Edwards et al., Curr Opin Struct Biol 2007

Riboswitches: small-molecule recognition by gene
regulatory RNAs
Thomas E Edwards, Daniel J Klein and Adrian R Ferré-D’Amaré
Riboswitches demonstrate the ability of highly structured RNA
molecules to recognize small-molecule metabolites with high
specificity and subsequently harness the binding energy for the
control of gene expression. Crystal structures have now been
determined for the metabolite-binding domains of riboswitches
that respond to purines, thiamine pyrophosphate and
S-adenosylmethionine, as well as for the glmS ribozyme,
a catalytic riboswitch that is activated by the metabolite
glucosamine-6-phosphate. In addition to these riboswitch
structures, a solution NMR structure has been reported for a
ribosensor that regulates heat shock genes in response to
changes in temperature. These studies reveal the structural
basis of the remarkable selectivity of riboswitches and, in
conjunction with biochemical and biophysical measurements,
provide a framework for detailed mechanistic understanding of
riboswitch-mediated modulation of gene expression.
Addresses
Division of Basic Sciences, Fred Hutchinson Cancer Research
Center, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA
Corresponding author: Ferré-D’Amaré, Adrian R (aferre@fhcrc.org)
Current Opinion in Structural Biology 2007, 17:273–279
This review comes from a themed issue on
Nucleic acids
Edited by Dinshaw J Patel and Eric Westhof
Available online 15th June 2007
0959-440X/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.sbi.2007.05.004
Introduction
Riboswitches are cis-acting RNA elements that control
gene expression by directly sensing the levels of specific
small-molecule metabolites [1–4]. Here, we review advances
in the structural characterization of these remarkable
RNAs during the preceding two years.
Formally, riboswitches consist of two domains [5]. A
metabolite-binding or ‘aptamer’ domain recognizes the
cognate small molecule. Binding is signaled to an effector
domain or ‘expression platform’ that interfaces with the
transcriptional, translational or post-transcriptional RNA
modification machinery to modulate gene expression. This
formal distinction is less clear-cut at the molecular level.
Most known riboswitches are part of untranslated regions
(UTRs) of mRNAs that appear to function by fluctuating
between different conformations. The metabolite-bound
conformation of the riboswitch sequesters an adjacent
UTR segment into a tightly folded RNA domain. In the
metabolite-free conformation, the same segment participates
in the control of translation (e.g. the segment interacts
with or contains a Shine–Dalgarno sequence) or
transcription (e.g. the segment forms a transcriptional
terminator or anti-terminator stem). Thus, the aptamer
and expression platform are not ‘domains’ that can co-exist,
but different structural states of an RNA segment. The
distinction also breaks down for the glmS ribozyme-riboswitch,
for which metabolite binding activates a latent
catalytic activity.
Overview of riboswitch structures
Crystal structures have been reported for metabolitebound
domains of the 5 0 -UTRs of bacterial mRNAs containing
the guanine [6,7] and the adenine [7] variants of the
purine riboswitch. The RNA folds into three helices or
paired regions, P1, P2 and P3, which are arranged as an
inverted ‘h’ (Figure 1a,b). P1 stacks coaxially under P3, and
P2 and P3 pack side-by-side. This overall arrangement,
stabilized by the interaction of the terminal loops of P2 and
P3, is reminiscent of the structure of the hammerhead
ribozyme ([8]; see the review by Scott in this issue). The
purine ligand is buried in a solvent-inaccessible pocket at
the three-way junction of P1, P2 and P3. The 3 0 strand of P1
(red in Figure 1a,b) forms part of an alternative secondary
structure within the expression platform of the metabolitefree
conformational state of the 5 0 -UTR. Direct interactions
between the 3 0 strand of the P1 ‘switch helix’
and both the purine and the J2/3 interhelical junction
explain the ligand dependence of P1 formation.
