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Nucleic Acids Research, 2002, Vol. 30, No. 24 5360-5368
© 2002 Oxford University Press

Interaction of C5 protein with RNA aptamers selected by SELEX

June Hyung Lee, Hana Kim, Jaehyeong Ko and Younghoon Lee*

Department of Chemistry, Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea

*To whom correspondence should be addressed. Tel: +82 42 869 2832; Fax: +82 42 869 2810; Email: ylee{at}mail.kaist.ac.kr

Received as resubmission September 30, 2002; Revised and Accepted October 24, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RNA aptamers binding to C5 protein, the protein component of Escherichia coli RNase P, were selected and characterized as an initial step in elucidating the mechanism of action of C5 protein as an RNA-binding protein. Sequence analyses of the RNA aptamers suggest that C5 protein binds various RNA molecules with dissociation constants comparable to that of M1 RNA, the RNA component of RNase P. The dominant sequence, W2, was chosen for further study. Interactions between W2 and C5 protein were independent of Mg2+, in contrast to the Mg2+ dependency of M1 RNA–C5 protein interactions. The affinity of W2 for C5 protein increased with increasing concentration of monovalent NH4+, suggesting interactions via hydrophobic attraction. W2 forms a fairly stable complex with C5 protein, although the stability of this complex is lower than that of the complex of M1 RNA with C5 protein. The core RNA motif essential for interaction with C5 protein was identified as a stem–loop structure, comprising a 5 bp stem and a 20 nt loop. Our results strongly imply that C5 protein is an interacting partner protein of some cellular RNA species apart from M1 RNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RNase P is a ubiquitous RNA processing enzyme that removes 5' leader sequences from precursor tRNA to generate mature 5' termini. RNase P derived from prokaryotes, eukaryotes and mitochondria contains both essential RNA and protein components (1,2). The Escherichia coli holoenzyme consists of two subunits, specifically a large RNA subunit (M1 RNA, 377 nt) and a small basic protein (C5 protein, 119 amino acids). Although RNase P functions as a ribonucleoprotein in vivo, enzymatic activity is associated with M1 RNA. M1 RNA alone performs the cleavage reaction in vitro and is capable of processing a variety of substrates, including tRNAs, 4.5S RNA, 10Sa RNA, small-model substrates and plant virus RNA (3–7). Although the roles of M1 RNA have been well documented, our understanding of the function of C5 protein in vivo remains relatively limited. C5 protein is not required for in vitro cleavage by M1 RNA, but both components are essential for RNase P activity in vivo (8). Accumulating evidence suggests that C5 protein is important for the RNase P reaction (8–12). The protein decreases the dependence of the reaction on magnesium ion, stabilizes M1 RNA structure by maintaining the catalytically active conformation, promotes substrate affinity by altering the structure of M1 RNA or directly interacting with the 5' leader sequences of substrates, reduces the deleterious effects of mutations in M1 RNA, and broadens substrate specificity of the enzyme.

The three-dimensional structure of C5 protein is yet to be resolved. However, the structures of RNase P protein from Bacillus subtilis and Staphylococcus aureus have been determined by X-ray crystallography and NMR spectroscopy, respectively (13,14). The RNase P protein has two RNA-binding motifs at an unusual left-handed ß{alpha}ß crossover connection and a large central cleft in its three-dimensional structure. Additionally, a metal binding loop may form the third RNA-binding site. Footprinting experiments suggest that M1 RNA has three main regions interacting with C5 protein (15,16). Non-contiguous binding regions are widely distributed in the secondary structure of M1 RNA. This is distinct from the simpler system exemplified as RNA recognition by MS2 coat protein or HIV tat protein (17,18), in which a cognate protein recognizes a short RNA motif structure. Due to the complexity of C5 protein–M1 RNA binding, the structural features of this interaction have remained poorly understood to date.

C5 protein was originally identified as the protein component of RNase P. It is possible that C5 protein participates in other cellular metabolic reactions as an RNA-binding protein, due to the high content of positively charged side-chains. Various RNA molecules in addition to M1 RNA were co-isolated with C5 protein during purification (19); these molecules are yet to be identified.

