Structural features of metabolite-sensing riboswitches

Abstract

Riboswitches, metabolite-sensing RNA elements that are present in untranslated regions of the transcripts that they regulate, possess extensive tertiary structure to couple metabolite binding to genetic control. Here we discuss recently published structures from four riboswitch classes and compare these natural RNA structures to those of in-vitro-selected RNA aptamers, which bind ligands similar to those of the riboswitches. In addition, we examine the glmS riboswitch - the first example of a ribozyme-based riboswitch. This RNA provides the latest twist in the riboswitch field and portends exciting advances in the coming years. Our knowledge of the mechanisms underlying genetic regulation by riboswitches has increased mightily in recent years and will continue to grow as new riboswitch classes and ligands are discovered and structurally characterized.

Figure 1

Secondary structure and genetic control mechanisms of structurally characterized riboswitches. The secondary structures and mechanisms of genetic control for each riboswitch class highlighted in this review are shown. Diversity in RNA-based genetic control mechanisms is illustrated herein. The guanine and SAM riboswitches highlighted in this figure employ transcription attenuation via mutually exclusive terminator (T)-antiterminator (AT) pairing elements for genetic regulation. The adenine and TPP riboswitches shown in the figure exert regulatory control over translation inhibition. Finally, the glmS RNA is a metabolite-sensing ribozyme that utilizes GlcN6P for activation of cis-cleavage of transcripts and is likely to control mRNA stability. Comparison of the guanine and adenine riboswitches shown in (a) and (b) also highlight how cis-acting regulatory RNAs can repress or activate gene expression. For all riboswitch classes except the glmS ribozyme, the mechanism of genetic control can vary pending on the microorganism. Color-coding of individual nucleotides matches colors for three-dimensional structures shown in Figure 2. Dashed red lines denote nucleotide positions that form hydrogen bonds to the metabolite ligands. `Off' and `on' indicate conditions wherein expression of downstream genes is reduced or increased, respectively. Pseudoknot interactions in (a) – (c) are depicted by the linked boxes. Gray boxes in (b) and (d) denote the ribosome binding site for the downstream gene. The blue arrow shown in (e) indicates the site of ribozyme self-cleavage that occurs in response to binding of GlcN6P. Pairing elements (helices) within aptamer domains are assigned the letter `P' and numbered with respect to their proximity to the 5′ end of the RNA transcript. Similarly, terminal loops are designated by the letter `L'. The source microorganisms for the riboswitches shown in (a) – (e) are Bacillus subtilis, Vibrio vulnificus, Thermoanaerobacter tencongensis, Escherichia coli, and Thermoanaerobacter tencongensis, respectively.

Figure 2

Global visualization of riboswitch structure and ligand binding. (a) to (e) Representative structures are presented for each structurally characterized riboswitch class. The natural ligand and PDB accession codes are listed in parentheses along with number of nucleotides (nt) and source organism. RNAs shown are bound to their natural ligand (shown as spheres) except the GlcN6P-binding ribozyme (e), which is bound to glucose-6-phosphate. Additional structures of the guanine- and GlcN6P-sensing riboswitches bound to hypoxanthine and GlcN6P, respectively, are deposited online at http://www.rcsb.org with accession codes 1U8D and 2NZ4. (f) To gain some perspective on the size of riboswitch aptamer domains, the structure of a classic RNA, tRNA-Phe, is shown [67]. Most structurally characterized riboswitches are approximately the same size as tRNAs and should not be considered trivial, small molecules even when compared to a large bacterial RNA polymerase, also shown [68]. (>300 kDa). All panels in this figure are on the same scale.

Figure 3

Stereo views of riboswitch aptamer ligand binding sites. The ligand binding sites from the (a) T. tencongensis SAM-I (b) E. coli TPP, and (c) T. tencongensis glmS riboswitches are shown with bases that make direct hydrogen bonding interactions to their respective ligands. Any Mg²⁺ that makes inner sphere contacts to the ligand are also shown as green spheres. RNA is shown as sticks and ligands are represented as ball-and-sticks.

Figure 4

Overview of ligand binding to in vitro evolved RNA aptamers. Non-natural RNA aptamer structures are shown as sticks with a ribbon drawn through the riboses of the RNA. The ligands are listed along with the PDB accession codes in parentheses and the number of nucleotides (nt). Ligands are shown as ball-and-stick and are colored gray. A secondary structure diagram is shown below each structure. (a) The ATP-binding aptamer bound to AMP showing contacts primarily to the adenine ring and a couple of contacts to the ribose. This RNA binds ligands with micromolar dissociation constants and promiscuously binds similar molecules with adenine-like moieties including ATP and NAD due to lack of extensive contacts on the solvent exposed side of the ligand. (b) The recently determined GTP aptamer [57] shows the most hallmarks of ligand binding to natural aptamers. The ligand is largely buried in its binding pocket with a K_d ~75 nM. Similarly to riboswitches, this RNA also has more tertiary interactions that are not directly involved in ligand binding, a possible reason for the increased affinity. The vitamin B12 aptamer (c) and the FMN aptamer (d) structures allow speculation on potential modes of binding of B12 and FMN to naturally occuring riboswitches. In the case of the B12 aptamer, the ligand is bound peripherally to the surface of the aptamer, a binding mode likely not utilized by the B12-sensing riboswitch based on evidence of high specificity of B12 binding to the natural aptamer. The FMN aptamer, on the other hand, might share features with the natural FMN-sensing riboswitch, particularly for the isoalloxazine ring contacts. FMN intercalates into the RNA and forms hydrogen bonds to bases via the edge of this ring, a binding mode that could easily be recapitulated in the FMN-sensing riboswitch. However, based on features of riboswitch structures, the natural FMN-aptamer will likely incorporate more tertiary interactions to indirectly facilitate FMN binding and also encapsulate the ligand more fully into a binding pocket. Abbreviations: AMP – adenosine-monophosphate; ATP – adenosine-triphosphate; GTP – guanosine-monophosphate; FMN – flavin mononucleotide; NAD – nicotinamide adenine dinucleotide.