From the Norris Cancer Center/Keck School of
Medicine, University of Southern California, Los Angeles, California
90089-9176 and the § Center for Biomolecular Interaction
Analysis, University of Utah School of Medicine, Salt Lake City, Utah
84132
Received for publication, February 21, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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Binding of the U1A protein to its RNA
target U1 hairpin II has been extensively studied as a model for a high
affinity RNA/protein interaction. However, the mechanism and kinetics
by which this complex is formed remain largely unknown. Here we use
real-time biomolecular interaction analysis to dissect the roles
various protein and RNA structural elements play in the formation of
the U1A·U1 hairpin II complex. We show that neutralization of
positive charges on the protein or increasing the salt concentration
slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within
the RNA/protein interface, or reducing the size of the RNA loop,
dramatically destabilizes the complex, as seen by a strong increase in
the dissociation rate. Our data support a binding mechanism consisting
of a rapid initial association based on electrostatic interactions and
a subsequent locking step based on close-range interactions that occur
during the induced fit of RNA and protein. Remarkably, these two steps
can be clearly distinguished using U1A mutants containing single amino
acid substitutions. Our observations explain the extraordinary affinity
of U1A for its target and may suggest a general mechanism for high
affinity RNA/protein interactions.
To execute their widely differing functions, RNA-binding proteins
must be able to bind to their correct RNA targets with appropriate kinetics, affinities, and specificities (1). In contrast to most
DNA-binding proteins, which are presented with a double-stranded B-form
helix of uniform structure in which bases can be contacted through the
major groove, RNA-binding proteins must be able to bind targets with
widely differing structures. Because the steep and narrow groove of
double-stranded RNA does not provide proteins easy access to the bases
for sequence-specific recognition, most RNA-binding proteins recognize
single-stranded regions or distorted double-stranded regions in which
the major groove has been widened by bulges, hairpins, or loops (2).
The natural variety of RNA targets is bound by a limited collection of
RNA-binding motifs (1, 2). The most common of these motifs is the
ribonucleoprotein (RNP)1
consensus domain or the RNA-binding domain, also referred to as
the RNA recognition motif (RRM). This motif is characterized by two
conserved stretches of eight and six amino acid residues (RNP-1 and
RNP-2) and a The U1A/U1hpII interaction has been used as a paradigm for RNA binding
by a single RRM and has been the subject of a multitude of biochemical
and structural analyses (4). Despite these extensive studies, little is
known to date about the mechanism and kinetics of this protein/RNA
interaction. Using the previously solved structure of the U1A·U1hpII
complex, we have engineered a series of mutants designed to
individually examine the roles of electrostatics, hydrogen bonding,
aromatic stacking, and RNA loop length, all of which have been
implicated in formation of the U1A·U1hpII complex (5-16). The
effects of these mutations on the binding dynamics were studied using a
surface plasmon resonance-based biosensor (BIACORE), which permits the
real-time monitoring of complex formation and dissociation (17-19).
Our analyses show that complex formation occurs by two clearly
distinguishable steps. First, well placed positively charged residues
on the protein allow it to rapidly associate with the RNA. Next,
close-range interactions at the RNA/protein interface allow the
formation of a very stable complex. Together, these steps result in the
high affinity of U1A for its U1 hairpin II RNA target
(KD ~32 pM). A similar two-step mechanism may play a role in many high affinity RNA/protein interactions.
Construction of the U1A Mutants and Protein
Purification--
The expression plasmid for the human recombinant U1A
protein (amino acids 1-101) was described previously (20). Using this plasmid, a U1A clone with a collection of engineered restriction sites
throughout the coding region (U1A-MSHEB) was made by
site-directed mutagenesis. All engineered restriction sites were silent
at the amino acid level, except a BssHII site that resulted
in a Lys88 Gel Shifts--
U1hpII RNA for the gel shift was made as
described previously (20), and gel shifts were carried out in a 10-µl
final volume of binding buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.5% Triton X-100, 0.25 mg ml Biosensor Analysis--
Surface plasmon resonance was used to
monitor the interactions of a set of variant U1A proteins binding to a
variety of RNA targets under different buffer conditions. Kinetic
experiments were performed on both BIACORE 2000 and BIACORE 3000 biosensors (Biacore, Inc., Piscataway, NJ). RNA targets were chemically
synthesized (Dharmacon Research, Boulder, CO) with a 5'-biotin tag to
allow the capturing of RNA molecules on streptavidin-coated sensor
chips. RNA was diluted to 1 µM in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) heated at 80 °C for 10 min, cooled to room temperature to allow annealing of the stem, diluted
500-fold in running buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 5% glycerol, 62.5 µg ml Equilibrium Analysis of the U1A/U1hpII Interaction--
The U1A
protein, which has a structural role in U1snRNP, has two RRMs (25).
