssRNA is cooperative. This cooperative binding is only
dependent on the CspA concentration (2.7 × 105
M) and not dependent on the molar ratio of CspA to RNA.
This was demonstrated by the following experiment; when 1 µg of CspA, which could not cause the supershift in lane 2, was used in
a 5-µl reaction mixture (one-third of the reaction volume used in the
experiment in lane 2), the supershift was observed as shown in lane 5. In this reaction, the CspA concentration became
identical to that with 3 µg of CspA in the 15-µl reaction mixture.
This critical concentration can be calculated to be approximately
2.7 × 10
5 M. Note that the
concentration of CspA inside the cold-shocked cell is estimated at
10
4 M (6), which is higher than the critical
concentration obtained above.
CspA Binding to ssDNA
It is known that most Y-box proteins
that contain a cold-shock domain are capable of nucleic acid binding
(for review, see Refs. 14, 15, 16). Some of them bind to RNA, while others bind to DNA. Therefore, it was interesting to investigate whether CspA
is also able to bind to ssDNA. To test this, a dsDNA (fragment A) was
generated from the template DNA used to generate fragment A RNA (see
"Experimental Procedures"). Fragment A
was labeled by
[
-32P]dATP at the 3
-end of the minus strand by Klenow
fill-in. As shown in Fig. 5, CspA failed to bind the
dsDNA fragment A
(lanes 1 and 2). When this
dsDNA was heat-denatured, the labeled minus strand was separated from
the plus strand and migrated at position b (lane
3). The addition of CspA caused a minor shift of the minus strand
(compare lane 4 with lane 3). As a result of a
minor shift of the minus strand in the presence of CspA, it migrated at
the same position as double-stranded fragment A
at position
c. Therefore, we subsequently performed the following
experiment. In addition to the 3
-end labeling, the 5
-ends of both
strands were also labeled with [
-32P]ATP by T4 kinase.
When this dsDNA was heat-denatured, it yielded two distinct bands at
positions a and b (lane 5). The band
at position b corresponded to the minus strand according to
lane 3, where only the minus strand was labeled. Thus, the
band at position a corresponded to the plus strand. When
CspA was added to the heat-denatured fragment A
, two bands at
positions c and d appeared (lane 6).
The band at position c appears to correspond to the bands
observed in lane 4, resulted from a minor shift of the minus
band caused by CspA. Therefore, the band at position d was
caused by a supershift, which resulted from the CspA binding to the
plus strand, the band at position a in lane 5.
These results indicate that CspA is able to bind to both ssRNA and
ssDNA with the same sequence specificity. However, CspA cannot bind to
dsDNA.
CspA Functions as an RNA Chaperone
"RNA chaperone" refers
to proteins that prevent RNA misfolding and resolve misfolded RNAs,
thereby ensuring their biological functions (24). Based on RNA binding
characteristics of CspA presented above, which include low sequence
specificity, cooperative binding, and low binding affinity to RNA
(2.7 × 105 M), it is speculated that
CspA may function as an RNA chaperone.
First, we attempted to examine whether CspA interacts with secondary
structures in RNA. For this purpose, we tested the effect of CspA on
non-heat-denatured fragment B. As shown in Fig. 2, CspA does not
interact with heat-denatured fragment B. When fragment B was analyzed
by acrylamide gel electrophoresis without heat denaturation, two
additional bands appeared at positions b and d
(Fig. 6A, lane 1), in addition to
the band at position a, which is considered to be the same
as heat-denatured fragment B (lane 2). RNAs at positions
b and d are likely to be dimers and trimers of
fragment B, respectively, which resulted from intermolecular hybrid
formation using a number of short inverted repeats in fragment B. Interestingly, when increased amounts of CspA were added to non-heat-denatured fragment B (0.2, 0.5, 1, 2, and 3 µg for
lanes 3, 4, 5, 6, and
7, respectively), band b shifted to c
and band d disappeared with concomitant appearance of band
e. Band a hardly moved as previously shown in
Fig. 2, and the addition of the same amounts of BSA (1, 2 and 3 µg
for lanes 8, 9, and 10, respectively) did not change the pattern. In order to confirm that CspA did not
modify the RNA molecule, the same reaction mixtures used above were
loaded on a 7 M urea-10% acrylamide gel, and all gave a
single band at the same position as shown from lane 1 to
10 in Fig. 6B. These results indicate that CspA
can also interact with certain structures formed by intermolecular
interaction between RNA molecules.
