High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking
© Carmel et al; licensee BioMed Central Ltd. 2010
Received: 19 February 2010
Accepted: 23 June 2010
Published: 23 June 2010
STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5' or 3' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for S TAR protein b inding e lement. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1.
Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins.
The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA binding by STAR proteins will help facilitate the identification of novel regulatory targets in vivo.
Gene expression is regulated at the post-transcriptional level as a means of ensuring the proper localization and timing of developmental processes in eukaryotic organisms . In a diverse set of pathways, a central feature of this post-transcriptional control involves specific binding to target RNA sequences by proteins belonging to the S ignal T ransduction and A ctivation of R NA/G RP33, S am68, G LD-1 (STAR/GSG) family. STAR protein-RNA interactions are important for translational silencing of genes necessary for germline fate in hermaphrodite C. elegans worms by the regulatory STAR protein GLD-1 [2, 3], mRNA localization and subsequent development of central nervous system components in mice by Quaking (Qk-1) [4–6] and pre-mRNA binding by yeast BBP (B ranchpoint sequence B inding P rotein) and mammalian s plicing f actor 1 (SF-1) proteins as precursors for splicing into mature mRNA [7, 8].
In C. elegans, GLD-1 initiates the formation of a multi-protein repression complex that silences tra-2 translation by binding the 3' untranslated region (UTR) of tra-2 mRNA within 28-nt regulatory elements called TGEs (T RA-2 and G LI E lements) [2, 17, 18]. GLD-1 optimally recognizes a 5'-UACUCA-3' consensus sequence in the TGE plus an upstream dinucleotide contributes to the overall binding energy . A comprehensive mutational analysis further identified other permissible nucleotide sequences that GLD-1 binds with slightly lower affinity , and numerous potential targets for GLD-1 mediated regulation have since been identified by the presence of a relaxed consensus hexamer with the sequence 5'-(U > G > C/A)A(C > A)U(C/A)A-3' in the 5' or 3' UTRs of these mRNAs.
A similar hexameric target was also identified for the mouse Quaking protein (Qk-1) having the sequence 5'-NA(A > C)U(A>>C)A-3' . In vivo, Qk-1 facilitates the proper sub-cellular localization and expression of m yelin b asic p rotein (MBP) in glial cells as a direct result of interactions with consensus binding sites in MBP mRNA [4, 6]. Qk-1 binds substantially tighter to a 5'-UAAUAA-3' consensus, different than GLD-1 in the strong preference for adenosine at both the third and fifth positions . Subsequent reports expanded the Qk-1 binding site to include additional upstream or downstream partial consensus sequences, although the role of these elements in Qk-1 binding remains less well understood [20, 21].
SF-1 is another well-characterized STAR protein which recognizes a 5'-UACUAAC-3' consensus in the b ranch p oint s equence (BPS) RNA that is identical within the sequence parameters delineated for GLD-1 and Qk-1 . Additionally, Sam68 and the Sam68-like proteins SLM-1 and SLM-2 recognize bipartite RNA elements that resemble the canonical consensus sequences bound tightly by GLD-1 and Qk-1 .
Based largely on sequence homology, novel STAR proteins have been identified whose functions and biological roles remain largely uncharacterized. One such protein called STAR-2 (also called ASD-2 for a lternative s plicing d efective-2) is thought to be a functional GLD-1 homolog expressed in C. elegans somatic tissues. In a recent report, STAR-2 was shown to play an important role in C. elegans development by facilitating alternative splicing patterns in let-2 mRNA and regulating expression of let-2 . Given the 67% sequence identity with GLD-1 in the STAR domain (Figure 1), it seemed likely that STAR-2 would bind similar RNA target sequences as GLD-1 and so may perform an analogous regulatory role. To test this, a combination of gel mobility shifts and fluorescence-polarization was used to assess whether STAR-2 binds TGE RNA with an affinity and specificity comparable to GLD-1. Interestingly, STAR-2 binds a similar consensus as GLD-1, but more closely resembles Qk-1 in the strong preference for adenosine at the third and fifth consensus positions. The relative competitive efficiency of mutant 12-mer RNA sequences containing a single hexamer site was then tested against STAR-2 bound to a modified TGE RNA in the more appropriate consensus site background.