We were particularly interested in identifying RNA motifs that bind to C5 protein in order to elucidate the mechanism of action of C5 protein as an RNA-binding protein. We performed SELEX (systematic evolution of ligands by exponential enrichment), a powerful tool for isolating rare nucleic acid molecules with either affinity for a target molecule or a specific function, from complex pools of random sequences by iterative rounds of selection and amplification (20). RNA aptamers that bound to C5 protein were selected and characterized from a synthetic RNA pool containing random sequences of 42 bases. Selected RNA aptamers displayed diverse sequences non-homologous to M1 RNA bound C5 protein with dissociation constants comparable to that of M1 RNA. This finding suggests that C5 proteins interact with various RNA motifs. Further selection from a doped library of the dominant sequence, designated W2, identified a stem–loop structure comprising a 5 bp stem and a 20 nt loop, as a core RNA motif. We additionally compared W2 with M1 RNA in terms of interaction with C5 protein. The stem–loop structure of W2 provides a small RNA motif that may be employed to elucidate the mechanism of interaction of C5 protein with RNA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Library construction
The RNA library used for selection was generated by in vitro transcription, using T7 RNA polymerase and a 138 bp DNA template containing 42 random nucleotides. The DNA template was created by ligation of three oligonucleotides, as described by Schneider et al. (21). A doped RNA library was prepared by doping the dominant RNA aptamer, W2, with 30% mutations at each position of the randomized sequence. All RNA samples were gel-purified under denaturing conditions, as described previously (22).

Purification of C5 protein and immobilization on amylose resin
C5 protein was purified as described previously (3). C5 protein was also expressed in E.coli as a fusion to maltose-binding protein (MBP–C5 protein), as described previously (23). The total proteins of cells overexpressing MBP–C5 protein were applied to an amylose column, which was extensively washed with washing buffer (10 mM Tris–HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA, pH 8.0, 0.1 M 2-mercaptoethanol and 1 mM NaN3). MBP–C5 protein immobilized on amylose resin was used for selection. Alternatively, MBP–C5 protein was eluted with the above buffer containing 10 mM maltose, according to the manufacturer’s instructions (New England Biolabs).

SELEX
SELEX was performed principally as described by Tuerk and Gold (24). In the first round of selection, random RNA (30 µg, 5 x 1014 molecules) in binding buffer containing 20 mM K–HEPES, pH 8.0, 400 mM NH4OAc, 10 mM Mg(OAc)2, 0.01% (v/v) Nonidet P-40 and 5% (v/v) glycerol was passed through an MBP-immobilized amylose column for negative selection. The pass-through fraction was mixed with MBP–C5 protein-immobilized amylose resin. The binding reaction was performed by slowly agitating the mixture at 37°C for 1 h. The resin was washed three times with 1.5 ml binding buffer. Finally, the MBP–C5 protein was eluted from the amylose resin by incubating with 500 µl binding buffer supplemented with 10 mM maltose. RNA ligands binding to MBP–C5 protein were recovered from the supernatant by phenol– chloroform extraction and ethanol precipitation, reverse-transcribed with AMV reverse transcriptase (Promega), amplified by PCR with Taq DNA polymerase (Promega), and used for additional rounds of selection. After the 15th round, cDNA was amplified by PCR, as described previously (21), digested with BamHI and HindIII, and cloned into pUC9. Individual clones were identified by DNA sequencing.

Gel-mobility shift assays
A gel-mobility shift assay was performed according to the procedure of Talbot and Altman (16) with minor modifications. Approximately 0.1 nM RNA internally labeled with [{alpha}-32P]CTP was incubated in standard binding buffer containing 20 mM K–HEPES, pH 8.0, 400 mM NH4OAc, 10 mM Mg(OAc)2, 0.01% (v/v) Nonidet P-40 and 5% glycerol at 37°C for 10 min in the presence of a 100-fold excess of unlabeled competitor tRNA. Where necessary, the concentrations of NH4OAc and Mg(OAc)2 in binding buffer were varied. Excess amounts of competitor tRNA were used to prevent non-specific interactions of target RNA with protein. MBP–C5 protein or C5 proteins were serially diluted to the desired concentrations. The binding assay was initiated by adding protein to the RNA-containing solution at a final volume of 20 µl. After a 10 min incubation at 37°C, 10 µl of each reaction mixture was loaded onto a 5% non-denaturing polyacrylamide gel (acrylamide:bisacrylamide = 29:1). Electrophoresis was performed at a constant current of 25 mA at 4°C with running buffer containing 50 mM K–HEPES, pH 8.0, 1 mM Mg(OAc)2 and 0.01% (v/v) Nonidet P-40. Where necessary, 0.25x TBE (1x TBE comprises 90 mM Tris–borate containing 2 mM EDTA) was used as running buffer. Following electrophoresis, the gel was dried and analyzed with Bas-1500 (Fuji).

