Multiple metal-binding cores are required for metalloregulation by M-box riboswitch RNAs

Abstract

Riboswitches are regulatory RNAs that control downstream gene expression in response to direct association with intracellular metabolites or metals. Typically, riboswitch aptamer domains bind to a single small-molecule metabolite. In contrast, an X-ray crystallographic structural model for the M-box riboswitch aptamer revealed the absence of an organic metabolite ligand but the presence of at least six tightly associated magnesiums. This observation agrees well with the proposed role of the M-box riboswitch in functioning as a sensor of intracellular magnesium, although additional nonspecific metal interactions are also undoubtedly required for these purposes. To gain greater functional insight into the metalloregulatory capabilities of M-box RNAs, we sought to determine whether all or a subset of the RNA-chelated magnesium ions were required for riboswitch function. To accomplish this task, each magnesium-binding site was simultaneously yet individually perturbed through random incorporation of phosphorothioate nucleotide analogues, and RNA molecules were investigated for their ability to fold in varying levels of magnesium. These data revealed that all of the magnesium ions observed in the structural model are important for magnesium-dependent tertiary structure formation. Additionally, these functional data revealed a new core of potential metal-binding sites that are likely to assist formation of key tertiary interactions and were previously unobserved in the structural model. It is clear from these data that M-box RNAs require specific binding of a network of metal ions for partial fulfillment of their metalloregulatory functions.

Figure 1

Separation of extended and compact M-box RNAs (a) Schematic showing the transition from extended to compact conformations for the M-box. Nucleotide stretches labeled 1–4 are regions that form base-pairing interactions such that downstream expression is `on' in the unbound state and `off' in the magnesium-bound state. Control of gene expression is achieved through mutually exclusive formation of either an intrinsic transcription terminator helix (“T”) or an antiterminator helix (“AT”). The portion of the RNA colored black represents the aptamer domain that is responsible for ligand binding. RNAP = RNA polymerase. (b) Representative elution profile for size exclusion chromatography in buffer containing 0.5 mM magnesium for M-box RNAs randomly modified at adenosines with phosphorothioate substitutions. (c) Non-denaturing gel electrophoresis of extended and compacted M-box species in the presence of 0.5 mM magnesium. WT (wild-type) and M3 mark the compact and extended states, respectively. A faint band labeled `unknown' was observed to migrate between extended and compact RNA species. This newly identified species may require a distinct subset of the magnesium binding sites (data not shown) and may represent a folding intermediate or a misfolded conformation.

Figure 2

Quantification of interference values for extended and compact M-box RNAs separated under (a) 0.5 mM and (b) 1.0 mM magnesium conditions. The dashed line indicates an arbitrary cutoff for interference values greater than 2.

Figure 3

Sites of phosphorothioate interferences at established magnesium-binding sites (a) Secondary structure diagram for the B. subtilis mgtE M-box RNA. Positions identified in the structural model as coordinating to magnesiums (Mg1–6) via phosphate oxygens are denoted by different symbols. The different helical elements and terminal loops are indicated with colored labels according to a color scheme utilized by the other figures in the manuscript. Red letters denote positions of phosphorothioate interferences. (b) Representative data showing sites of phosphorothioate interferences for RNA molecules in 0.5 mM magnesium. P = Parental, unselected transcription reaction. E = Extended RNA molecules. C = Compact RNAs. In most cases, individual phosphorothioate interferences for compact RNAs correlated with phosphorothioate enhancements for extended RNAs relative to the parental transcription pool. Symbols corresponding to Mg1–6 are grouped into columns based on whether the metal coordinates to either the pro-S_p or pro-R_p oxygen in the structural model.

Figure 4

Phosphorothioate interferences at positions not previously identified as magnesium binding sites. (a) Representative data showing sites of phosphorothioate interferences for RNA molecules in 0.5 mM magnesium. P = Parental, unselected transcription reaction. E = Extended RNA molecules. C = Compact RNAs. Strong interferences are marked by large circles, moderate by medium-sized circles, and weak by small circles. (b) Phosphorothioate modifications (shown with red spheres) at A101 and A105 are to likely interfere with formation of structural features required for the Mg1 binding site and interactions between L5 and P2. Black dashed lines denote putative hydrogen bonding interactions disrupted by phosphorothioate substitutions. The green sphere represents Mg1. (c) Structural context for the phosphorothioate interference at the pro-R_p of U119. The pro-S_p phosphate oxygens of A117 and A118 and the U119 pro-R_p oxygen appear to coordinate formation of a candidate metal binding site (M7). (d) Structural context for the phosphorothioate interferences at the pro-R_ps of C89 and G91, which may participate in formation of a metal binding site (M8). (e) Structural context for phosphorothioate interferences at the pro-R_ps of C35 and C36, which may participate in formation of a metal binding site (M9). Phosphate oxygens of C143 also contribute to electronegativity near the C35 and C36 backbone.

Figure 5

Metal binding sites can be grouped into three separate cores. (a) Secondary structure diagram showing long-range base interactions (dashed lines), A-minor motif interactions (colored boxes connected by dashed lines), sites of phosphorothioate interferences (red letters) and magnesium cores (gray shaded boxes). (b) Residues contacting metals or involved in long-range base interactions are shown within the structural model. These residues cluster within the three magnesium cores postulated herein. (c) The magnesiums of Core 2 assist in orienting bases to stack on either the top or bottom half of the P4 helix, while introducing a kinked orientation of the P4 terminal loop (L4). This orientation is necessary for formation of tertiary contacts between L4, L5 and P2. Observation of phosphorothioate interferences at the Mg5 and Mg6 sites indicates that Core 2 is functionally important. (d) Residues within Core 3. Specific hydrogen bonding interactions are highlighted with dashed lines. Key A-minor motifs are labeled accordingly. Regions corresponding to the newly identified candidate metal sites (M7–9) are indicated. These sites appear to assist formation of key tertiary interactions, interhelical bonds and stacking interactions.