Gel mobility shifts and fluorescence-polarization were then used to further detail the contributions of individual hexamer consensus positions for high-affinity binding by GLD-1 and Qk-1. Because wild-type TGE does not contain the consensus sequence most preferred by Qk-1, a modified TGE RNA with the tightest binding Qk-1 consensus was used as a probe in a competition fluorescence-polarization assay to better define the sequence requirements for high-affinity RNA interactions. The role of full and partial consensus elements situated upstream or downstream of a canonical hexamer was also examined in a variety of RNA backgrounds. These results indicate that a single consensus hexamer is sufficient for tight binding by STAR proteins and additional upstream or downstream consensus elements may enhance binding depending on the sequence and positioning.
STAR-2 binds TGE RNA with high affinity
Determination of STAR-2 binding specificity
Competition fluorescence-polarization was used to probe the nucleotide sequence identity of the consensus recognized by STAR-2 with the same 12-mer TGE RNA library used previously for GLD-1 . Each 12-mer RNA, containing a single nucleotide substitution in the wild-type 5'-UACUCA-3' consensus, was tested for it's ability to interfere with a STAR-2-STAR/TGE complex. Of the 18 RNAs, only two point mutations in the 12-mer library, C19A and C21A, bind as tight or tighter than wild-type TGE half-site, 4-fold and 20-fold for each substitution (relative IC50 half site/C19A = 4.2; relative IC50 half-site/C21A = 20), respectively (Figure 2D, 2E).
Interestingly, because GLD-1 binds only slightly tighter to the 12-mer C21A variant (data not shown), the 20-fold preference by STAR-2 for an adenosine at the fifth position more closely resembles Qk-1, which binds the C21A half-site 41-fold tighter by this method . Similarly, wild-type 12-mer competes relatively poorly for binding to STAR-2-STAR, 7-fold weaker than the 28-nt TGE self-competition (relative IC50 TGE/wild-type half-site = 0.13) while the C21A 12-mer binds nearly 3 fold better than the full-length TGE (relative IC50 TGE/C21A 12-mer = 2.7) just as with Qk-1. Furthermore, STAR-2-STAR binds the C19A 12-mer 4-fold tighter than the wild-type half-site, similar to that seen for this RNA by Qk-1, while GLD-1 has a slight preference for cytidine at this position over adenosine. STAR-2 binds most tightly to RNA sequences containing the consensus 5'-UA(A>C)U(A>>C)A-3', identical to the high-affinity consensus binding site for Qk-1, but still within the sequence parameters of the relaxed consensus identified for GLD-1.
Summary of Qk-1 binding to 28-mer RNAs
Kd (nM) EMSA
Kd (nM) FPA
49 ± 2
63 ± 4
1.2 ± 0.2
13 ± 2
C21A 5' Mutant
7.1 ± 1
To more effectively define the contribution of each nucleotide for high-affinity STAR-2 binding, a competitor library of 18 oligonucleotides, synthesized in a background containing the 5'-UACUAA-3' consensus sequence optimal for STAR-2, was used for competition fluorescence-polarization (Figure 3C, 3D). In this assay, no single point mutation competes effectively with the 28-nt C21A TGE for binding STAR-2-STAR except for the A21 half-site 12-mer. Neither the wild-type TGE half-site with the A to C "reversion" mutation or C19A was found to bind with high-affinity when the bound probe contains adenosine at the fifth position. In addition, STAR-2 prefers uracil at the first position, but all other nucleotide substitutions compete within 5-fold affinity of the A21 half-site.
Qk-1 and STAR-2 bind identical hexanucleotide consensus sequences
Competition fluorescence-polarization was then used to measure the ability of each mutant 12-mer RNA, representing all 18 point mutations in the preferred 5'-UACUAA-3' consensus, to compete against the 28-nt C21A TGE variant in complex with Qk-1-STAR. In this assay, Qk-1-STAR has a slight preference for uracil at the first position (U17) but will tolerate any of the other three nucleotides nearly as well (Figure 4C, 4D). Interestingly, no other single mutant 12-mer competes measurably with the C21A TGE RNA, including A21C (wild-type TGE half-site) and C19A, the only effective competitor being the A21 half-site 12-mer RNA (5'-AUCUACUAA UAU-3'; Kiapp = 46 nM). These results highlight that the strongest determinant for high-affinity binding is a hexameric sequence with adenosine at the fifth position, as even C19A does not confer any additional binding specificity in this context.
