Folding of the SAM Aptamer is Determined by the Formation of a K-turn-dependent Pseudoknot

Abstract

The S-adenosylmethionine (SAM) riboswitch is one of the most recurrent riboswitches found in bacteria and has three known different natural aptamers. The Bacillus subtilis yitJ SAM riboswitch aptamer is organized around a four-way junction which is characterized by the presence of a pseudoknot and a K-turn motif. By replacing the adenine involved in a Watson-Crick base pair at position 138 in the core region of the aptamer with the fluorescent analogue 2-aminopurine (2AP), we show that the ligand-induced reorganization of the aptamer strongly attenuates 2AP fluorescence. The fluorescence quenching process is specific to SAM on the basis of the observation that the structural analogue S-adenosylhomocysteine does not promote a similar effect. We find that the pseudoknot is important for the reorganization of the core domain and that the K-turn motif also has a marked influence on the core domain reorganization, most probably through its important role in pseudoknot formation. Finally, we show that SAM riboswitch ligand binding is facilitated by the L7Ae K-turn binding protein, which suggests that K-turn motifs may be protein anchor sites used by riboswitches to promote RNA folding.

Folding of the SAM-I riboswitch

“…23 In agreement with this model, it was previously found that the folding of the kink-turn motif located in the P2 helical domain is crucial for the structure and ligand binding activity of the SAM-I aptamer. 46,50 Our study also revealed that SAM binding to the aptamer produces a second folding transition involving the stacking of stems P1 and P4. 23 SAM binding was also previously shown to reorganize regions J1/2 and J3/4, 33,54 consistent with the formation of the native RNA-ligand complex as observed in the crystal structure.…”

“…A K-turn motif located in the P2 stem has been shown to be important for the structure and activity of the riboswitch. 45,46 The K-turn has been originally observed in the 23 rRNA and is characterized by the presence of a sharp bend in the phosphate backbone, which is important to mediate RNA tertiary structure interactions and protein…”

Modulation of quaternary structure and enhancement of ligand binding by the K-turn of tandem glycine riboswitches

“…Those studies (Blouin and Lafontaine 2007;Heppell and Lafontaine 2008) utilized the archaeal protein L7Ae. We recently found crystallographically that the B. subtilis protein YbxF is structurally homologous to L7Ae and that it binds to K-turns (Baird et al 2012).…”

“…The motif causes the helical path to bend by ∼120°a nd is often the binding site for proteins (Klein et al 2001). K-turns are present in several riboswitch classes, including the Class-I S-adenosylmethionine (SAM-I) and lysine riboswitches, where it has been shown that K-turn integrity is essential for productive folding and ligand recognition (Blouin and Lafontaine 2007;Heppell and Lafontaine 2008). Kladwang et al (2012) and Sherman et al (2012) analyzed the effect that the presence of P0 has on the glycine riboswitch at the local, single-nucleotide level through chemical probing techniques.…”

A structural database for k-turn motifs in RNA

“…Further examples have been found in mRNA (Mao et al 1999;Winkler et al 2001;Allmang et al 2002;White et al 2004) and in riboswitches (Montange and Batey 2006;Blouin and Lafontaine 2007;Heppell and Lafontaine 2008). They are very commonly found in C/D and H/ACA guide snoRNAs and in U3 snoRNA species (Kuhn et al 2002;Watkins et al 2002;Bortolin et al 2003;Marmier-Gourrier et al 2003;Rozhdestvensky et al 2003;Hamma and Ferré-D'Amaré 2004;Moore et al 2004;Szewczak et al 2005;Youssef et al 2007).…”