Dissociation kinetics
C5 protein–RNA complexes were generated under standard binding conditions at 23, 30 and 37°C for 10 min. The concentration of 32P-labeled RNA employed was ~0.1 nM, and that of MBP–C5 protein was sufficient for complex formation by 50–90% input RNA. Excess unlabeled M1 RNA was added to a final concentration of 50 nM at zero time. Aliquots were withdrawn at time intervals and loaded immediately onto a running gel for gel retardation assays.

Boundary determination analysis
RNA was labeled with 32P, either at the 5' terminus with T4 polynucleotide kinase and [{gamma}-32P]ATP or the 3' terminus with T4 RNA ligase and [32P]pCp. Labeled RNA and 5 µg unlabeled yeast tRNA were suspended in alkaline hydrolysis buffer (50 mM NaHCO3/Na2CO3, pH 9.5), and subjected to partial cleavage by incubation at 95°C for 5 min. Hydrolyzed RNA fragments were ethanol precipitated and resuspended in binding buffer containing MBP–C5 protein-amylose beads (1 µg protein). RNA fragments binding to C5 protein were isolated with the selection procedure described above, and recovered by phenol–chloroform extraction and ethanol precipitation. The RNA fragments were fractionated on an 8% polyacrylamide gel containing 7 M urea.

Footprinting of W2 using Fe(II)-EDTA/H2O2
5' End labeled W2 RNA (1 nM) and various concentrations of protein were preincubated at 20°C for 10 min in 20 µl binding buffer lacking glycerol and Nonidet P-40. H2O2 (0.3%), 4.2 mM sodium ascorbate, Fe-EDTA as 1.4 mM Fe(NH4)2 (SO4)2 and 2.8 mM EDTA were separately added to the reaction tube. Following a 10 min incubation at 20°C, the reaction was quenched with thiourea (final concentration of 20 mM). Cleavage products were separated on an 8% denaturing polyacrylamide gel.

RNase mapping
W2 was labeled at the 5' end with [{gamma}-32P]ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (New England Biolabs). We incubated labeled RNA in 45 µl binding buffer for 10 min at 37 or 23°C. Next, S1 nuclease (Promega), RNase V1 (Pierce) or RNase T1 (Industrial Research) was added to the above mixture, and the reaction volume was adjusted to 50 µl with binding buffer, including an additional 10 mM ZnCl2 for nuclease S1 cleavage. The reaction mixture was incubated for another 30 min at 37 or 23°C, respectively. Cleavage products were electrophoresed on an 8% polyacrylamide gel containing 7 M urea.

Mutagenesis
W2-1, W2-2, W2{Delta}L5 and W2{Delta}R5 were generated by site-directed mutagenesis of W2 plasmid DNA, using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Mini-W2, mini-W2{Delta}L5 and mini-W2{Delta}L5 were prepared by PCR amplification, using the W2 sequence as a template. Sequences of the primers used for preparing the DNA templates are shown in Table 1.


