Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation

Abstract

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2-loop L3 and helix P2-loop L4)--all of this for the creation of a specific lysine binding site. We have determined that the interaction P2-L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2-L3 and P2-L4 allows both interactions to fold at lower magnesium ion concentrations. The P2-L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem-loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

Figure 1.

Secondary structure and activity of the lysine riboswitch as a function of lysine. (A) Secondary structure representing the lysine riboswitch. Regions involved in the formation of the kink–turn, loop E and terminator are indicated (16–21). Loop–loop L2–L3 and helix–loop P2–L4 interactions are shown by dotted lines and regions involved in the formation of the antiterminator are boxed and indicated by an arrow. Position 194 where a 2AP is introduced is shown by a star. The tertiary structure arrangement is shown in the inset, where discontinuities in the single-stranded regions are represented by dashed lines. The P2–L4 interaction is represented by vertical lines. (B) Single-round in vitro transcriptions performed as a function of P1 length in absence (–) or in presence (+) of 5 mM lysine. Variations in the total number of base pairs in P1 are indicated. Readthrough and prematurely terminated transcripts are indicated on the right and the percentage of termination is indicated below each reaction lane. Both structures representing the ON and OFF states are shown in the inset where the P1 stem is stabilized in presence of lysine.

Figure 2.

The P2–L4 interaction is important for the folding and activity of the riboswitch. (A) Secondary structure of the 4 bp and the 19 bp P4 stem mutants. (B) Single-round in vitro transcription assays made using selected P4 riboswitch mutants in absence (–) or in presence (+) of 5 mM lysine. Reactions were performed for the A156-deleted variant (ΔA156), A156C/A157C (L4), G98C/G99C (P2), P4 (4 bp) and P4 (19 bp). Readthrough (R) and prematurely terminated (T) transcripts are indicated on the right and the percentage of termination (%T) is indicated below each reaction lane. Vertical bars separate results obtained from different gels. (C) Non-denaturing gel electrophoresis of the wild-type (WT) and lysine aptamer mutants in the presence of 1 mM magnesium ions. The mutants L2 (G72C/A73U/U74A/G75C/G76C), P2 and L4 disrupt one of either L2–L3 or P2–L4 tertiary interactions, which results in a slower electrophoretic migration rate. (D and E) SHAPE modification of the lysine riboswitch performed at various magnesium ion concentrations. SHAPE data show that disruption of the P2–L4 interaction increases the magnesium requirement to induce the L2–L3 interaction (D) and that the disruption of the L2–L3 interaction increases the magnesium requirement to form the P2–L4 interaction (E). Arrows show SHAPE-reactive sites that are significantly modulated by magnesium ions.

Figure 3.

The G39C mutation inhibits binding without disrupting the global folding of the riboswitch. (A) Non-denaturing gel electrophoresis of the wild-type (WT) and G39C mutant in presence of 1 mM magnesium ions. The G39C mutant exhibits a migration similar to the wild-type, indicating that it does not perturb the global folding of the riboswitch. Note that both lanes were taken from the same gel but were manually juxtaposed to clearly indicate the co-migration of both aptamers. (B) SHAPE modification of the lysine riboswitch performed on the wild-type aptamer (WT). Reactions were performed at 0.5 mM MgCl₂, in absence (–) and in presence (+) of 5 mM L-lysine. N and A represent unreacted RNA and an adenine sequencing lane, respectively. Nucleotides of the wild-type aptamer exhibiting a lysine-induced modification are indicated on the right of the gel. The histogram on the left represents the relative density of bands corresponding to nucleotides 156–194, where filled and empty bars represent quantifications in absence and in presence of lysine, respectively. Positions showing significant difference in lysine-induced modification are marked by an asterisk. The inset represents a quantification of the reactivity of nucleotides G111 and A112. (C) SHAPE modification of the lysine riboswitch performed for the G39C aptamer mutant. Reactions were performed at 0.5 mM MgCl₂, in absence (–) and in presence (+) of 5 mM l-lysine. N and A represent unreacted RNA and an adenine sequencing lane, respectively.

Figure 4.