Only the N-terminal RRM domain, however, is required for binding to
U1hpII RNA (26-28). The same RRM also mediates binding of U1A to two
adjacent target sites in the 3'-untranslated region of its own
mRNA, thereby autoregulating U1A expression by preventing
polyadenylation (29). We used a 101-amino acid N-terminal U1A fragment
(referred to here as U1A; previously shown to be required and
sufficient for specific, high affinity binding to U1hpII RNA (27, 28);
see Fig. 1A). Before
initiating kinetic analyses on the biosensor, we assessed the
equilibrium binding affinity of the recombinant human U1A polypeptide
using traditional gel shift experiments (Fig.
2A). Triplicate experiments
yielded an equilibrium binding constant (KD) of 4.7 ± 0.7 × 10 Kinetics of the U1A/U1hpII Interaction--
To study the kinetics
of the U1A/U1hpII interaction on the biosensor, chemically synthesized
5'-biotinylated U1hpII (Fig. 1B) was captured on one BIACORE
chip flow cell, whereas a second, unmodified flow cell served as a
reference surface. A representative data set for the U1A/U1hpII
interaction is shown in Fig. 2B. The overlay of triplicate
injections of each U1A concentration demonstrates that the biosensor
assay is highly reproducible. As expected, the responses during the
association phase are concentration-dependent. The dissociation is
slow, demonstrating the stability of the U1A·U1hpII complex over
time. No binding was detected when a mutated target (in which the order
of the loop residues had been inverted) or an unrelated RRM (RRM3 from
yeast poly(A)-binding protein) were used (data not shown). The kinetic
data in Fig. 2B were modeled using a simple 1:1 Langmuir
interaction that included a term for mass transport (23) and were
analyzed using global analysis (24). The use of the 1:1 interaction
model resulted in an excellent fit to the data, as evidenced by the
overlay of the simulated curve (red lines in Fig.
2B) and the experimental results. The entire biosensor
experiment was repeated three times using individually prepared samples
and sensor surfaces. The kinetic results obtained from these
independent studies were very similar (see Table
I), further demonstrating the
reproducibility of the U1A biological system and the biosensor
technology.
The kinetics of the U1A/U1hp interaction are marked by a fast
association rate (ka = 1.1 ± 0.2 × 107 M Positively Charged Residues Facilitate Rapid Association--
To
dissect the role of electrostatic interactions in U1A·U1hpII complex
formation, we used the U1A·U1hpII co-crystal structure (5) to
identify positively charged residues that are located near the RNA
binding pocket but are not implicated in hydrogen bonding interactions.
