A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

Abstract

The lysine riboswitch is associated to the lysC gene in Bacillus subtilis, and the binding of lysine modulates the RNA structure to allow the formation of an intrinsic terminator presumably involved in transcription attenuation. The complex secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that undergoes structural changes upon ligand binding. Using single-round transcription assays, we show that a loop-loop interaction is important for lysine-induced termination of transcription. Moreover, upon close inspection of the secondary structure, we find that an unconventional kinkturn motif is present in one of the stems participating in the loop-loop interaction. We show that the K-turn adopts a pronounced kink and that it binds the K-turn-binding protein L7Ae of Archaeoglobus fulgidus in the low nanomolar range. The functional importance of this K-turn motif is revealed from single-round transcription assays, which show its importance for efficient transcription termination. This motif is essential for the loop-loop interaction, and consequently, for lysine binding. Taken together, our results depict for the first time the importance of a K-turn-dependent loop-loop interaction for the transcription regulation of a lysine riboswitch.

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…Reactions were stopped using an equal volume of formamide loading dye. K switch values were obtained using a procedure similar to that used for T 50 (26,27). The quantification assumes a simple 1:1 stoichiometry between the aptamer and AdoCbl as expected from in-line probing data (10,16).…”

Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

“…2-Aminopurine Fluorescence Spectroscopy-Fluorescence spectroscopy was performed as described previously (21). The fluorescence data were fitted to a simple two-state model that assumes an all-or-none conformational transition induced by the binding of magnesium ions, with a Hill coefficient n and an apparent association constant K A .…”

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

“…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.…”