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  		Table 1. Sequences of primers used for preparing W2 derivatives
 
Pb2+-induced cleavage of W2
5' End labeled W2 RNA was preincubated at 37°C for 10 min in 20 µl binding buffer containing various concentrations of Mg2+. Cleavage was initiated by the addition of freshly prepared Pb(OAc)2 at a final concentration of 0–10 mM. The reaction mixture was incubated for 1 h at 37°C. To terminate the reaction, 0.5 M EDTA (1 µl) was added to the mixture. Cleavage products were separated on an 8% denaturing polyacrylamide gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of RNA aptamers
SELEX was employed to select RNA aptamers as high-affinity C5 protein binding sequences from an RNA library of 108mers. The RNA library contained a central stretch of 42 random nucleotides. We used C5 protein fused to maltose-binding protein (MBP–C5 protein) instead of C5 protein, since the purification of high concentrations of functional C5 protein is difficult as a result of low solubility (3,15,23). The number of unique RNA molecules present in the initial pool was ~5 x 1014. A binding buffer of relatively high ionic strength (400 mM ammonium ion) was employed to reduce non-specific electrostatic interactions between C5 protein and target RNA molecules (15). Prior to selection for C5 protein, we preferentially removed the RNA species binding to MBP or amylose resin by passing the RNA pool through an MBP–amylose affinity column.

After 10 rounds of affinity selection, the RNA species binding to MBP–C5 protein significantly increased (Fig. 1). The resulting pool was subjected to five additional rounds of selection and amplification by PCR. Following 15 rounds, 101 sequences were cloned and sequenced (Table 2). Fifty-eight clones displayed identical sequences (designated W2), while seven differed from W2 by one or two bases. We identified 18 clones with unique sequences after eliminating minor derivatives. Sequence analyses revealed that little homology existed in the primary sequence among the clones. In addition, no common secondary structural motifs were detected while predicting secondary structures with an RNA folding algorithm. The RNA aptamers were not related to M1 RNA in terms of sequence and secondary structure. Furthermore, the GC content of the RNA aptamers was relatively low (~45%) compared with that of M1 RNA (63% GC).



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  		Figure 1. Selection of RNA aptamers binding to MBP–C5 protein by SELEX. Mixtures of internally labeled RNA pools with [{alpha}-32P]CTP and MBP–C5 protein were incubated in binding buffer at 37°C for 10 min. The mixtures were electrophoresed on 5% non-denaturing polyacrylamide gels. RNA concentration was 5 nM, while the protein concentrations of 5 nM (lane 9), 10 nM (lanes 1, 5 and 10), 20 nM (lanes 2, 6 and 11), 50 nM (lanes 3, 7 and 12) and 100 nM (lanes 4 and 8) were employed. C, complex. F, free RNA.

 

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  		Table 2. Primary sequences of selected RNAs
 
We determined dissociation constants of complexes between RNA aptamers and MBP–C5 protein using a gel-mobility shift assay. The dissociation constants varied from 3 to 9 nM (Table 3). Surprisingly, some aptamers (e.g., W14 and W29) did not bind well to either MBP–C5 protein or MBP alone (data not shown). It is likely that these sequences were selected as RNA species binding to minor proteins associated with MBP–C5 protein. This possibility is supported by the detection of interacting proteins in MBP–C5 protein purified from cells that were not induced with IPTG (23) (S.Lee, unpublished results). W2 was chosen as a representative aptamer for further study. This dominant aptamer contained 40 nt in the randomized region instead of the initial 42 nt. The decrease in number may be due to deletion during the selection-PCR cycle. W2 bound efficiently to both C5 protein and MBP–C5 protein (Fig. 2A and B), but not MBP (data not shown), suggesting that the aptamer binds the C5 protein moiety of MBP–C5 protein. Since C5 protein was purified under denaturing conditions, it was difficult to estimate the active proportion in the refolded protein population. Accordingly, in subsequent experiments, we examined protein–RNA interactions using MBP–C5 protein instead of C5 protein. The dissociation constant of the W2 complex was 3.6 nM, while that of M1 RNA was 1.8 nM under the same conditions [The Kd value of the M1 RNA–C5 protein complex previously determined by Vioque et al. (15) is 0.4 nM]. This binding differs from non-specific interactions (Kd = ~10–7 M) of C5 protein with other RNA samples, such as rRNA (15). The addition of a 100-fold excess of competitor tRNA to the binding assay reaction had no effect on W2 binding (data not shown). These data collectively suggest that W2 specifically interacts with C5 protein.