Consequently, we excluded a number of residues that interact with RNA
bases in the splayed-out loop (Arg47 to G11,
Arg52 to A1, Lys80 to U3, Arg83 to
U3, and Lys88 to C5 (5, 7, 11)). In addition, we avoided
mutating Lys96 and Lys98, because the
C-terminal region of the RRM had been reported to be required for high
affinity binding (13, 14). This left the positively charged residues
Lys20, Lys22, and Lys50, all of
which are conserved in U1A from mammals (25, 35), Drosophila
(36), Xenopus (X57953), and plants (37) and are also present
in the related RNA-binding protein U2B, which binds to a similar stem
loop in U2snRNP (25, 38). In the RNA·protein complex,
Lys20 and Lys22 lie near the base of the RNA
stem, in an area between Increasing the NaCl Concentration Reduces the Association
Rate--
If the roles of the lysine residues are to promote
electrostatic interactions with the phosphate backbone, it would be
expected that increasing the salt concentration in the buffer would
lead to a loss in binding affinity of U1A for U1hpII. Indeed, filter binding experiments showed a hundredfold loss in U1A·U1hpII
equilibrium binding affinity as the NaCl concentration was increased
from 150 to 500 mM (31). To assess how the increased NaCl
concentration affects the reaction kinetics, we analyzed the U1A/U1hpII
interaction at NaCl concentrations of 150 mM, 275 mM, 500 mM, and 1 M (Fig. 4). In agreement with results from filter
binding assays, we observed a hundredfold increase in the
KD as the NaCl concentration was raised from 150 to
500 mM (Table I). Binding was completely abolished in 1 M NaCl (data not shown). From the analysis of the kinetic
data we determined that the loss in affinity was attributable to a
decrease in the association rate, which dropped 59-fold as the NaCl
concentration was increased to 500 mM. In contrast, the dissociation rate remained relatively constant, varying less than 3-fold across this NaCl concentration range (Table I).
The marked effect of NaCl concentration on the association rate
strongly suggests that the initial interaction of U1A with its RNA
target is based on electrostatic interactions, which may play a role in
prolonging the time the molecules collide, as well as in enhancing the
probability of correct alignment (34). If this assumption is correct,
mutation of positively charged residues involved purely in
electrostatic interactions should diminish the effect of the NaCl
concentration. We measured the effect of NaCl concentration on the
association rate of the Lys20,22Ala and Lys50Ala mutants and compared
them to that obtained for wild type U1A (see Fig. 4D and
Table I). The slopes of the log(ka) versus log[NaCl] plots were reduced from Aromatic Stacking and Hydrogen Bonding Interactions Stabilize the
Complex--
We next examined the kinetic effects of mutations that
would prevent stacking or hydrogen bonding interactions that occur in
the U1A·U1hpII RNA interface. To this end, the interaction between
U1A mutant Phe56Ala and wild type U1hpII and U1hpII mutant G4C (Fig.
1B) and wild type U1A were studied. Phe56 stacks
on base A6 in the RNA loop, which in turn stacks on base C7 and
Asp92 (Fig. 5A).
In the free protein, Phe56 is hidden from the solvent and
covered by Ile93. The Phe56:A6 stacking must
therefore be accompanied by rearrangements in the protein (12). Base G4
stacks on amino acid Gln54 and also makes hydrogen bond
contacts with residues Asn15 and Glu19 (Fig.
5C). Mutation of G4 to C would cause loss of these hydrogen bonds whereas the ability of the base to stack on Gln54
would be maintained. A G4 to A mutation had been previously reported to
decrease the affinity three to four orders of magnitude (14, 15). Based
on previous structural analyses, both the mutant protein and the mutant
RNA would be predicted to show strong effects on the dissociation
kinetics of the complex, because they are involved in short range
interactions that form during the induced fit of RNA and protein.
Kinetic analyses of the binding interactions showed that Phe56Ala
exhibited a 1400-fold increase in dissociation rate, while showing a
less than 5-fold decrease in association rate (see Fig. 5B
and Table I). Similarly, the U1hpIIG4C RNA showed a 2500-fold increase
in dissociation rate but displayed a less than 4-fold decrease in
association rate (see Fig. 5D and Table I). Our observations
support the idea that aromatic stacking and hydrogen bonding
interactions that mediate the intimate contact of the RNA binding
surface and the splayed-out bases do not play a strong role in the
initial step of association but are critical for the ability to form a
stable complex.
