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 kink-turn 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.

FIGURE 1.

Sequence and secondary structure of the lysC lysine riboswitch of Bacillus subtilis (Grundy et al. 2003; Sudarsan et al. 2003a). The represented secondary structure is shown with the transcription terminator structure (shaded region). Regions involved in the formation of the antiterminator helix are boxed and indicated by an arrow. The loop–loop interaction is indicated by a dotted line. The K-turn is shown in the P2 helical domain in a box. The mutations studied in this work are represented by boxed sequences. The chemical structure of lysine is shown in the inset.

FIGURE 2.

Lysine-induced riboswitch transcription termination. (A) Schematic showing constructs used in these experiments. The terminated (252 nt) and readthrough (347 nt) transcription products are indicated by arrows. Lysine is indicated by a rounded rectangle. While the premature termination product is expected to occur in the presence of ligand, transcription readthrough is expected in its absence. (B) Single-round transcriptions performed in the presence of 1 μM, 10 μM, 100 μM, 500 μM, 1 mM, 2.5 mM, 5 mM, 10 mM, 20 mM, 25 mM, and 30 mM L-lysine and resolved on 5% denaturing polyacrylamide gels. Readthrough and terminated products are indicated on the right. (C) Percentage of termination (%T) is plotted as a function of lysine concentration. The line shows a two-state model from which a T_1/2 value = 2.1 mM lysine was calculated.

FIGURE 3.

A loop–loop interaction is important for transcription termination. Single-round transcriptions were carried out in absence (−) and in presence (+) of 5 mM lysine for each riboswitch variant. Readthrough and terminated products were separated by gel electrophoresis and are indicated on the right. Reactions were performed for the natural sequence (WT), the G39C core variant, P2 and P3 loop–loop-deficient variants, and for the P2 + P3 variant. The percentage of terminated product for each reaction is indicated below the gel.

FIGURE 4.

A K-turn motif is present in the lysC aptamer. (A) The sequence and predicted secondary structure of B. subitlis lysC K-turn motif (left). The secondary structure has been written to match the established K-turn consensus (right) (Klein et al. 2001). Watson–Crick and purine base pairs are shown by vertical lines and circles, respectively. The star shows the nucleotide A67, known to be important for the adoption of the K-turn motif (Goody et al. 2004). R denotes a purine consensus. (B) Electrophoretic migration of the lysC K-turn motif in presence of magnesium ions. Comparison of electrophoretic mobility of RNA duplexes containing a centrally located lysC K-turn motif (K-turn) or variants (A_n) with duplexes kinked by virtue of a central oligoadenine bulge. Note that the lysC K-turn sequence migrates similarly to the five-adenine bulge species. Introduction of an A67C mutation (Kt′ variant) completely abolishes the K-turn structure, as previously observed for the Kt-7 variant (Goody et al. 2004). (C) Gel mobility shift assay of lysine aptamer as a function of the K-turn binding L7Ae protein. The aptamer was incubated in the presence of increasing concentrations of L7Ae, and the complexes analyzed by nondenaturing polyacrylamide gel electrophoresis. The aptamer was incubated in the absence (−) and in the presence of 0.8 nM, 2 nM, 5 nM, 6.7 nM, 10 nM, 12.5 nM, 16 nM, 50 nM, and 100 nM L7Ae. The free (F) and L7Ae-complexed aptamer (C) are indicated on the right. (D) Graphical representation of the K-turn motif obtained from 32 lysine aptamer sequences. The logo has been arranged to match the secondary structure of the K-turn motif. A proportional representation for each residues is shown. Watson–Crick and purine base pairs are shown by vertical lines and circles, respectively. A star indicates the location of A67. Nucleotide positions are indicated below each representation.

FIGURE 5.

The kink-turn motif is important for loop–loop formation and transcription control. (A) Partial digestions of end-labeled wild-type and Kt′ variant aptamers by RNase T1 show that G72, G75, and G76 present in the P2 helical domain are protected from cleavage by the addition of magnesium for the wild-type molecule (left panel). However, no protection is observed when the Kt′ mutant is introduced in the aptamer (right panel). Lane NR contains unreacted RNA, while lane L contains RNA subjected to partial alkaline digestion. Positions of strong cleavage are indicated on the left. A schematic is shown on the right representing the three observed guanines and the loop–loop interaction. (B) Single-round transcriptions of the Kt′ mutant performed in the absence (−) and in the presence (+) of 5 mM lysine. Readthrough and terminated products were separated by gel electrophoresis and are indicated on the right. The percentage of terminated product is indicated below the gel for each reaction.

FIGURE 6.

Formation of the loop–loop interaction is important for the reorganization of the aptamer core region. The insert shows fluorescence emission spectra (λex = 300 nm) from 330 nm to 420 nm for each magnesium ion concentration. An increase of fluorescence is observed when the fluorescent lysine aptamer is incubated by the addition of magnesium ions. The normalized 2AP fluorescence intensity is plotted as a function of magnesium ions for the natural sequence (WT), the loop–loop-deficient variants P2 (squares) and P3 (diamonds), the doubly substituted aptamer P2 + P3 (crosses) and the K-turn-compromised variant (triangles). The experimental data were fitted (line) by regression to a simple two-state model where the binding of metal ions to the aptamer induces a structural change.