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Flipping Off the Riboswitch: RNA Structures That Control Gene Expression Dipali G. Sashital and Samuel E. Butcher* Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, Wisconsin 53706
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ne of the overriding themes in biology is the need for exquisite control and precision in the regulation of gene expression for the normal growth and development of organisms. Regulated gene expression has been discovered at every level, from transcription of the genetic code to post-translational protein modification. In its simplest terms, genetic regulation is the process by which the cell recognizes its metabolic needs and acts to modulate the levels of certain gene products on the basis of these requirements. Traditionally, these functions were thought to be controlled exclusively by proteins. However, it is now known that ⬎2% of bacterial genes are regulated by metabolitesensing RNAs without the assistance of proteins (1–3). These “riboswitches” form within the leader sequences of messenger RNAs (mRNAs) and bind their target metabolites with the affinity and specificity required for the precise regulation of gene expression. Metabolite binding to the riboswitch effects an allosteric conformational change that modulates gene expression at the transcriptional or translational level. In the past, our knowledge of how riboswitches could transfer the binding energy of specific metabolites into optimal genetic control was limited by a shortage of 3D structural data. A new flood of riboswitch crystal structures from the laboratories of Batey (4), Ban (5), and Patel (6) now presents a clearer picture of the structural complexity required for these functions (Figure 1). www.acschemicalbiology.org
Similar to RNA aptamers selected in vitro to bind small molecules (7, 8), riboswitches A B S T R A C T Riboswitches are metabolitehave evolved in vivo to bind a remarkably sensing RNA structures that have been discovered wide range of molecules with extremely high in regulatory regions of messenger RNA (mRNA). They have the remarkable ability to shut off the affinity, including several diverse metabotranscription or translation of their own mRNAs in lites and coenzymes such as guanine response to binding a specific metabolite. In other and adenine (9–12), L-lysine (13, 14), words, riboswitches regulate their own genes thiamine pyrophosphate (TPP) (15), and using RNA instead of protein. Three new crystal S-adenosylmethionine (SAM) (16–18) (for structures reveal how S-adenosylmethionine and review, see refs 1–3). This observation is not thiamine pyrophosphate riboswitches accomplish wholly surprising, given the numerous types this task. of artificial RNA aptamers that had been previously isolated in vitro (7, 8, 19). One of the truly fascinating and unique features of riboswitches is the elegant way in which they utilize a variety of ligand-induced structural rearrangements to modulate gene expression. This mechanism depends on the riboswitch’s intricate architecture, which uses both secondary and tertiary structures with far greater complexity than is typically seen in aptamers selected in vitro (Figure 1) (20). Within a riboswitch, the ligand-binding aptamer domain is coupled to an “expression platform” whose conformation controls gene expression through a variety of methods. Transcription can be turned off or on through either the formation or the preclusion of a terminator stem (Figure 2, panel a), translation can be inhibited by the *Corresponding author, sequestration of the Shine–Dalgarno (SD) sequence within a structure (Figure 2, panel
[email protected]. b), or the mRNA can be cleaved through enzymatic activity of the riboswitch itself Published online July 21, 2006 (1–3, 21). 10.1021/cb002465 CCC: $33.50 Riboswitches, like their metabolitesensing protein counterparts, must be able © 2006 by American Chemical Society VOL.1 NO.6 • ACS CHEMICAL BIOLOGY
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Figure 1. Structures of TPP-sensing riboswitches from E. coli and A. thaliana and a SAM-sensing riboswitch. Overall folding schemes for a) TPP and b) SAM riboswitches. Crystal structures of E. coli and A. thaliana c) TPP riboswitches and d) a SAM riboswitch. Ligands (green) are bound at the interface of parallel helices in all three structures. Helix P1, the ligand, and the PK are labeled. Surface representation of e) TPP and f) SAM riboswitches reveals the significant burial of the ligand within the globular RNA fold.