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  		Table 3. Dissociation constants of selected RNA aptamers
 


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  		Figure 2. Complex formation of W2 and its derivatives with C5 protein. 32P-labeled W2 (0.1 nM) was incubated with increasing concentrations of C5 protein or MBP–C5 protein at 37°C for 10 min. The mixture was analyzed on 5% non-denaturing polyacrylamide gels. Protein concentrations used are indicated above each lane. F and C signify free RNA and complex, respectively. (A) Complex formation of W2 with C5 protein, (B) W2 with MBP–C5 protein, (C) W2-1 with MBP–C5 protein, (D) W2-2 with MBP–C5 protein. C, complex: C(u), upper band; C(l), lower band. F, free RNA.

 
Consensus RNA motif
We generated a pool of variants by doping W2 with 30% mutations at each position of the randomized 40 nt. SELEX was repeated with 6 x 1013 RNA molecules. After the seventh round, we sub-cloned and sequenced selected RNA samples (Fig. 3). RNA aptamers from the doped pool had the consensus sequence C(/G)GUCCCC(/A)A-N8-GUGUUG-N4-G(/A)GGA(/C)C. Interestingly, W2 contains a sequence motif similar to the consensus sequence (Fig. 3). A comparison of W2 and RNA aptamers selected from the doped pool reveals that, in both cases, the RNA sequences encompass a GUCCC-N20-GGGAC sequence in which the distance between two pentanucleotides is fixed at 20 nt. The homologous sequences of W2 and the RNA aptamers selected from the doped pool are located differently within the randomized region, thus confirming that this sequence does not result from biased selection. The two pentanucleotides are able to base-pair with each other to form a stem. W2 variants with minor substitutions did not display any change in pentanucleotides, except RNA W111. RNA W111 contains GUUCC in the first pentanucleotide, which can base-pair with the second pentanucleotide, GGGAC, through G-U base pairing (Table 2).



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  		Figure 3. A representative set of RNA aptamers selected from a doped pool of W2. The predicted consensus motif showing the conserved positions is underlined in the sequences. The consensus nucleotides observed in W2 are depicted in boxes.

 
Minimal binding domain of W2
To determine the minimal binding domain of W2, we first performed a boundary determination analysis. Partially hydrolyzed W2 was incubated with MBP–C5 protein immobilized on amylose resin, and RNA fragments binding to the protein were analyzed. Figure 4A shows that the RNA region spanning 35 nt from A26 to C60 is responsible for binding to C5 protein. The consensus sequence of W2 is located within this minimal binding domain. A possible secondary structure of the minimal binding domain predicted using the RNA folding algorithm comprises a stem–loop structure with a 5 bp stem and a 20 nt internal loop (Fig. 4D). A footprinting analysis also showed that MBP–C5 protein protected U47–U54 in the loop from cleavage by Fe-EDTA/H2O2 (Fig. 4C). We generated a mini-W2 construct comprising 39 nt, including the minimal binding region of 35 nt. Mini-W2 complexed with C5 protein exhibited a comparable dissociation constant (Kd = 9.5 nM) to W2 (Table 3).



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  		Figure 4. Predicted secondary structure of W2 and its minimal binding domain essential for interaction with C5 protein. (A) Boundary determination analysis. W2 was labeled with 32P, either at the 5' or 3' end. Labeled W2 was partially digested by alkaline hydrolysis and incubated with MBP–C5 protein immobilized on amylose resin. RNA fragments binding to MBP–C5 protein were analyzed on an 8% denaturing polyacrylamide gel. The solid line to the right of the figure represents the essential domains in W2 for binding to C5 protein. P, protein-bound RNA fragments. OH, partial alkaline hydrolytic products of W2. G, RNase T1 digests of W2. (B) RNase mapping of W2. Nuclease S1, ribonuclease V1 and RNase T1 treatments are indicated by S1, V1 and T1, respectively. G, G-specific cleavage products by RNase T1. OH, alkaline ladders. (C) Footprinting of W2 using Fe(II)-EDTA/H2O2. 5' End labeled W2 RNA was preincubated with the indicated concentrations of MBP–C5 protein at 20°C for 10 min. Cleavage products were separated on 8% denaturing polyacrylamide gels. A major protected region is indicated by the solid line to the right of the figure. (D) Predicted secondary structure of W2. The consensus nucleotides capable of forming a stem are represented by the rectangle. The nuclease S1 cleavage sites (representing single-stranded regions) are specified with either strong (open arrows) or weak (open arrowheads) bands. RNase V1 cleavage sites (representing double-stranded regions) are also specified with either strong (filled arrows) or weak (filled arrowheads) bands. End boundaries of the binding domain for interaction with C5 protein are indicated by thick arrows. Asterisks represent cleavage sites by Pb(II).