Upstream elements contribute to high-affinity binding by Qk-1
In addition to a hexanucleotide consensus, GLD-1 recognizes an upstream 5'-UA sequence in the TGE (Figure 2A) . Partial consensus elements situated upstream or downstream of a canonical hexamer in RNA sequences bound tightly by Qk-1 have also been noted previously, although the quantitative importance of the upstream 5'-UA for Qk-1 binding to TGE RNA has not been determined [19–21]. A modified TGE RNA in the 5'-UACUAA-3' consensus background was created by substituting 5'-CUC for the upstream partial consensus 5'-UAA element (Figure 2A). This RNA was used in a gel mobility shift assay to measure the binding affinity to Qk-1-STAR by direct titration (Figure 4B). Qk-1-STAR binds the 5' mutant TGE RNA with roughly 6-fold reduced affinity compared to the C21A TGE (7.1 ± 1 vs. 1.2 ± 0.2 nM; Table 1) but still considerably tighter than to wild-type TGE RNA. This result is consistent with the modest contribution of this upstream element for GLD-1 binding to TGE RNA and further indicates that a 5'-UA(A/C)UAA-3' hexanucleotide consensus is the most important feature for high-affinity binding by Qk-1.
Alternative mutations in MBP consensus restore high-affinity binding by Qk-1
Previously, Ryder et al. described the GLD-1 and Qk-1 high-affinity binding sites as consensus hexamer sequences [12, 19]. In a subsequent report based on in vitro selection, the Qk-1 binding site was expanded to include a "half-site" positioned upstream or downstream of a consensus hexamer that further defined the STAR binding consensus as a bipartite element . In that report, nucleotides surrounding the hexanucleotide consensus were viewed as crucial for Qk-1 recognition because binding was virtually abolished when either the core hexamer or "half-site" was mutated in one of the high-affinity binding sites found in myelin basic protein (MBP) mRNA. This led the authors to conclude that both the core and "half site" must be critical for high-affinity binding if mutations in either one rendered Qk-1 unable to bind. However, we propose as an alternative that these particular core and "half site" mutations caused unforeseen secondary structure changes in MBP RNA that prevented Qk-1 from binding by potentially masking the high-affinity hexameric consensus site.
These results seemingly contradict the idea of a hexanucleotide consensus as the primary specificity determinant for Qk-1 binding. Both MBP:2-5'm1 and MBP:2-3'm1 have valid hexanucleotide elements, but it seemed possible that introduction of multiple G residues in an A/U rich RNA sequence might result in the formation of secondary structures that inadvertently sequester the hexanucleotide element. Indeed, RNA secondary structure predictions using mfold [23, 24] suggest that both MBP:2-5'm1 and MBP:2-3'm1 contain highly stable structured regions that mask the high-affinity binding sites in these RNAs (data not shown). Furthermore, tight binding to these sequences is restored when more conservative mutations are made in either the 5' or 3' sites. Replacing the 5' half-site with the sequence 5'-CU CUCU C-3' (MBP:2-5'm2) results in nearly 5-fold tighter binding (Kd = 100 nM) when compared to MBP:2-5'm1. Similarly, Qk-1-STAR binds tightly to MBP:2-3'm2 and the two distinct complexes seen in the gel have measured Kd values of 69 nM and 199 nM for the first and second shifts, respectively. These values are nearly identical to that for wild-type MBP:2 RNA and neither of these mutant RNAs contain the stable secondary structures in mfold predictions as seen for MBP:2-5'm1 or MBP:2-3'm1. In addition, by simply adding multiple guanosine residues to the 5' end of the TGE in order to facilitate in vitro transcription using T7 polymerase, GLD-1 binding was essentially abolished, presumably due to formation of stable secondary structures in the RNA (data not shown). Taken together, these results suggest that MBP:2 RNA contains two independent consensus binding sites and that only one is necessary for Qk-1 to bind with high-affinity. In addition, Qk-1 is indifferent to the positioning of the consensus site as it bound the full-length MBP RNA with roughly the same affinity when either the 5' or 3' consensus sites were mutated.