The structures of the metabolite-binding domain of the
thiamine pyrophosphate (TPP) riboswitch from the eukaryote
Arabidopsis thaliana [9 ] and the bacterium Escherichia
coli [10 ,11 ] are essentially identical. Like the purine
riboswitch, the metabolite-bound TPP riboswitch adopts a
compact, inverted-h architecture with two parallel sets of
coaxially stacked helices (P1-P2-P3 and P4-P5) joined by a
three-way junction (Figure 1c,d). An A-minor interaction
between L5 and P3, functionally analogous to the loop–
loop interaction of the purine riboswitch, stabilizes this
arrangement. Unlike the purine riboswitch, TPP is not
buried at the three-way helical junction; instead, TPP
bridges the two parallel helical stacks. This places TPP
25 Å away from the P1 switch helix, suggesting that
metabolite binding only indirectly stabilizes P1.
Three distinct classes of S-adenosylmethionine (SAM)
riboswitches have been identified [12–14]. The crystal
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274 Nucleic acids
Figure 1
Crystal structures of riboswitch–effector complexes, and schematic depiction of conserved primary and secondary structures. (a,b) Purine
riboswitch (PDB code 1U8D), (c,d) TPP riboswitch (PDB code 2GDI), (e,f) SAM riboswitch (PDB code 2GIS) and (g,h) glmS ribozyme-riboswitch
(PDB code 2H0Z). The 3 0 half of the P1 helices, which participate in the genetic switch event, is colored red, and tertiary interactions thought
to be important in folding and RNA–metabolite complex stability are green. Bound metabolites are shown in blue; PK and KT denote
pseudoknots and K-turns, respectively; ‘var’ indicates an RNA segment of phylogenetically variable length and composition. Filled spheres
indicate that the sequence is not conserved, but the Watson–Crick pairing is.
Current Opinion in Structural Biology 2007, 17:273–279
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Metabolite recognition by riboswitches Edwards, Klein and Ferré-D’Amaré 275
structure of the metabolite-binding domain of the first
class to be discovered (the SAM-I riboswitch) reveals
an architecture that is distinctly different from the
inverted-h fold of the purine and TPP riboswitches
(Figure 1e,f) [15 ]. The SAM-I riboswitch contains two
stacks (P1-P4 and P2a-P3) that, rather than packing sideby-side,
cross at an angle of 708. A pseudoknot coupled
to a kink-turn [16] atop P2 appears to stabilize this
fold. The SAM-binding site is located at the interface
of the minor grooves of P1 and P3, and has features
reminiscent of the metabolite-binding sites of both the
TPP and the purine riboswitches. Like TPP, SAM
bridges two helical stacks. Like the purine riboswitches,
SAM makes van der Waals contact with the 3 0 strand of
the P1 switch helix.
In many Gram-positive bacteria, the glmS ribozyme-riboswitch
is part of the 5 0 -UTR of the mRNA that encodes
glucosamine-6-phosphate (GlcN6P) synthetase [17]. This
riboswitch has a self-cleavage activity that becomes activated
when it binds GlcN6P. The structure of the glmS
ribozyme [18 ,19 ] consists of three parallel helical stacks
(Figure 1g,h). A doubly pseudoknotted core (P1-P2-P2.1-
P2.2) is buttressed by a peripheral RNA domain (P4-P4.1).
The solvent-exposed GlcN6P-binding pocket is composed
of two highly distorted major grooves and abuts the site of
self-cleavage, reflecting the coenzyme function of GlcN6P
(see the review by Scott in this issue). Among currently
characterized riboswitches, the glmS ribozyme is unique
because it adopts its active structure in the absence of its
metabolite ligand [18 ,20 ]. As other ribozymes, such as
the natural hairpin ribozyme [21,22] and the in vitro
selected Diels–Alderase [23], also assemble rigid active
sites, this disparity might reflect the different constraints
under which ribozymes and RNAs that function by alternative
folding evolved.