RNA Loop Size Is Important for Stable Complex
Formation--
Several features of U1hpII RNA are critical for
recognition, including the presence of a stem, the identity of the
closing base pair, and the identity of the first seven of ten loop
nucleotides (AUUGCAC) (15, 26, 39). The last three loop nucleotides are
thought to function as a spacer and can be replaced by a polyethylene glycol linker without loss of binding affinity (30). Indeed, in the
3'-untranslated region targets, which are very similar in structure and
sequence, two of these three nucleotides form part of a stem linking
the two targets (40). The need for the spacer nucleotides is linked to
the fact that the loop between
In addition to the dramatic increase in the dissociation rate, a
15-fold loss in the association rate was also seen with the U1hpII A Multistep Model for Binding--
Our kinetic analyses, combined
with structural information about the free and bound protein and RNA,
suggests that formation of the U1A·U1hpII complex proceeds in at
least two steps, which we call "lure" and "lock". First, the
protein and RNA are electrostatically attracted through well placed
positive charges on the protein and negative charges on the RNA (the
phosphate backbone). This initial interaction is followed by a rapid
induced-fit event, which locks the RNA and protein into a stable
complex. The presence of positively charged residues surrounding the
RNA binding pocket supports this notion. These positive charges could
aid association by increasing the time that the free RNA and protein
remain close together following a random collision, thereby increasing
the odds that during subsequent collisions, both molecules will adopt an orientation compatible with locking (34, 41). In this scenario, flexibility of the free RNA loop would facilitate establishment of the
initial electrostatic contacts, allowing the RNA backbone to "mold"
onto the RNA binding site. As soon as the orientations of the RNA and
protein are compatible, close-range interactions could initiate between
the two molecules, resulting in interactions that require
rearrangements in protein and RNA (such as stacking of
Phe56 on A6). An induced-fit mechanism in which the RNA,
the protein, or both adapt during complex formation appears to play a
role in many RNA/protein interactions (42). Our observation that this
induced fit (lock) is preceded by an electrostatically-mediated binding step (lure) warrants detailed kinetic investigations of other
RNA·protein complexes. The distribution of positively charged residues along the RNA binding tract of poly(A) binding protein (43),
Sex-lethal (44), and nucleolin (45), three multi-RRM proteins, suggests
these proteins bind RNA by a similar two-step mechanism. The initial
electrostatically based association step may offer a way of engineering
RNA-binding proteins with increased affinity for their targets, through
the introduction of more positively charged residues near the RNA
binding area, leading to an increase in the association rate.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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REFERENCES
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secondary structure (see Fig. 1A) (3, 4). RRMs fold into a baseball glove-like structure in which the
-sheet and the surrounding regions form the RNA binding
surface. Proteins containing one or more RRMs recognize a variety of
RNA sequences and structures (3, 4). An RRM that binds very tightly to
its RNA target is the N-terminal RRM of the spliceosomal protein U1A,
which binds to an RNA hairpin in the U1 small nucleolar (sn) RNP
(U1 hairpin (hp) II or U1hpII) (see Fig. 1).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Arg88 substitution. Proteins
from both plasmids had identical binding properties (data not shown).
The MSHEB plasmid was used to generate the mutants used here by
digesting the plasmid with the unique restriction sites flanking the
amino acid to be mutated and replacing the released fragment with
annealed complementary oligonucleotides encoding the specific
substitution (in addition to translationally silent restriction sites
included for easy identity verification). The mutation in each of the
clones was confirmed by sequencing and/or restriction digests. All of
the clones contained a C-terminally fused MYC tag and a hexahistidine
tag used in protein purification. Constructs were transformed into
Escherichia coli strain BL21/DE3 (Novagen, Madison,
WI). Proteins were expressed and purified as described previously (20,
21), with the following modification: a reduced NaCl concentration in
the sonication and elution buffers (150 mM NaCl). The
active concentration of each protein preparation was determined as
described by Christensen (22).
1
bovine serum albumin, 1 mM dithiothreitol, 0.5 mg
ml
1 tRNA, and 10% glycerol) as described previously
(21). Dried gels were analyzed using a Molecular Dynamics
PhosphorImager, and bands were quantitated with the ImageQuant software
(Amersham Pharmacia Biotech). The KD value
was calculated by plotting the logarithm of the ratio of the
complexed/free RNA against the logarithm of the protein concentration
(20). The final KD value given is an average of
three independent experiments.
1 bovine
serum albumin, 125 µg ml
1 tRNA, 1 mM
dithiothreitol, and 0.05% surfactant P20), and injected at 10 µl
min
1. For U1hpII, 25-35 resonance units of RNA were
captured on the streptavidin-coated sensor chip, whereas for the mutant
RNAs 100-125 resonance units were captured, as binding to these
mutants was significantly weaker, and therefore more RNA was required
to generate a reliable binding response. To study the U1A/U1hpII
interactions, the proteins were diluted in running buffer and injected
at the concentrations indicated in the sensorgrams. In the experiments aimed at determining the effect of the NaCl concentration, the running
buffer contained NaCl at 150, 275, 500, and 1000 mM.