to discriminate against other small molecules in order to be viable gene regulators. Accordingly, riboswitches are exceptionally specific and can reject even subtle variations of the natural ligand. For example, riboswitches distinguish between molecules on the basis of the presence or absence of functional groups (15, 18, 22), atomic charge (23), and stereochemistry (14, 24). This ligand selectivity has led to several seemingly paradoxical observations about the types of molecules that are bound. In several cases, including the TPP riboswitch, negatively charged phosphate groups on the ligand are required for optimal binding to the polyanionic RNA (15, 21, 22); this reveals another rather puzzling facet to the extraordinary ligand specificity displayed by riboswitches. 342
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So how do riboswitches translate a binding event into a conformational rearrangement dramatic enough to turn off a gene? And how is a binding pocket created that can comfortably house negatively charged molecules that would normally be repelled by RNA? New crystal structures of the TPP- and SAM-sensing riboswitches offer insights into these questions (4–6). The TPP-responsive riboswitch is located in the 5= untranslated region of mRNAs involved in thiamine biosynthesis, controlling the expression of these genes by inhibiting translation, in the case of the crystallized TPP riboswitch, or transcription, as has been observed for other known TPP riboswitches (15). Interestingly, this riboswitch is one of the most ubiquitous of the known classes, with representatives identified in all SASHITAL AND BUTCHER
three domains of life (25). Appropriately, the structures of both a prokaryotic (Escherichia coli) and a eukaryotic (Arabidopsis thaliana) TPP riboswitch are revealed in publications from the Patel and Ban laboratories, respectively; this allows for an evolutionary comparison (Figure 1, panels a, c, and e) (5, 6). The Batey laboratory unveiled the structure of a SAM-responsive riboswitch, a common bacterial regulator that modulates the expression of genes involved in sulfur metabolism and methionine biosynthesis through transcription termination (Figure 1, panels b, d, and f) (4, 16–18). One of the most striking features of the riboswitches is their elaborate tertiary structures, in which the RNAs adopt globular folds that surround and bury their ligands within their cores (Figure 1, panels c–f). Complex RNA structures are made up of preformed secondary structural elements, including helices, loops, and junction regions that organize into tertiary folds through RNA–RNA stacking or hydrogenbonding interactions (Figure 1, panels a and b) (26). Intriguingly, in the riboswitch structures, the ligands appear to be the catalysts for the formation of many of the tertiary contacts. Binding of the ligand in each structure occurs at the interface between two parallel helices, creating intricate hydrogen-bonding and electrostatic networks between the RNA, bound Mg2⫹ ions, and the small molecule (Figure 3). These interactions drive the induced-fit binding mechanism of the riboswitch, with the ligand serving to juxtapose and tether separate domains that form tertiary contacts only upon ligand binding. The resulting compaction is instrumental in the formation of a crucial helix (P1) that turns off both riboswitches through the sequestration of the SD sequence (Figure 2, panel a) or the formation of a terminator stem (Figure 2, panel b). Along with helical packing, ligand binding triggers the organization of single-stranded regions of the RNA that would likely be diswww.acschemicalbiology.org
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VIEW Figure 2. Genetic control by riboswitches is achieved by the coupling of aptamer and expression platform domains. a) Translational and b) transcriptional methods for genetic regulation are shown, as they apply to the TPP and SAM riboswitches, respectively. a) In the absence of TPP, the riboswitch contains little tertiary structure, and helix P8* forms, leaving the SD sequence free to interact with the ribosome. Upon binding of TPP, the aptamer domain folds into a compact, globular structure, forming helix P1. Helix P8 forms within the expression platform, sequestering the SD, thus inhibiting translation. b) The SAM riboswitch forms a PK interaction in the absence of ligand, forming a partial binding pocket for the ligand. An anti-terminator stem forms, portions of which are mutually exclusive with helix P1. Ligand binding stabilizes the formation of P1, thus disrupting the anti-terminator and allowing a terminator stem to form.
ordered in the absence of the small molecule. Many of these unpaired strands are located at the junctions between helices and must be folded in order for helical stacking to occur (Figure 1, panels a and b). This is especially true in the TPP-sensing riboswitch, in which the pyrimidine ring of the ligand interacts extensively with a singlestranded junction between two helices, resulting in a helical stack that forms onehalf of the parallel helical motif that dominates the structure (Figure 1, panel c). In general, the globular structure of the TPP riboswitch appears to form only upon ligand binding. Structure-probing experiments presented by Serganov et al. (6) suggest that the tertiary contacts observed in the structure only occur when the ligand is bound. In contrast, the tertiary structure in the SAM-sensing riboswitch is partially preformed (18, 23) through a pseudoknot (PK) interaction (27) that occurs between a loop region of one stem-loop and the junction region between two other helices (Figure 1, panels b and d). In the SAM riboswitch, the PK preserves the global architecture of the structure, including a helical stack and a kink-turn motif (28) (Figure 1, panel b, and Figure 2, panel b). The partial formation of the SAM riboswitch structure likely aids in ligand recognition (4). www.acschemicalbiology.org
