Mg(2+)-induced conformational changes in the btuB riboswitch from E. coli

Abstract

Mg(2+) has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B12 (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B12 transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg(2+) and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg(2+). With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg(2+) (or low Mg(2+) concentration) and the other appearing above ∼0.5 mM Mg(2+). Distinct regions of the riboswitch exhibit different dissociation constants toward Mg(2+), indicating a stepwise folding of the btuB RNA. Increasing the Mg(2+) concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg(2+) defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B12, and switch the RNA conformation. Moreover, raising the Mg(2+) concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg(2+) indicates a crucial role played by Mg(2+) for defining an active conformation of the riboswitch.

Keywords: Mg2+; RNA; coenzyme B12; folding; riboswitch.

FIGURE 1.

Mg²⁺-induced conformational changes in the btuB aptamer. (A) In-line probing of the btuB aptamer in the absence of AdoCbl by varying the Mg²⁺ concentration from 0 to 20 mM in the absence and presence of 100 mM KCl. Lane 14 with 20 mM Mg²⁺ and 500 μM AdoCbl serves as a reference to map the AdoCbl interacting sites 1–8. Nucleotides 19–183 indicate the sites of the most visible changes at Mg²⁺ > 0.1 mM. (NR) btuB in plain water, (T1) RNase T1 ladder, (OH) alkaline hydrolysis ladder. Structural changes detected by in-line probing experiments in the absence of AdoCbl are mapped on the secondary structure of the btuB aptamer in the absence (B) and in the presence of 2 mM Mg²⁺ (C). Shown are the nucleotides undergoing strong cleavage (green), intermediate cleavage (orange), and complete protection (black). Nucleotides in gray could not be mapped from the gel. Black arrows in C indicate the sites modulated by AdoCbl.

FIGURE 2.

Structural changes in the btuB aptamer induced by K⁺ and Mg²⁺. (A) Mg²⁺-induced stabilization of the btuB riboswitch folded in 100 mM KCl as indicated by the increase of the melting temperature from 52.8 ± 0.13 (no Mg²⁺, black curve) to 63.3 ± 0.10 in the presence of 1 mM Mg²⁺ (red curve). (B) CD spectra of the btuB aptamer renatured in 100 mM KCl and 0 mM (black), 0.1 mM (red), 1.5 mM (green), or 20 mM (blue) MgCl₂.

FIGURE 3.

Mg²⁺-induced folding transition for the btuB aptamer. (A) Partial RNaseT1 digestion of the btuB aptamer subjected to renaturation in varying Mg²⁺ concentrations (0–20 mM). Arrows indicate the corresponding guanine residues. (NR) btuB in plain water, (T1) RNaseT1 ladder under denaturing conditions, (OH) alkaline hydrolysis ladder. (B) The relative intensity of cleavage as detected in the RNase T1 assay is plotted (▵) against the Mg²⁺ concentration, and the data points were fitted according to a 1:1 binding model (—) to obtain the respective dissociation constants K_D (Sigel et al. 2000). The K_D values for G94 and G105 are not considered due to the large error associated with the values. (C) The proposed folding pathway for the btuB aptamer as a function of the Mg²⁺ concentration indicated by the five stages of folding as I (blue), II (orange), III (yellow), IV (pink), and V (green). This pathway is based on categorizing the K_D values of the individual guanine residues toward Mg²⁺ into the following five categories: 0.1–0.3 mM, 0.3–0.4 mM, 0.4–0.5 mM, 0.5–0.75 mM, 0.75–1 mM. The folding stages for the regions indicated in gray could not be predicted with certainty, e.g., for the 3′ tail, only the data for G188 could be fit (see also Table 1).

FIGURE 4.

Detection of the binding competent conformation of the btuB aptamer. (A) In-line probing of the btuB aptamer in the presence of 500 μM AdoCbl and 100 mM KCl by varying the Mg²⁺ concentration (0–20 mM). Sites 1–8 represent nucleotides modulated by AdoCbl. (NR) btuB in plain water, (T1) RNase T1 ladder, (OH) alkaline hydrolysis ladder. (B) Mg²⁺-dependent relative maximal changes at the sites (1–8) modulated by AdoCbl. The differences between the intensity changes in the presence and absence of AdoCbl at each Mg²⁺ concentration were normalized to the difference at 20 mM Mg²⁺. Data for site 5 and site 7 could not be evaluated. (C) Structural changes induced by AdoCbl along with Mg²⁺ (>0.1 mM). Superposition of structural changes detected by an in-line probing experiment in the presence of AdoCbl on the secondary structure of the btuB aptamer indicating nucleotides undergoing strong cleavage (green), intermediate cleavage (orange), or complete protection (black). Residues marked in red indicate the position of nucleotides undergoing enhanced cleavage by AdoCbl. Nucleotides in gray could not be mapped from the gel. (D) The native gel electrophoresis of the btuB riboswitch indicating the faster migration of the RNA species incubated with AdoCbl (100 μM) (red arrow) compared to the one in the absence of AdoCbl (yellow arrow).

FIGURE 5.

A model pathway for the folding and interaction between the btuB riboswitch and AdoCbl. The unfolded RNA begins to fold, leading to the formation of a binding-incompetent conformation (gray) at a Mg²⁺ concentration <0.5 mM (Pathway I). A binding-competent conformation of the RNA (black) is achieved at Mg²⁺ concentrations >0.5 mM (Pathway II). AdoCbl (pink) may or may not bind to the binding-incompetent conformation (gray) (Pathway III), but it does switch the RNA conformation at Mg²⁺ > 0.5 mM (Pathway IV). AdoCbl not only binds the binding-competent conformation but also switches its conformation, making it more compact (blue) (Pathway V).

FIGURE 6.

Mapping of Mg²⁺-induced changes on the AdoCbl aptamer consensus sequence depicted on the btuB sequence. The nucleotides undergoing Mg²⁺-induced conformational changes as observed in in-line probing experiments are indicated by black circles. The nucleotides in red are conserved in 90% of the representatives of AdoCbl aptamer sequences (Nahvi et al. 2004). The nucleotides in green represent the consensus sequence of the btuB aptamer and the AdoCbl riboswitches from the crystal structures (Johnson et al. 2012; Peselis and Serganov 2012). The nucleotides of the btuB aptamer shown in blue are common to one of the two sequences from the crystallized AdoCbl riboswitches (Johnson et al. 2012; Peselis and Serganov 2012).