Binding experiments were carried out at 20 °C and a flow rate of 50 µl min
1. Any protein that remained bound after a 5-min
dissociation phase was removed by injecting 2 M NaCl for
60 s at 20 µl min
1, which regenerated the RNA
surface completely. Analysis of each protein concentration was repeated
at least twice, and samples were run in random order. Any background
signal from a streptavidin-only reference flow cell was subtracted from
every data set. Data were fit to a simple 1:1 Langmuir interaction
model with a correction for mass transport (23) using the global data
analysis program CLAMP (24).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
11 M, which agreed well with
published values (13, 30, 31). Although equilibrium analysis provides
information about the affinity of a molecular interaction, it provides
no insight into the kinetics underlying the binding mechanism. To
obtain kinetic data for the U1A/U1hpII interaction, a BIACORE surface
plasmon resonance-based biosensor was used to monitor the formation of
the complexes in real time (32, 33).
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Fig. 1.
U1A protein and U1hpII RNA.
A, amino acid sequence of the N-terminal RRM domain (amino
acids 1-101) of the human U1A protein. Residues whose interaction with
U1hpII were studied are indicated in bold typeface. Residues
that were mutated are Lys20 and Lys22
(Lys20,22Ala), Lys50 (K50A), and Phe56 (F56A).
Asn15, Glu19, and Gln54 interact
with nucleotide G4 in U1hpII. The RNP-1 and -2 consensus sequences are
marked by an overline. Secondary structure features are
marked below the sequence (underline).
B, sequence of the U1hpII RNA used for the biosensor
analyses. Nucleotides U 5 to G15 are identical to the wild type
sequence. G4 (underlined) was mutated to C in the U1hpIIG4C
variant. The "spacer" nucleotides, whose identity is unimportant
for U1A binding, are U8-C10. The molecule is biotinylated at the
5'-end.
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Fig. 2.
Binding studies of U1A with U1hpII RNA.
A, gel shift analysis of U1A with U1hpII.
Radiolabeled U1hpII RNA was incubated with increasing concentrations of
U1A protein (given in pM below the
lanes). Free radiolabeled U1hpII is indicated by
F whereas C represents the shifted complex. The
experiment was performed in triplicate. B, BIACORE analysis
of the U1A/U1hpII interaction. Biotinylated U1hpII RNA was captured on
a streptavidin-coated sensor chip, and increasing concentrations of
protein were injected over the surface. The black lines
represent protein injections performed in triplicate at the indicated
concentration. The red lines represent the global fit of the
entire data set to a single site interaction model including a term for
mass transport component. Injections were performed for 60 s
followed by 5 min of buffer flow. The kinetic parameters for each of
three independent experiments are shown in Table I.
Kinetic and affinity constants for U1A/U1hpII interaction
1 s
1), as well
as a slow dissociation rate (kd = 3.6 ± 1 × 10
4 s
1), resulting in
a high affinity complex (KD = 32 ± 7 pM; see Table I). The close agreement of the
KD value obtained using BIACORE with that obtained
by gel shift analysis indicates that attachment of the RNA to the
BIACORE sensor chip surface does not perturb the reaction
thermodynamics. The fast association rate is consistent with the need
to include a transport step in the data analysis (23). The association
rate surpasses the expected diffusion-based rate constant for two
macromolecules in solution (~106
M
1 s
1 (34)), suggesting that
association may be influenced by electrostatic interactions that
increase the odds of productive collisions between the molecules.
-strand 1 and
-helix 1 that follows the
curve of the double-stranded stem. Lys20 and
Lys22 could play a role in drawing in the RNA by
interacting with the phosphate moiety of nucleotides A
4, U
3, and
C
2 (Fig. 3A).
Lys50 lies in the
2-
3 loop region and points into
solution in the free protein, whereas in the complex it protrudes
through the RNA loop (Fig. 3C). Thus it appears to be well
positioned to play a role in attracting the RNA to the binding pocket.