Next, the ribonuclease assay was carried out to examine whether CspA binding to RNA secondary structure facilitates the RNA susceptibility to ribonuclease A. In the absence of CspA, fragment B was degraded by RNase A into two major products, indicated as X and Y (Fig. 6B, lane 11). However, when 3 µg of CspA was added to fragment B followed by the addition of RNase A, product Y was further degraded and migrated to the bottom of the gel (not shown in Fig. 6B), while product X was unchanged (lane 12). Note that CspA itself did not contain any ribonuclease activity (Fig. 6B, lanes 3-10), this result indicates that fragment B becomes more sensitive to the RNase A in the presence of CspA. When the equal amount of BSA was added with RNase A, no enhancement of RNase A digestion was observed (lane 13).
On the basis of the results obtained above, we next used a longer RNA
from the cspA mRNA, which has a long 5-UTR consisting of 159 bases (6). Out of the 159-base 5
-UTR, the RNA from +1 to +142
was produced under a T7 promoter in the presence of [
-32P]CTP. As shown in Fig. 7, this RNA
migrated at position h in 8% acrylamide gel electrophoresis
(lane 1). When CspA (3 µg in the final volume of 15 µl)
was added, it was supershifted to position j (lane
2). When 0.5 µg of RNase A was added to the RNA and the mixture
was incubated for 5 min at 15 °C, five RNase A-resistant bands
appeared at positions a, b, c,
d, and e (lane 3). As the concentration of RNase was reduced to 0.25 and 0.125 µg, the density of band e increased, while the density of band d
decreased (lanes 4 and 5, respectively). Further
reduction of RNase A to 0.063 µg resulted in substantially long RNA
fragments at positions f and g without producing
shorter fragments at positions c, d, and e (lane 6). However, when 3 µg of CspA was
added before the addition of RNase A, RNase-resistant bands at
positions c, d, and e disappeared as
shown in lanes 7, 8, and 9,
respectively, leaving mainly bands a and b. At
the lowest RNase A concentration in the presence of CspA (lane
10), a broad, supershifted band at position i as
well as very broad bands around position f were
observed.
Similar results were obtained with RNase T1; in the absence of CspA, digestion of the RNA with 0.4, 0.2, and 0.1 µg of RNase T1 (lanes 11, 12, and 13, respectively) resulted in the formation of a stable RNA fragment at positions o and l. In the presence of 0.4 µg of RNase T1, part of the band at position o was further digested to produce a band at position m. When CspA was added into the reaction mixture, the band at position o completely disappeared, leaving a very stable band at position l in all concentrations of RNase T1 (lanes 14-16). It should be noted that in the presence of 0.4 µg of RNase T1 (lane 14), the background in the region higher than band l was very low, and a faint band at position n observed in the absence of CspA (lanes 11-13) was not detected. Instead, another stable band at position k appeared. It should also be noted that CspA itself did not have RNase activity as shown in lane 2 of Fig. 7, where RNA was quantitatively supershifted.
The results described above demonstrate that CspA indeed enhances
susceptibility of RNA to RNases; in the case of RNase A, it was
estimated that CspA stimulates the RNase activity approximately 10-fold
(compare lane 9 with lane 3 in Fig. 7).
RNase-resistant bands observed in Fig. 7 are considered to be due to
secondary structures formed in the RNA molecule. Fig. 8
shows a predicted secondary structure of the 5-UTR of the
cspA mRNA from +1 to +142. In such a structure, loop
regions are sensitive to RNase cleavage, which generates a number of
fragments of different sizes like bands a, b,
c, d, and e in RNase A digestion and
k, l, m, and n in RNase T1
digestion (cleavage at G residues circled in Fig. 8). The
fact that these bands disappeared in the presence of CspA indicates
that CspA destabilizes the secondary structures existing in RNA
molecules to further digest them into smaller fragments. We, therefore,
propose that CspA functions as an RNA chaperone.