 
To determine whether the stem–loop structure of W2 is essential for binding to C5 protein, we disrupted the stem by changing the first pentanucleotide, GUCCC, to GCCCG to generate W2-1. W2-1 did not bind to C5 protein. Upon regenerating the stem structure by compensatory mutation of the second pentanucleotide to CGGGC in W2-2, binding ability was recovered (Fig. 2C and D). We additionally altered the loop size. The deletion of 5 nt adjacent to either the first or second pentanucleotide in W2{Delta}L5 and W2{Delta}R5, respectively, abolished binding (data not shown). The loss of binding ability of the deletion derivatives may have resulted from alteration of the secondary structure. To exclude this possibility, we introduced the same deletions in mini-W2 to generate mini-W2{Delta}L5 and mini-W2{Delta}R5, since these deletion derivatives of mini-W2 still maintained a stem–loop in the predicted secondary structures. Mini-W2{Delta}L5 and mini-W2{Delta}R5 also did not bind C5 protein (data not shown). Our data clearly show that both the stem and the size of the loop are crucial for binding to C5 protein. However, the conserved sequence, GUGUUG, detected in the loop sequence of RNA aptamers selected from the doped pool of W2, indicates that the loop sequence may also be important for binding.

Secondary structure of W2
To determine the secondary structure of W2, we performed RNase mappings with RNase V1 (specific for double-stranded regions and stacked nucleotides next to a helix) and S1 nuclease (specific for single-stranded regions) (Fig. 4B), and compared these with the predicted structure (Fig. 4D). RNase V1 treatment led to cleavage at G30-C37 and U54-G56, suggesting that these regions are involved in the formation of a stem structure. The predicted 5 bp stem of W2 or mini-W2 lies in these regions. Nucleotides G38-C39 and U47-A51 present in the predicted 20 nt loop were cleaved with S1 nuclease. RNase T1 cleaved G nucleotides within the 20 nt loop better than those in the 5 nt stem. RNase mapping data correlate well with the predicted structure depicted in Figure 4D. Additionally, cleavages by single- and double-strand specific nucleases were mutually exclusive and cleavage patterns were the same at 23 and 37°C (Fig. 4B). These findings suggest that W2 exists in one major conformation.

Comparison of W2 and M1 RNA interactions with C5 protein
To compare W2 and M1 RNA interactions with MBP–C5 protein, we determined the half-life of the W2–MBP–C5 protein complex. We initially examined the half-life of mini-W2 instead of intact W2, since the W2 complex contained at least two bands (upper and lower bands; see Fig. 2) that may complicate the analysis. The upper band complex might be formed by extra non-specific binding of MBP–C5 proteins at high protein concentrations. In contrast, mini-W2 displayed one complex band. When complex formation between 32P-labeled mini-W2 and MBP–C5 protein was competed out with a 500-fold excess of unlabeled M1 RNA, the half-life of the complex was 0.53 min at 37°C. This value is very short compared to the half-life of 115 min observed with M1 RNA–C5 protein complex at 37°C (25). Lowering the binding temperatures increased the half-life of the complex. The half-life of the mini-W2 complex was 1.1 and 10.1 min at 30 and 23°C, respectively (Fig. 5). In the case of W2, the upper-band complex of W2 was very rapidly disrupted by competition, but the half-lives of the lower-band complex were similar to those of the complex of mini-W2 (data not shown).



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  		Figure 5. Analysis of the stability of the mini-W2–C5 protein complex. Mini-W2–MBP–C5 protein complex formation was competed out with 500-fold excess M1 RNA at 37, 30 and 23°C. Aliquots were withdrawn at indicated times and analyzed on a 5% non-denaturing polyacrylamide gel. (A) Autoradiogram of the gel. –, no protein. C, complex. F, free RNA. (B) The remaining fractions of the complex were plotted as a function of time.