To investigate the role of these lysine residues in electrostatic
interactions, we replaced them with alanine. Lys20 and
Lys22 were altered together (Lys20,22Ala mutant),
because they appeared to be making similar contacts with the phosphate
backbone. Kinetic data for Lys20,22Ala and Lys50Ala binding to
immobilized U1hpII were fit well by a simple 1:1 bimolecular
interaction model (Fig. 3, B and D). The
Lys20,22Ala and Lys50Ala mutations resulted in a 39- and 16-fold loss
of affinity, respectively (Table I). In both cases an ~10-fold
decrease in the association rate contributed inordinately to this loss.
The Lys50Ala mutation had a minimal effect on dissociation of the
complex, indicating that the primary role of this positively charged
residue is likely to be in the initial positioning of the RNA loop,
possibly through interaction with the exposed phosphate backbone of the
free RNA loop. Besides the reduction in its association rate, the
Lys20,22Ala mutant also showed an ~4-fold increase in its
dissociation rate, indicating that Lys20 and
Lys22 also play a moderate role in complex stability.
However, for both Lys20,22Ala and Lys50Ala, the major effect on binding
resulted from the reduced association rate, indicating the importance
of these residues in bringing RNA and protein together.
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Fig. 3.
The role of electrostatic interactions in the
U1A·U1hpII complex. A, U1A·U1hpII complex as seen
from the back. The RNA loop is splayed out on the -sheet surface,
which is facing away. Lys20 and Lys22
(indicated in blue) in the U1A N-terminal RRM
(gray) lie close to the phosphates groups
(orange) of stem nucleotides A
4-C
2 of the RNA
(purple). B, BIACORE analysis of the interaction
of Lys20,22Ala with U1hpII (also see the legend for Fig. 2).
C, U1A·U1hpII complex seen from the front.
Lys50 (blue) is located in the protein loop
connecting
-strands 2 and 3 and protrudes through the RNA loop
(purple). D, BIACORE analysis of the interaction
of Lys50Ala with U1hpII (also see the legend for Fig. 2).
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Fig. 4.
The effect of salt concentration on
U1A/U1hpII interaction. Sensorgrams show the binding curves for
U1A/U1hpII interaction in buffer containing 150 mM NaCl
(A), 275 mM NaCl (B), and 500 mM NaCl (C). Protein concentrations used were as
follows: 0.1, 0.3, 0.9, 2.7, 8, 24, and 73 nM (in
A and B), and in C they were 1, 3, 9, 27, 81, and 245 nM. No binding was detected at 1 M NaCl (data not shown). All NaCl concentrations were
assayed on the same RNA surface. D, effect of the NaCl
concentration on ka. Experiments similar to
those shown in A-C were performed for the Lys20,22Ala and
Lys50Ala mutants, using the same U1hpII RNA surface.
Log(ka) versus log[NaCl] plots for
wild type U1A ( ), Lys20,22Ala (
), and Lys50Ala (
, solid
line) show a linear relationship.
3.3 (U1A wild
type) to
2.8 for Lys50Ala and
2.4 for Lys20,22Ala. Although both
mutants remained sensitive to the salt concentration (which is not
unexpected, because the remaining positively charged residues were left
intact), the reduction in this effect provides support for a model in
which electrostatic interactions play an important role in the rapid association of U1A and U1hpII.
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Fig. 5.
The role of stacking and hydrogen bonding
interactions in the U1A·U1hpII complex. A, diagram of
the position of Phe56 in the complex. Phe56
(green) stacks on A6 (orange), which in turn
stacks on C7 (purple), and Asp92 (dark
gray) within the protein. Other parts of the RNA and protein are
indicated by smaller size sticks in purple and
gray, respectively. B, BIACORE analysis of the
interaction of Phe56Ala with U1hpII (also see legend for Fig. 2).
C, diagram of the position of nucleotide G4 in the complex.
G4 (orange) stacks onto Gln54 (green)
and forms hydrogen bonds with Asn15 (yellow) and
Glu19 (blue). Other parts of the RNA and protein
are indicated by smaller size sticks in purple
and gray, respectively. D, BIACORE analysis of
the interaction of U1A with U1hpIIG4C (also see legend for Fig. 2). Due
to the weak interaction between the mutated RNA and the protein, an RNA
surface with higher capacity was used to obtain enough information for
the kinetic analysis.