CspA shows 43% identity with the CSD of eukaryotic Y-box proteins
that have RNA or DNA binding activities (for review, see Refs. 14, 15, 16).
In previous studies, it has been suggested that CspA binds to DNA and
is involved in gene regulation (3, 25). In addition, the
three-dimensional structure of CspA strongly indicates that CspA is a
nucleic acid-binding protein (12). In this paper, we have demonstrated
that CspA binds cooperatively to heat-denatured ssRNA as well as ssDNA
with a low sequence specificity, while CspA cannot bind to dsDNA. A
certain length of ssRNA (74 nucleotides) and the minimal concentration
of CspA at 2.7 × 105 M are required for
the supershift of the RNA caused by CspA. Since the CspA concentration
in cold-shocked cells was estimated to be 10
4
M (6), CspA in cold-shocked cells is able to cooperatively bind ssRNA. In addition to CspA binding to heat-denatured ssRNA, we
demonstrated that CspA is able to interact with RNA secondary structures formed either intermolecularly or intramolecularly. As a
result, RNA becomes more susceptible to RNase digestion, and
RNase-resistant double-stranded RNA fragments disappear. All of these
properties of CspA support a notion that CspA functions as an RNA
chaperone, which weakly interacts with RNA to affect RNA folding. Such
a property is considered to be essential for efficient translation of
mRNA at low temperatures (1). Recently, a new cold-shock protein,
CsdA, was found, which is exclusively associated with ribosomes and has
double-stranded RNA unwinding activity (5). It is possible that CsdA
unwinds stable secondary structures formed in mRNAs at low
temperatures and that CspA, as an RNA chaperone, binds to unwound
mRNAs. Alternatively, CspA first binds to RNA to assist the CsdA
activity to unwind folded RNA. The weak binding of CspA to mRNAs,
however, probably does not interfere with the translational activity of
ribosomes, but rather may facilitate mRNA translation.
It is possible that CspA may share some functional similarities with a
Xenopus Y-box protein, FRGY2, which is known to mask mRNA in oocytes (26, 27, 28). FRGY2, however, binds to a 95-base RNA
having a specific 6-base sequence at a Kd value of
2 × 108 M, 1000-fold lower than the
concentration of CspA required for its binding to RNA (18). Its CSD of
77 residues by itself was shown to still retain a reasonably low
Kd value (about 10
7 M)
with the sequence-specific RNA binding ability. In contrast, our
attempts to determine the specific CspA-binding sequences by SELEX (23)
were unsuccessful. At present, it is not known what accounts for the
differences in RNA binding and sequence specificity between CspA and
the FRGY2 CSD. One can speculate that FRGY2 acquired the
sequence-specific binding ability as well as the ability to bind to RNA
more tightly during evolution. Also, other domains of FRGY2 may
contribute to the higher affinity to RNA as well as the sequence
specificity for its binding. It is important to note that FRGY2 is a
major component of masked maternal mRNA in Xenopus
oocytes, while CspA is speculated in the present paper to be an RNA
chaperone to facilitate mRNA translation at low temperatures.
Nevertheless, these two proteins also share some similar properties
such as the size requirement of RNA and the ability to bind nonspecific
RNA (Ref. 18 and this study). It has been speculated that nonspecific
binding of CspA to RNA may occur through the hydrophobic interactions
between nucleotide bases and the seven aromatic side chains regularly
aligned on the surface of the CspA molecule (12). Mutations of these
aromatic residues were indeed shown to affect the interaction between
RNA and proteins for CspB from Bacillus subtilis (29) and
for FRGY2 (18).
We thank Li Fang for helpful assistance and discussions throughout this work. We also thank Drs. Guy Montelione and Beate Schwer for advice.