Role of lysine binding residues in the global folding of the lysC riboswitch

Abstract

Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.

Keywords: RNA folding; Riboswitch; helical junction; lysine; metabolite-sensing.

Figure 1.

Structure and function of the lysine riboswitch. (A) Schematic representation of the lysine riboswitch regulation mechanism. In absence of lysine, the lysine lysC riboswitch adopts a conformation in which transcription readthrough is allowed. In this configuration, the P1 stem is presumably not formed in the aptamer domain. However, upon lysine (LYS) binding, the P1 stem is stabilized, which in turn allow the formation of a transcription terminator. Tertiary interactions involving L2-L3 and P2-L4 are shown. (B) Structure of the lysine riboswitch aptamer domain. The crystal structure of the lysine aptamer bound to lysine is shown. Lysine is represented in violet and selected nucleotides involved in the lysine-binding site are shown. The insert shows base pair G40-C110 and the wobble pair G144•U170 in yellow and green, respectively. G111 is represented in magenta. The nucleotide nomenclature is based on previous studies. (C) Representation of the lysine-binding site. The base pair G40-C110 and the wobble pair G144•U170 are shown in yellow and green, respectively, and G111 is represented in magenta. While pairs G40-C110 and G144•U170 interact with the carboxyl end of lysine, G111 is involved in the recognition of the ε-ammonium group. Lysine is shown in violet. A potassium ion (K⁺) coordinating lysine is shown in green. Hydrogen bond interactions are shown as dotted lines. Nitrogen and oxygen atoms are shown in blue and red, respectively.

Figure 2.

Lysine-induced transcription termination of riboswitch junction core variants. (A) Single-round transcriptions as a function of lysine concentration. Transcriptions were performed in presence of 1 µM, 10 µM, 50 µM, 500 µM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 50 mM and 75 mM of lysine. Readthrough (R) and prematurely terminated (T) products are shown on the right. Reactions were quantified and plotted as a function of lysine concentrations. Fitting the data to a 2-state model gave a T₅₀ value of 3.8 ± 0.7 mM. (B) Junction core residues are important for riboswitch transcription termination. Single-round transcriptions were performed in absence (-) and presence (+) of 10 mM lysine for each riboswitch variant. Readthrough (R) and prematurely terminated (T) products are shown on the right. The percentage of terminated product for each reaction is indicated below.

Figure 3.

Folding of the P1-P5 transition monitored by FRET assays. (A) Schematic of the lysC aptamer construct used to monitor the P1-P5 FRET vector. Donor (fluorescein; F) and acceptor (Cy3; C) fluorophores are shown as hexagons linked to the 3′ and 5′ ends of P1 and P5 stems, respectively. The construct is based on a previous study that has shown that the removal of the P5 loop does not perturb riboswitch folding. The location of both fluorophores was chosen according to crystal structures. (B) Efficiency of FRET (E_FRET) as a function of Mg²⁺ concentration in absence (black circles) or presence (white circles) of 5 mM lysine. The fluorescence data were fitted (lines) by regression using a 2-state model where Mg²⁺ binding to the RNA induces P1-P5 folding. (C) Efficiency of FRET (E_FRET) as a function of lysine in the context of 10 mM Mg²⁺ ions. Data were fitted to a 2-state model assuming that lysine binding to the aptamer domain induces the folding of the P1-P5 FRET vector.

Figure 4.

Importance of junction core residues for P1-P5 folding transition. (A) Efficiency of FRET (E_FRET) of the G39A riboswitch mutant as a function of Mg²⁺ concentration in absence (black circles) or presence (white circles) of 5 mM lysine. The fluorescence data were fitted (lines) by regression using a 2-state model where Mg²⁺ binding to RNA induces P1-P5 folding. (B, C and D) Efficiency of FRET (E_FRET) as a function of Mg²⁺ concentration in absence (black circles) or presence (white circles) of 5 mM lysine. Experiments were carried out for the G111U (B), G144A (C) and U170C (D) riboswitch variants. Data analysis as performed as indicated in (A).

Figure 5.

Junction core mutations are not important for the global folding of the aptamer. (A) Secondary structure representing the lysC aptamer. The regions participating in the formation of L2-L3 and P2-L4 interactions are indicated (dotted lines). Arrows indicate nucleotides involved in the corresponding tertiary interactions. Residues mutated are shown in black circles. The nucleotide nomenclature is based on previous studies. (B, C, D and E) SHAPE modification of selected lysC aptamers performed in the context of 10 mM NaCl. Experiments were done as a function of 10 mM Mg²⁺ and 5 mM lysine and analysis was performed using the wild-type aptamer (B) and variants G111U (C), G144A (D) and U170C (E). In each case, cytosine (C) and uracil (U) sequencing lanes were added to resolve the sequence. NMIA was replaced by DMSO (D) in control reactions. Nucleotides or regions exhibiting changes either in Mg²⁺ or lysine are indicated on the right.

Figure 6.

Proposed folding pathway and ligand sensing mechanism of the lysC riboswitch aptamer. In absence of Mg²⁺ ions, the lysC aptamer is assumed to adopt an unfolded structure (unfolded) in which only secondary structure elements (P1 to P5 stems) are formed. The presence of black braces pointing outside the aptamer domain indicates that junction core residues are not folded at this stage. Upon Mg²⁺ ions binding, the aptamer is reorganized into a structure (F_Mg) exhibiting long-range tertiary interactions L2-L3 and P2-L4 (dotted lines), as observed in crystal structures. Mg²⁺ ions binding also promote the formation of the P1-P5 folding transition (double arrow). Addition of lysine (LYS, black circle) results in the adoption of the native state (F_NS) that consists of an additional P1-P5 transition as well as the reorganization of the junction core residues (inward orientation of black braces).