One of the most surprising aspects of the TPP riboswitch is its ability to select for negatively charged phosphate groups. Previous studies of the TPP riboswitch revealed that the aptamer also binds thiamine and thiamine phosphate, albeit with decreasing affinity; this suggests that the presence and the length of the phosphate functional group are instrumental in binding specificity (15). The structures of the TPP riboswitch confirm this hypothesis, because the RNA forms two binding pockets: one for the pyrimidine ring present in all three compounds and one for the pyrophosphate found solely in TPP (Figure 3, panels a and b). In the first pocket, the pyrimidine ring of thiaminecontaining compounds forms several hydrogen bonds with nucleotides within the riboswitch. Additionally, the pyrimidine ring is sandwiched between two purines, creating a very snug fit for the ligand (Figure 4, panel b). However, for the two helical domains of the riboswitch to be bridged, the pyrophosphatebinding pocket must also be occupied. Binding of the TPP pyrophosphate moiety occurs in a large pocket formed at a junction between two helices, which also coordinates Mg2⫹ ions that shield the negative charge of the phosphate groups (Figure 3, panels a and b). Binding of the TPP pyrophosphate moiety occurs in a large pocket
formed at a junction between two helices. The helices also coordinate the Mg2⫹ ions that shield the negative charge of the phosphate groups (Figure 3, panels a and b). The E. coli structure contains two partially hydrated Mg2⫹ ions, which directly coordinate to the pyrophosphate and form watermediated hydrogen bonds between the pyrophosphate and the RNA (Figure 3, panel a). The A. thaliana structure also shows an identically bound Mg2⫹ ion (Figure 3, panel b). The second Mg2⫹ ion was not observed in the A. thaliana structure, possibly because of differences in the data resolution or the occupancy of the second ion. In both structures, the nonbridging oxygen atoms of the pyrophosphates hydrogen-bond to nucleotides within the junction. The structures highlight the importance of Mg2⫹ in neutralizing repulsive forces between the ligand and RNA; this process allows the pyrophosphate to hydrogen-bond within the binding pocket. Like the TPP riboswitch, the SAM riboswitch integrates the polar functionalities of the ligand into an extensive hydrogenbonding network (Figure 3, panel c). The methionine amino acid group of SAM stacks on top of its own adenine ring, and both the amino acid and the adenosyl moieties of SAM are recognized by three or four hydroVOL.1 NO.6 • 341–345 • 2006
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Figure 3. Close-up views of ligand-binding pockets reveal extensive hydrogen-bonding and electrostatic interactions. a) and b) The TPP riboswitch forms two binding pockets for the pyrimidine ring and pyrophosphate groups. The pyrimidine ring forms three hydrogen bonds with two nucleotides in both TPP riboswitch structures. a) The E. coli pyrophosphate-binding pocket contains two Mg2ⴙ ions (purple spheres) that coordinate (blue dashed lines) the RNA, the ligand, and the water molecules (blue spheres). Several water-mediated and direct ligand–RNA hydrogen bonds (black dashed lines) also form to facilitate pyrophosphate binding. b) The A. thaliana structure only contains one Mg2ⴙ ion in the pyrophosphate-binding region. The hydrogen bonds occur directly between the ligand and the RNA. c) The adenosyl and amino acid moieties of SAM hydrogen-bond extensively with the RNA, securing the ligand in a compact conformation. Two carbonyl oxygens in the RNA mediate selectivity for SAM through favorable electrostatic interactions with the positively charged sulfonium ion (indicated by arrows).
gen bonds with adjacent residues within the nearby helix. As a result of these RNA–ligand interactions, the molecule is held in a compact conformation in which its positively charged sulfonium ion is juxtaposed with car344
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bonyl oxygens in the minor groove of helix P1 (Figure 3, panel c). This arrangement of the ligand creates a unique electrostatic basis for recognition of SAM over the analogous molecule S-adenosylhomocysteine, which lacks the positively charged sulfonium ion (18). The TPP and SAM riboswitches reveal a variety of strategies with which RNA can selectively bind small molecules. However, information about features that are not recognized by riboswitches may also be informative. For example, the TPP riboswitch does not recognize the TPP thiazole ring through any specific contacts; this may explain why pyrithiamine pyrophosphate (PTPP) is an effective antimicrobial compound (5, 6, 29). In PTPP, a pyridine ring replaces the thiazole ring, but the compound binds the riboswitch with similar affinity to TPP and subsequently can turn off gene expression in a vital metabolic pathway. This example elegantly demonstrates how understanding the structures of bacterial-riboswitch-binding pockets can contribute significantly to our search for novel antimicrobial agents. Given the ubiquity of riboswitches in bacteria, it is somewhat surprising how few have been found in eukaryotes. The one
exception is the TPP riboswitch, which has been identified in fungi and plants and appears to retain many of the characteristics of the prokaryotic riboswitch (25). The structures of the E. coli and A. thaliana riboswitches from the Patel and Ban groups confirm this conservation, revealing very few differences between the prokaryotic and eukaryotic RNAs (Figure 4). The divergences occur mainly in regions that do not contact the ligand (Figure 4, panel a). Therefore, ligand binding and specificity are achieved nearly identically in the two structures (Figure 4, panels b and c). These observations support the hypothesis that the TPP riboswitch is a relic of an ancient “RNA world”, in which RNA controlled all the processes of life. The known riboswitches appear to have survived because of the economy that they provide the cell. Given that this efficiency is inherent in a selfregulating mRNA, it is tempting to speculate that modern riboswitches may have evolved in eukaryotes that are not present in prokaryotes. The multitude of additional mRNA processing steps unique to eukaryotes affords a variety of targets for RNA-based genetic control. Further research into potential eukaryotic riboswitches will help to shed
Figure 4. A comparison of prokaryotic and eukaryotic riboswitch structures. a) Overall folding of the E. coli (orange) and A. thaliana (purple) riboswitches. Regions with minimal divergence are highlighted in gray. b) A close-up of the TPP pyrimidine-ring-binding pocket reveals a high degree of similarity between the two structures. c) A close-up of the pyrophosphate-binding pocket. The RNA does not diverge significantly, although the positioning of the pyrophosphate moiety does. The Mg2ⴙ ion observed in the A. thaliana structure occupies the same space as one of the Mg2ⴙ ions in the E. coli structure. SASHITAL AND BUTCHER
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Point of
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