 
We additionally determined the ability of W2 to disrupt the M1 RNA–MBP–C5 protein complex. The M1 RNA–MBP–C5 protein complex was competed out with a 100-fold excess of unlabeled M1 RNA or W2, and competition abilities were compared. W2 disrupted interactions between M1 RNA and MBP–C5 protein although the competition by W2 was 48 and 95% less effective than the competition by M1 RNA at 37 and 23°C, respectively (data not shown).

We investigated the effect of Mg2+ on W2–C5 protein binding. W2 bound efficiently to C5 protein, regardless of the presence of Mg2+ (Fig. 6). One may argue that 1 mM Mg2+ present in the running buffer participates in interactions between W2 and C5 protein. However, a similar binding ability of W2 to C5 protein was observed in a gel-mobility shift assay with 0.25x TBE containing no Mg2+. Therefore, interactions between W2 and C5 protein do not require Mg2+. This finding is in direct contrast to the observed Mg2+ requirement of M1 RNA binding to C5 protein (25). Although the selection process was performed in the presence of Mg2+, none of the RNA aptamers tested demonstrated Mg2+ dependency on interactions with C5 protein (data not shown). Lead(II)-induced cleavage of W2 was also examined (Fig. 7) because a lead(II) coordination site was previously proposed to correspond to a structural motif for tight Mg2+ binding (26). Lead(II)-induced cleavage was observed only at high lead(II) concentrations (>3 mM), suggesting the absence of Mg2+ binding sites in W2. In addition, the cleavage sites, which are mostly in single-stranded regions, were not significantly affected by the presence of Mg2+.



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  		Figure 6. Effects of Mg2+ ion and ionic strength on the binding affinity of W2. Dissociation constants of W2 and mini-W2 were determined using binding buffer containing different concentrations of NH4+ (A) and Mg2+ (B). Dissociation constants were calculated with data from gel-mobility shift assays. All data are represented as means of at least three different determinations.

 


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  		Figure 7. Lead(II)-induced cleavage of W2. Cleavage of W2 by Pb(II) was performed at 37°C, as described in Materials and Methods. Cleavage sites are also represented by asterisks in Figure 4D.

 
We additionally determined the effects of ionic strength on W2–C5 protein interactions. Binding efficiency increased with the concentration of monovalent NH4+ (Fig. 6). The ammonium ion had a similar effect on M1 RNA–C5 protein interactions, in accordance with data reported by Talbot and Altman (25). These results suggest that the main interactions between the RNA aptamer and C5 protein comprise hydrophobic attractions, similar to M1 RNA–C5 protein binding.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that C5 protein interacts with various RNA aptamers displaying diverse sequences that lack homology to M1 RNA, the known interacting partner of C5 protein in vivo. Moreover, this interaction occurs through specific attractions, with dissociation constants comparable to that of M1 RNA. We extensively examined W2, the dominant RNA aptamer. Interactions between W2 and C5 protein were independent of Mg2+ (Fig. 6), suggesting that Mg2+ does not affect the RNA-binding ability of the C5 protein. The absence of Mg2+ requirement in W2–C5 protein interactions is distinct from the binding of M1 RNA to C5 protein for which Mg2+ is essential (25). M1 RNA requires Mg2+ to form its tertiary structure (27). Therefore, Mg2+ may only play a role in the formation of a high-ordered structure that is essential for binding to C5 protein.

W2 forms a fairly stable complex with C5 protein with a half-life of 10 min, particularly at 23°C. This stability is notable, compared to the common RNA–protein complexes displaying transient interactions with half-lives of <1 min even at 2°C (28). Similarly, M1 RNA forms a complex with C5 protein displaying a much higher stability, with the half-life of 115 min at 37°C (25). The binding of M1 RNA to C5 protein may induce structural changes, especially on the complex tertiary structure of M1 RNA, which in turn increases the stability of the ribonucleoprotein. In contrast, the structure of W2 is relatively simple, resulting in the formation of a less stable complex between W2 and C5 protein than M1 RNA and C5 protein at 37°C. The W2 complex is stabilized by lowering the temperature (Fig. 5), which is possibly accomplished by enhancing the formation of higher-ordered structures of W2 at lower temperatures. In this respect, we propose that the interaction mode of W2 with C5 protein is similar to that of M1 RNA. We additionally observed that the W2–C5 protein interactions increased with ionic strength (Fig. 6), comparable to M1 RNA–C5 protein binding (25). This suggests that the main driving force in the formation of RNA–C5 protein complex is hydrophobic attraction, thus supporting the hypothesis that W2 is similar to M1 RNA in forming ribonucleoproteins with C5 protein.

