Interaction of C5 protein with RNA aptamers selected by SELEX

Abstract

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.

Figure 1

Selection of RNA aptamers binding to MBP–C5 protein by SELEX. Mixtures of internally labeled RNA pools with [α-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.

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.

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.

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).

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.

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.

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.