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Corresponding author: Ite A. Laird-Offringa, Ph.D. Tel. (323) 865-0655 Fax: (323) 865-0158 E-mail: ilaird@hsc.usc.edu

TITLE:

Two functionally distinct steps mediate high affinity binding

of U1A protein to U1 hairpin II RNA

Phinikoula S. Katsamba1, David G. Myszka2 and Ite A. Laird-Offringa1,3

Running title: Two steps in binding of U1A protein to U1 hairpin II RNA

1Norris Cancer Center/University of Southern California, Keck School of Medicine, Los

Angeles, California 90089-9176, USA. 2Center for Biomolecular Interaction Analysis,

University of Utah, School of Medicine, Salt Lake City, Utah 84132, USA.

3Corresponding author: e-mail: ilaird@hsc.usc.edu

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 10, 2001 as Manuscript M101624200 by guest on June 11, 2018

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SUMMARY

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.

Keywords: BIACORE / RNA-binding protein / RNA-protein interaction / RRM / U1A

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INTRODUCTION

In order 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. Since

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) consensus domain or the RNA-binding domain (RBD), also

referred to as the RNA recognition motif (RRM). This motif is characterized by two

conserved stretches of 8 and 6 amino acid residues (RNP-1 and RNP-2) and a β−α−β−β−α−β

secondary structure (Fig. 1A) (3,4). RRMs fold into a baseball glove-like structure in which

the β-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 U1snRNP (U1 hairpin II or U1hpII) (Fig. 1).

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). In spite of 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

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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.

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EXPERIMENTAL PROCEDURES

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 which resulted in a

Lys88 to 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 E.

coli strain BL21/DE3 (Novagen, Madison, WI). Proteins were expressed and purified as

described previously (21), with only one 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).

Gel shifts - U1hpII RNA for the gel shift was made as described previously (20) and gel

shifts were carried out in 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-1 bovine serum albumin, 1 mM DTT, 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

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software (Amersham Pharmacia Biotech Inc., Piscataway NJ). 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 3 independent

experiments.

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 (SA) 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-1 bovine serum albumin, 125 µg ml-1 tRNA, 1 mM DTT and 0.05% surfactant

P20), and injected at 10 µl min-1. For U1hpII, 25-35 resonance units (RU) of RNA were

captured on the SA sensor chip, while for the mutant RNAs 100-125 RU 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 five-minute dissociation phase was removed by injecting 2 M NaCl for 60 sec at 20

µl min-1, which regenerated the RNA surface completely. Analysis of each protein

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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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RESULTS AND DISCUSSION

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); 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

x 10-11 M, which agreed well with published values (13,30,31). While equilibrium analysis

provides information about the affinity of a molecular interaction, it provides no insight into

the kinetics underlying the binding mechanism. In order 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).

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, while 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

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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.A),

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 x 107 M-1 s-1) as well as a slow dissociation rate (kd = 3.6+1 x 10-4 s-1), resulting in a

high affinity complex (KD = 32+7 pM, Table I.A). 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.

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

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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 stemloop in U2snRNP (25,38). In the

RNA/protein complex, Lys20 and Lys22 lie near the base of the RNA stem, in an area

between β-strand 1 and α-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 β2-β3 loop region and

points into solution in the free protein, while 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. In order 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), since 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. 3B and D). The Lys20,22Ala

and Lys50Ala mutations resulted in a 39- and 16-fold loss of affinity, respectively (Table

I.B). In both cases a ~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

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backbone of the free RNA loop. Besides the reduction in its association rate, the

Lys20,22Ala mutant also showed a ~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.

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 hundred-fold loss

in U1A/U1hpII equilibrium binding affinity as the NaCl concentration was increased from

150 to 500 mM (31). In order 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 1M (Fig. 4). In agreement with results from filter-binding

assays, we observed a hundred-fold increase in the KD as the NaCl concentration was raised

from 150 mM to 500 mM (Table I.C). 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 three-fold across this NaCl concentration range (Table

1.C).

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

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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 (Fig. 4D and Table I). The slopes of the log(ka) vs. log[NaCl] plots were reduced

from -3.3 (U1A wild type) to -2.8 for Lys50Ala and -2.4 for Lys20,22Ala. While both

mutants remained sensitive to the salt concentration (which is not unexpected since 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.

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 while 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

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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 less than 5-fold decrease in association rate (Fig. 5B and Table I.D). Similarly, the

U1hpIIG4C RNA showed a 2500-fold increase in dissociation rate, but displayed a less than

4-fold decrease in association rate (Fig. 5D and Table I.D). 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'UTR

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 β-strands 2 and 3 of the protein protrudes through the RNA

loop, where it appears to aid in the splaying out of the loop bases so that contacts can be

made with the protein β-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). While 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

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in order 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 approximately 17Å, a distance that could not

be bridged by less than two nucleotides. Optimally, 3 nucleotides may be required to

comfortably accommodate the protein β2-β3 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. In order 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 (Fig. 6A and Table

I.E). 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 (Fig. 6B and Table I.E). 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 β2-β3 loop bulge.