GLD-1-STAR binds tighter to RNA with two consensus hexamers
One poorly understood aspect of this system is the mode that homodimeric STAR proteins employ when binding target RNA sites, since structural models are based largely on the solution structure of monomeric human SF-1 in complex with Branch Point Sequence (BPS) RNA . SF-1 lacks the Qua1 region and does not dimerize, making the structure only illustrative as a model for RNA binding by a single STAR monomer . Furthermore GLD-1, Qk-1 and STAR-2 bind much tighter to their target RNA sequences than SF-1 does to its consensus RNA in vitro, and this difference may be due in large part to the extra binding energy attained from recognition of additional RNA elements by the second protomer.
Competition fluorescence-polarization was used to address this issue by measuring the affinity of GLD-1-STAR for various TGE RNAs, truncated so as to contain zero, one or two consensus hexamers (Figure 6A). The tra-2 mRNA 3'-UTR contains two 28-nt TGE repeats each with a single consensus hexamer, plus an additional hexamer encompassing part of the conserved 5'-CUCA-3' in the linker separating the two TGEs. Neither of the two TGE truncations lacking a consensus element (1-18 and 56-67) compete well with the fluorescently-labeled 28-nt TGE for GLD-1-STAR binding. When compared to self-competition with the 28-nt TGE, 1-18 and 56-67 bind 42 and 32-fold weaker, respectively, underscoring the necessity of a hexamer consensus for high-affinity binding.
GLD-1-STAR binds the TGE half-site RNA (labeled 14-25) containing one consensus hexamer roughly 9-fold weaker than the TGE. This is consistent with the previously measured values for this RNA by both Fp and EMSA [12, 19]. Weaker binding to the half-site is likely due to the lack of any additional recognition elements in the shorter 12-nt RNA since tight binding is restored with the longer 46-67 RNA, which has both a consensus hexamer and a downstream UAA element. GLD-1-STAR binds this RNA nearly ten-fold tighter compared to the half-site and binds as tightly as to the full 28-nt TGE (10.7 ± 2 nM versus 11.1 ± 4 nM). Among these sequences, GLD-1-STAR binds the tightest to the 14-35 RNA containing two consensus hexamers (Kiapp = 4.6 ± 0.8 nM), binding this RNA nearly 2.5-fold tighter than the TGE 28-mer.
Consensus site spacing is acceptable over a broad range
As a homodimer, both protomers should be equally competent for binding a consensus element in the context of a dual hexamer RNA. However, it remained unclear whether enhanced binding occurs only when the consensus sites are spaced within a certain permissible range. To this end, a library of RNA constructs was developed that placed either one or two canonical hexamers at different locations within a poly-uridine background and were tested for binding to GLD-1-STAR by competition fluorescence-polarization (Figure 6B). The 28-nt TGE sequence is 54% uridine (15 of 28 bases) and a 28-mer RNA consisting entirely of uridine does not compete for GLD-1-STAR binding (Figure 6B).
Addition of one 5'-UACUCA-3' hexamer in the poly-U background at either the 5' or 3' end resulted in a Kiapp of 110.3 ± 22 nM or 83.5 ± 6 nM, respectively, which is similar to that observed for the half-site 12-mer (14-25) containing a single hexamer and also consistent with the affinity of GLD-1-STAR for TGE RNA with mutations in the upstream UA element . Addition of a second hexamer restores tighter binding to a similar level as that of the 28-nt TGE when there is a 4-nt spacing between hexamers (4-nt, Kiapp = 12.2 ± 2 nM). Binding affinity is 2-fold weaker than TGE RNA when the hexamers are directly adjacent to each other (0-nt, Kiapp = 25.3 ± 6 nM) or with a 2-nt spacer (2-nt, Kiapp = 22.7 ± 6 nM). A spacing of six to twelve nucleotides between hexamers was optimal for tight binding with Kiapp values of 4.8 ± 0.8 nM and 4.8 ± 0.4 nM for a 6-nt and 12-nt spacing, respectively.