-strands 2 and 3 of the protein
protrudes through the RNA loop, where it appears to aid in the splaying
of the loop bases so that contacts can be made with the protein
-sheet surface (5). Previous studies of the 10-nucleotide RNA loop
had shown that the length of this loop is important for optimal binding
(30). Although the identity of the last three loop nucleotides of the RNA target is irrelevant (39), removal of one or more of these nucleotides strongly reduced the binding affinity. We were curious as
to how much of this effect was due to the inability of the RNA and
protein to achieve initial association and how much of it to the
inability to form a stable complex. It is clear from the co-crystal
structure of the U1A·U1hpII complex that a minimal length of the loop
is needed to link the last conserved nucleotide (C7) with the top of
the RNA stem. The distance between C7 and the top of the stem is ~17
Å, a distance that could not be bridged by less than two nucleotides.
Optimally, 3 nucleotides may be required to comfortably accommodate the
protein
2-
3 loop. Reducing the loop size by too much would
clearly prevent the final complex from forming. On the other hand, it
could be argued that reducing the size of the loop may affect the
structure of the free RNA in solution and may therefore change the way
the RNA is presented to the protein. To distinguish between these
possibilities, we analyzed the kinetics of the interaction between U1A
and RNAs lacking C9 (U1hpII
C9) and U8-C9 (U1hpII
UC). Deletion of
a single C resulted in a loss of affinity of two orders of magnitude,
in accordance with previous equilibrium binding studies (30). Our kinetic analysis demonstrates that this could be attributed almost completely to a 70-fold increase in the dissociation rate of the complex (see Fig. 6A and Table
I). The association rate was decreased by less than 4-fold. These data
suggest that the role of the three linker nucleotides is indeed that of
a spacer, which allows the first seven loop nucleotides to be
accommodated on the protein surface. This is supported by the
observation that loop nucleotides 8-10 are not visible in the
co-crystal due to disorganization (5). Removal of two loop residues had
an even more pronounced effect; the KD increased by
over three orders of magnitude (see Fig. 6B and Table I).
Again most of this loss in affinity was due to a dramatic increase in
the rate of dissociation (~240-fold). Thus we conclude that a minimal
length of the loop is critical to allow assembly of a stable complex.
This is consistent with the requirement for the loop to circle the
protein
2-
3 loop bulge.
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Fig. 6.
Effects of RNA loop size reduction on U1A
binding. A, BIACORE analysis of the interaction of U1A
with U1hpII C9 RNA (also see legend for Fig. 2). B,
BIACORE analysis of the interaction of U1A with U1hpII
UC RNA (also
see legend for Fig. 2). Due to the weak interaction between the mutated
RNA and the protein, an RNA surface with higher capacity was used to
obtain enough information for the kinetic analysis.
UC
RNA, suggesting that too much shortening also affects the initial stage
of complex formation. Based on NMR studies (11) and molecular dynamics
simulations (16), nucleotides 4-10 of the free wild type RNA loop do
not appear to be strongly constrained. Perhaps the flexibility of the
loop is helpful in establishing initial contacts. This view is
supported by the observation that increasing the length of the loop by
replacement of U8-C10 with polyethylene glycol linkers two or
three times the natural length had a negligible effect on the
KD (30).
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ACKNOWLEDGEMENTS |
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We thank Ian Haworth, Huynh-Hoa Bui, Meline Bayramyan, and Peter Laird for useful comments and help with the structure analysis and figures, and we thank members of the Laird-Offringa laboratory for helpful criticism.
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FOOTNOTES |
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* 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.
We dedicate this work to the memory of Eri Mettler, who with his wife, Mary Lou, generously supported our work.
¶ To whom correspondence should be addressed. Tel.: 323-865-0655; Fax: 323-865-0158; E-mail: ilaird@hsc.usc.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101624200
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ABBREVIATIONS |
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The abbreviations used are: RNP, ribonucleoprotein; RRM, RNA recognition motif.
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