The crystal structure of RNase P of B.subtilis suggests that the protein has at least three RNA-binding motifs (13). Among these, RNR is an important motif for interactions with RNase P RNA (13,23). It is additionally reported that the 5' leader sequence of precursor tRNA and small homo-oligomers bind a site other than the RNR motif (10,14). However, it is unclear whether this binding is specific. The RNA aptamers with diverse sequences (Table 2) identified in this study may provide a tool to elucidate the mechanism of interactions of C5 protein with RNA.

The minimal binding motif of W2 was determined as a stem–loop structure consisting of a 5 bp stem and a large loop of 20 nt. Mini-W2 of 39 nt containing this small RNA motif also binds efficiently to C5 protein (Table 3 and Fig. 5). Therefore, it is likely that the stem–loop structure is the binding domain for C5 protein. It should be noted that the sequences surrounding the minimal binding domain affect binding efficiency, since the presence of additional flanking nucleotides in W2 slightly increased the binding efficiency (Table 3). The structural features affecting binding of W2 with C5 protein may be similar to those influencing interactions between U1A protein and stem–loop II of U1 snRNA (29). In the U1A-U1 snRNA complex, the U1A protein recognizes a specific AUUGCAC sequence present in a loop of 10 nt through sequence-specific RNA recognition (30,31). The largely unstructured feature of the hairpin loop in free RNA suggests that the RNA loop binds the U1A protein by an induced-fit mechanism (32). The binding affinity of a single-stranded RNA containing the AUUGCAC heptamer sequence to U1A protein is substantially lower (33). Thus, the intact, double-helical stem may be essential for binding. The AUUGCAC heptanucleotide is also conserved in U2 RNA hairpin IV, which is recognized by the closely related U2B" protein. However, U1A does not bind U2 RNA (34). It is noteworthy that the U2 hairpin IV loop is composed of 11 nt instead of 10. The interaction between W2 and C5 protein needs both the stem and the entire loop. Furthermore, the formation of a fairly stable complex of W2 with C5 protein implies the involvement of an induced-fit mechanism. A conserved hexanucleotide, GUGUUG, was also observed in the loop sequence of the RNA aptamers selected from the doped pool of W2. In this respect, C5 protein would evolve as an RNA-binding protein that possesses a binding mechanism similar to the U1A.

It remains to be determined whether the W2–C5 protein complex has a biological counterpart. Since the 5 bp stem and 20 nt loop structures are recognized by C5 protein, we searched sequences composed of two complementary pentanucleotides with a 20 nt spacer in E.coli and obtained 11 910 matching sequences. It is possible that some RNA sequences are recognized by C5 protein. We additionally identified various RNA motifs interacting with C5 protein through a genomic SELEX approach (J.H.Lee, H.Ryoo and Y.Lee, unpublished results). These results indicate that C5 protein interacts with cellular RNA species other than M1 RNA.

Since most studies to date have focused on the function of C5 protein as the protein component of RNase P, our understanding of C5 protein as an RNA-binding protein is quite limited. The characterization of RNA aptamers binding to C5 protein is a starting point towards understanding its function as an RNA-binding protein. In addition, the 5 bp stem and 20 nt loop structures identified as a small RNA motif specific for binding to C5 protein may be employed as model RNA for studying RNA–protein interactions. Elucidating the RNA-binding mechanism of C5 protein would provide a molecular basis for explaining the effects of the protein on RNase P function. In addition, this RNA motif may be used as an artificial inhibitor of bacterial RNase P.


   ACKNOWLEDGEMENT
 
This work was supported by a Korea Research Foundation Grant (KRF-2001-DP0349).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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