In the same 28-nt poly-U RNA background discussed above, various dinucleotide sequences were then inserted upstream of a single consensus hexamer and the contributions of these smaller sequence motifs to GLD-1 binding affinity were evaluated by competition fluorescence-polarization (Figure 6C). None of the partial motifs recapitulated the effect of adding a second consensus hexamer, although three combinations of dinucleotide substitutions reflect a portion of a relaxed consensus element. Adding an upstream AA or AC (A6A7, 5'-UAA UUU-3'; A6C7, 5'-UAC UUU-3') was the most preferred, with a Kiapp of 35.8 ± 5 nM and 38.0 ± 16 nM, respectively, but both are still nearly 8-fold reduced compared to the addition of a consensus hexamer (Kiapp = 4.8 ± 0.8 nM). Interestingly, RNA with the C9A10 substitution (5'-UUUUCA-3') does not bind tighter than 5'-UUUUUU-3' (Kiapp = 85.1 ± 22 nM versus Kiapp = 83.5 ± 6 nM) even though this substitution places half of a 5'-UACUCA-3' consensus upstream of the full hexamer. Of the other sequences tried, C7A10 (5'-UUC UUA-3') and A6C9 (5'-UA UUC U-3') compete with a Kiapp of 51.8 ± 18 and 116.2 ± 16 nM, respectively, indicating that these sequences do not enhance GLD-1 dimer binding when only a single consensus element is present.
Discussion and Conclusions
Optimal RNA hexamer consensus sequences for high-affinity STAR protein binding
5'-(U > G > C/A)A(C > A)U(C/A)A-3'
This has been useful for identifying binding sites in potential RNA regulatory targets and for helping us to initially characterize the RNA binding activity of newly recognized STAR proteins. For example, STAR-2 was initially identified as a potential GLD-1 homolog in C. elegans somatic tissues. In this report, it was established that STAR-2 and GLD-1 bind similar hexameric consensus sequences (Table 2) and so may regulate gene expression in an analogous fashion. Although STAR-2 binding is tightest to a consensus hexamer that more closely resembles the sequence preferred by Qk-1 rather than GLD-1, this is consistent with the model that most STAR proteins recognize similar specificity determinants in the 5' or 3' UTR regions of target RNAs.
Although the consensus sequence requirements vary for each individual STAR protein, all of those studied in detail necessitate a hexameric consensus element at a minimum in order to bind RNA with high-affinity. However, while not an absolute requirement for tight RNA binding, upstream or downstream partial consensus elements play an integral role in high-affinity STAR binding that remains somewhat less understood. For instance, mutations in partial consensus sequences found in TGE and MBP RNA have an adverse effect on both GLD-1 and Qk-1 binding, respectively . However, these results further validate the idea that only one hexameric consensus site is absolutely necessary and that upstream or downstream partial consensus elements play a secondary, but not essential role for high-affinity binding by STAR proteins.
Most of the competition experiments described here involved assessing binding affinities using 12-mer RNAs containing only a single hexamer. It is possible based on this model, in which each protomer is equally competent for binding a consensus hexamer, that a STAR dimer may simultaneously bind two RNA 12-mers with each protomer recognizing a consensus hexamer in a symmetric fashion (Figure 7D). This has been termed the symmetric hexamer binding model and it may also describe the mode of binding to a dual hexamer RNA, such as that employed by GLD-1. As learned in this report, GLD-1 binds tightly to poly-U RNA containing two consensus hexamer sequences and the permissible spacing between hexamers varies over a fairly wide range. Symmetric binding is possible in this case only if the intervening RNA sequence is of sufficient length to properly orient the two consensus binding sites. Since GLD-1 binds the tightest to RNA with two consensus hexamers spaced between 6 and 12-nt apart, it is possible in this context for the RNA to be oriented such that each protomer interacts with a consensus hexamer in an identical manner. These binding consensus studies should prove useful for defining potential targets for the individual STAR proteins.
Other possible models that may explain high-affinity binding must take into consideration the likely orientation of the STAR protomers and the positioning of RNA elements that may contribute differentially to the binding energy of the complex. This highlights the difficulty in describing the interaction between STAR dimers and target RNA sequences without the benefit of a high-resolution structure. Hopefully, efforts currently underway to describe the structure of a full STAR domain will provide more insight on the mode of high-affinity RNA binding by STAR proteins in general.
