Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis

Abstract

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

Expected and unexpected features of protein‐binding RNA aptamers

“…Recently, an RNA aptamer binding to the ribosomal protein S8 from Bacillus anthracis was described. In this case, the aptamer does not have any major sequence resemblance to the natural RNA ligands, but is still capable of mimicking the natural RNA–protein interactions . The bacterial ribosomal protein S8 (r‐protein S8) plays a structural role in the arrangement of 16S ribosomal RNA (16S rRNA) in the 30S ribosomal subunit.…”

“…Structural comparisons of unliganded and aptamerbound proteins reveal that the conformations of the target proteins are, in most cases, not significantly influenced by aptamer binding. 12,14,16,21,27,29,34 However, aptamer-protein interface residues may sometimes adopt novel orientations to accommodate RNA-protein interactions. 27,29 In the crystal structure of the NF-κB-aptamer complex, the two NF-κB p50 monomers of the homodimer adopt an open conformation relative to each other, compared with DNA-bound NF-κB.…”

“…While the structural changes of the NF‐κB aptamer following protein binding seem to stem from minor alterations to the secondary structure, the differences between the secondary structures of the free and protein‐bound ribosomal protein S8 aptamers appear more extensive . In the solution structure of the RNA aptamer, the internal loop, which primarily mediates protein S8 binding in the aptamer‐protein complex, largely adopts helical geometry with several non‐canonical base pairs formed (Figure ).…”

2-amino-1,3-benzothiazole-6-carboxamide Preferentially Binds the Tandem Mismatch Motif r(UY:GA)

“…All enzymes were purchased (Sigma) with the exception of T7 RNA polymerase which was prepared as described 18 The three-dimensional structures of A-D have been solved previously. 11,12,13,17 RNA molecules were prepared from 10 ml transcription reactions using 4 mM 5'-NTPs.…”

“…The tandem mismatch within the aptamer facilitates formation of a tertiary structure necessary for protein binding and may play a similar role in the rRNA L11 protein binding site 17 . Structural studies of RNAs containing the UU:GA mismatch also suggest that the identity of the flanking base pairs may influence the structure and dynamics of the mismatch [11][12][13]17 .…”