The formation of the P2–L4 interaction is important for riboswitch core folding. Normalized 2AP fluorescence intensity plotted as a function of magnesium ions for the WT (circles), the P2–L4-deficient L4 mutant (diamonds) and the G39C mutant (squares). The experimental data were fitted by regression to a simple two-state model where the binding of metal ions to the aptamer induces a structural change. Note that no significant change is observed when using either the L4 or G39 mutant.

Figure 5.

The P5 stem is important for lysine riboswitch activity. (A) Secondary structures of the 5 bp and the 16 bp P5 stem mutants. (B) Single-round in vitro transcription assays made using selected P5 riboswitch mutants in absence (–) or in presence (+) of 5 mM lysine. Reactions were performed for the wild-type riboswitch, the P5 (5 bp) and the P5 (16 bp) mutants. Readthrough (R) and prematurely terminated (T) transcripts are indicated on the right and the percentage of termination (%T) is indicated below each reaction lane.

Figure 6.

Lysine riboswitch folding monitored using a P1–P5 FRET vector. (A) Plot of the efficiency of FRET as a function of magnesium ions in the absence (squares) or the presence (circles) of 5 mM lysine. The FRET increase corresponds to a shortening of the distance between fluorophores attached to P1 and P5 stems. The data were fitted by regression to a simple two-state model in which the binding of Mg²⁺ to the aptamer induces a structural change. (B) Plot of the efficiency of FRET as a function of magnesium ion concentration for the WT molecule (squares), the L2 mutant (diamonds), the G39C mutant (circles) and the L4 mutant (triangles) in absence of lysine. Note that no significant change is observed when using the L4 riboswitch variant.

Figure 7.

The P5 stem can be used as an anti-antiterminator. (A) Schematic of the lysine riboswitch in which the expression platform is fused to the P5 stem. (B) Single-round in vitro transcriptions were performed using constructs with a P5 stem having either 6 or 10 bp in the absence (–) and the presence (+) of 5 mM lysine. Readthrough (R) and prematurely terminated (T) transcripts are indicated on the right and the percentage of termination (%T) is indicated below each reaction lane.

Figure 8.

The structure of the P6 stem is important for transcription termination. (A) Secondary structure of P6 and terminator mutants. Mutated sequences for the 0-, 4- and 7-bp P6 mutants are boxed and only replace the corresponding 5′-side of P6 enclosed between dotted lines. The 13 bp P6 stem is also boxed and replaces the entire wild-type P6 stem enclosed between dotted lines. The location and the additional base pairs introduced in the terminator stem (12 and 14 bp mutants) are also shown. (B) Single-round in vitro transcription assays were made using selected P6 mutants in absence (–) or in presence (+) of 5 mM lysine. Reactions were performed for the 0-, 4-, 7- and the 13-bp P6 mutants. (C) Single-rounds in vitro transcriptions assays were made using selected terminator stem mutants in absence (–) or in the presence (+) of 5 mM lysine. Reactions were performed for the 12-bp and 14-bp terminator stem mutants either in the context of the wild-type 10 bp (left panel) or the 13 bp (right panel) P6 stem. Readthrough (R) and prematurely terminated (T) transcripts are indicated on the right and the percentage of termination (%T) is indicated below each reaction lane.

Figure 9.

Folding of the lysine riboswitch as a function of magnesium ions and lysine. (A) Secondary structure of the lysine riboswitch summarizing NMIA reactivity obtained under different conditions. Regions involved in the formation of the kink–turn and loop E motif are boxed. L2–L3 and P2–L4 long-range tertiary interactions are indicated by dashed lines. Nucleotides reacting to NMIA upon increasing the concentration of magnesium ions or 5 mM l-lysine are indicated by black and empty circles, respectively. Nucleotides reacting in both conditions are indicated by gray circles. Nucleotides that are identified by a star show an increase in NMIA reaction. Nucleotides involved in the binding pocket and in the purine quartet are indicated in green and blue, respectively. (B) Representation of the lysine riboswitch crystal structure (19,20). Lysine is represented in red and nucleotides forming the purine quartet spanning stems P1 and P5 are indicated in blue. The inset shows the bottom junction layer where non-canonical base pairs G39•G193 and G171•A192 identified in blue are involved in the purine quartet. Note that numbering of the crystallized lysine riboswitch of Thermotoga maritima has been replaced by corresponding positions in the lysC lysine riboswitch of Bacillus subtilis (21).