Protein expression and purification
The STAR domains from GLD-1 and Qk-1 were overexpressed in E. coli as maltose binding protein (MBP) fusions and purified as described previously [12, 19]. Plasmid containing the coding sequence for full-length STAR-2 (Wormbase gene sequence #T21G5.5) was provided by Elizabeth Goodwin's laboratory (University of Wisconsin). The region corresponding to the STAR-2 STAR domain (residues 55-265) was amplified using PCR and inserted into pMal-c2x (NEB) plasmid for overexpression of STAR-2-STAR with an N-terminal MBP fusion. BL21(DE3) gold E. coli cells transformed with pMal-c2x-STAR-2 plasmid were grown in LB media to an OD600 of 0.6 whereby STAR-2 overexpression was induced by addition of 1 mM IPTG for 3 hours. Harvested cell pellets were resuspended and lysed by sonication followed by initial purification on an amylose resin (NEB) column. Pooled fractions containing STAR-2 were further purified by ion-exchange chromatography, first on a HiTrap Q column (Amersham Biosciences) followed by a HiTrap SP column. Pure STAR-2 protein was dialyzed against 4 L of storage buffer (20 mM Tris, pH 7.5, 20 mM NaCl, 2 mM DTT) and stored at 4°C for use in binding experiments.
All RNA constructs were chemically synthesized (Dharmacon). Lyophilized RNA pellets were resuspended in deprotection buffer and incubated at 60°C for 30 minutes. Deprotected RNAs were thoroughly dried in a SpeedVac, resuspended in deionized water and stored according to the manufacturer's protocol. 5'-Fluorescein-labeled RNA constructs used in Fp binding experiments were treated as above except all steps were conducted in the dark. RNAs used in gel mobility shift experiments were 5' radiolabeled by incubation with γ-32P-ATP (Perkin Elmer) and T4 polynucleotide kinase (NEB) for 1 hour at 37°C.
Fluorescence-polarization (Fp) and Electrophoretic Gel Mobility Shift (EMSA) RNA binding assays
Complex formation between the various STAR proteins and RNA was monitored by both fluorescence-polarization and EMSA. For direct titration experiments, individual STAR proteins at a range of concentrations were titrated into a constant concentration of labeled RNA (1 nM fluorescein-labeled RNA for Fp or 100-300 pM radiolabeled RNA for EMSA) in reaction buffer (10 mM Tris, pH 8.0, 25 mM NaCl, 0.1 mM EDTA, 0.1 mg/mL tRNA, 5 μg/mL heparin and 0.01% IGEPAL CA630) and the reactions equilibrated at room temperature for 3 hours. Prior to equilibration with protein, labeled RNA was heated at 65°C for 2 minutes and allowed to cool to room temperature to remove any residual secondary structure.
Fp samples were equilibrated in a total volume of 100 μL in 96 well plates (Grenier) and every plate successively measured 10 times in a Packard Fusion plate reader to obtain the average polarization and standard deviation values for each protein concentration. Samples for gel mobility shifts were equilibrated in 20 μL total volume. 5 μL of each sample was loaded on a pre-run 6% native gel (29:1 acrylamide/bis-acrylamide, 0.5 × TBE) after addition of loading dye (30% v/v glycerol, bromophenol blue, xylene cyanol) and the gel run at 600 V for 30 minutes at 4°C. Gels were dried and exposed to a phosphorimager screen overnight. ImageQuant software (Molecular Dynamics) was used to quantify the fraction of labeled RNA in complex with protein. Kd values for each RNA were calculated by fitting the binding data to a modified version of the Hill equation as described previously [19, 25].
R, P, C, Kd and Kc are the concentrations of fluorescent RNA, protein, competitor RNA, and the dissociation constants for the wild type and competitor RNAs, respectively.
The authors would like to thank Dr. Sean Ryder for experimental assistance and Dr. Stephen Edgcomb for critical reading of the manuscript. This work was supported by the National Institutes of Health [RO1-GM-53320 to J.R.W.]
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