In addition to the dramatic increase in the dissociation rate, a 15-fold loss in the

association rate was also seen with the U1hpII∆UC RNA, suggesting that too much

shortening also affects the initial stage of complex formation. Based on NMR studies (11)

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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).

A multi-step 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 be preceded by an electrostatically mediated

binding step ("lure") warrants detailed kinetic investigations of other RNA/protein

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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.

Acknowledgements - We thank Ian Haworth, Huynh-Hoa Bui, Meline Bayramyan, and Peter

Laird for useful comments, and help with the structure analysis and figures, and members of

the Laird-Offringa lab for helpful criticism. We dedicate this manuscript to the memory of

Eri Mettler, who with his wife Mary Lou, generously supported our work.

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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 22 (Lys20,22Ala), Lys50 (Lys50Ala) and Phe56 (Phe56Ala). 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.

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 in indicated by F while 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 seconds followed by a 5 minute of buffer flow. The kinetic parameters for

each of three independent experiments are shown in Table I.A.

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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 β-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 legend Fig. 2B). (C) U1A/U1hpII complex seen from the front. Lys50 (blue) is located

in the protein loop connecting β-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. 2B).

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, in (A) and (B):

0.1; 0.3; 0.9; 2.7; 8; 24 and 73 nM and in (C): 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 Lys 50Ala mutants, using the

same U1hpII RNA surface. Log(ka ) vs. log [NaCl] plots for wild type U1A (�),

Lys20,22Ala (∆) and Lys50Ala (�, solid line) show a linear relationship.

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

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respectively. (B) BIACORE analysis of the interaction of Phe56Ala with U1hpII (also see

legend Fig. 2B). (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 Fig. 2B). Due to the weak interaction between the mutated RNA and the protein, an

RNA surface with higher capacity was used in order to obtain enough information for the

kinetic analysis.

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 Fig. 2B). (B) BIACORE

analysis of the interaction of U1A with U1hpII∆UC RNA (also see legend Fig. 2B). Due to

the weak interaction between the mutated RNA and the protein, an RNA surface with higher

capacity was used in order to obtain enough information for the kinetic analysis.

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Table I Kinetic and affinity constants for U1A/U1hpII interaction

ka

1

(M-1 s-1)

fold

decrease2

kd

3

(s-1)

fold

increase2

KD

4

A. Wild type U1A and U1hpII

Experiment 1 0.958[3]x107 2.416[9]x10-4 25.2[1] pM

Experiment 2 1.290[3]x107 4.07[2]x10-4 31.6[2] pM

Experiment 3 1.061[3]x107 4.22[3]x10-4 39.8[3] pM

Average 1.1+0.2x107 3.6+1x10-4 32+7 pM

B. Lysine mutations

Lys20,22Ala 1.119[3]x106 9.8 1.397[5]x10-3 3.9 1.248[6] nM

Lys50Ala 1.081[2]x106 10 5.42[2]x10-4 1.5 0.502[2] nM

C. Effect of NaCl on binding

U1A 275 mM 1.311[6]x106 8.4 1.81[5]x10-4 0.5 138.2[7] pM

U1A 500 mM 1.87[1]x105 59 7.93[6]x10-4 2.2 4.23[2] nM

Lys20,22Ala 275 mM 2.53[3]x105 4.4* 2.59[3]x10-3 1.8* 10.2[1] nM

Lys20,22Ala 500 mM 7.92[3]x104 14* 1.059[3]x10-2 7.6* 134[6] nM

Lys50Ala 275 mM 4.59[2]x105 2.4* 1.64[3]x10-4 0.3* 0.357[1] nM

Lys50Ala 500 mM 5.75[3]x104 19* 3.94[1]x10-3 7.3* 68.5[4] nM

D. RNA/protein interface mutations

Phe56Ala 2.32[7]x106 4.7 5.0[1]x10-1 1400 0.213[9] µM

U1hpIIG4C 3.13[6]x106 3.5 9.1[1]x10-1 2500 0.291[7] µM

E. RNA loop deletions

U1hpII∆C9 3.102[9]x106 3.5 2.523[9]x10-2 70 8.13[4] nM

U1hpII∆UC 7.33[6]x105 15 8.54[2]x10-2 240 0.116[1] µM

1: the number in brackets represents the standard error in the last significant digit of the ka value from each experiment

(consisting of randomized injections repeated at least twice). 2: fold decrease or increase is given with respect to the

U1A/U1hpII interaction, using the average values given in A, except in C (where indicated with *), in which the values

were compared to the interaction of the same mutant protein with U1hpII in 150 mM NaCl. 3: the number in brackets

represents the standard error in the last significant digit of the kd value from each experiment. 4: the number in brackets

gives the calculated standard error in the last significant digit of the KD value (KD = kd/ka). The most prominent differences

are highlighted in italic bold typeface.

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Phinikoula S. Katsamba, David G. Myszka and Ite A. Laird-Offringahairpin II RNA

Two functionally distinct steps mediate high affinity binding of U1A protein to U1

published online April 10, 2001J. Biol. Chem. 

  10.1074/jbc.M101624200Access the most updated version of this article at doi:

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