Ligand recognition by riboswitches
The purine riboswitch recognizes its ligand almost exclusively
through hydrogen-bonding interactions that satisfy
nearly all possible acceptors and donors of the purine
(Figure 2a). Purine riboswitch structures have been
solved bound to 2,6-diaminopurine [24 ] and 2,4,6-triaminopyrimidine
[25], in addition to the biological activators
hypoxanthine [6], guanine [7] and adenine [7]. The
purine ligand is primarily recognized by residue 74 of the
riboswitch, a pyrimidine, through Watson–Crick pairing
[6,7,26]. In addition, U51 and the 2 0 -OH of U22 hydrogen
bond to the N3/N9 edge (corresponding to the sugar edge
of nucleotides) and the N7, respectively, of the purine.
Reliance on Watson–Crick (as opposed to Hoogsteen)
pairing for recognition enables the same RNA scaffold to
regulate either adenine or guanine metabolism by having
U74 or C74, respectively. Gilbert et al.[24 ] noted that the
purine ligand makes poor stacking interactions and proposed
that this enhances the discriminatory role of Watson–Crick
pairing with residue 74.
The two helical stacks of the thi-box riboswitch separately
recognize the aminopyrimidine and pyrophosphate moieties
of TPP. J3/2 of the ‘pyrimidine sensor helix’ (the P1-
P2-P3 stack) adopts a canonical T-loop fold [9 ,10 ,11 ].
Binding of the aminopyrimidine ring of TPP to G40 of this
T-loop (Figure 2b) mimics a tertiary interaction between
the D- and T-loops in the classic L-shaped fold of tRNA.
The aminopyrimidine of TPP and G40 replace G18 and
C55, respectively (purines and pyrimidines are reversed
between the riboswitch and the tRNA). Mimicry of an all-
RNA structure by an exogenous small molecule is reminiscent
of ATP binding by an in vitro selected aptamer
RNA, whereby the ATP completes a GNRA tetraloop
[27,28]. Rather than directly binding to the negatively
charged pyrophosphate of TPP, the ‘pyrophosphate sensor
helix’ (the P4-P5 stack) of the riboswitch coordinates the
pyrophosphate mostly through two solvated divalent
metals ions (the exception is G78) [9 ,10 ,11 ,29]. Thus,
the riboswitch effectively binds a positively charged TPP–
cation complex. Structures of the TPP riboswitch bound to
three metabolite analogs suggest that the pyrimidine sensor
helix is largely preformed, whereas the pyrophosphate
sensor helix becomes organized concomitant with binding
of the TPP–cation complex [11 ].
The SAM-I riboswitch sandwiches its ligand between two
parallel helices [15 ]. P1 recognizes the ribose–sulfur
backbone of SAM primarily through van der Waals contacts
(Figure 2c). By contrast, the P3 helix binds the
adenine ring and the amino acid by making several
hydrogen bonds and stacking interactions. Interestingly,
the RNA does not directly recognize the methionine
e-methyl group. Rather, the positively charged sulfur atom
makes a favorable electrostatic interaction with the partial
negative charge on O2 of U7. This might explain the
enhanced binding of SAM compared to S-adenosylhomocysteine
and other non-positively charged analogs [12,30].
Recognition of GlcN6P, a simple phosphorylated hexosamine
sugar, by the glmS ribozyme-riboswitch presents a
challenge for RNA that is distinct from those posed by
purines, TPP and SAM, all of which have nucleotide-like
substructures. Crystal structures of the glmS ribozymeriboswitch
were solved bound to glucose-6-phosphate
(Glc6P), an isosteric competitive inhibitor (antagonist)
of GlcN6P [18 ], and to the authentic activator GlcN6P
([19 ]; DJ Klein and AR Ferré-D’Amaré, unpublished),
revealing that GlcN6P and Glc6P are equivalently positioned
in the glmS ribozyme-riboswitch active site. The
sugar hydroxyl groups hydrogen bond to G1, C2, A50 and
G65 (Figure 2d). The amine of GlcN6P hydrogen bonds
to a water molecule, U51 and the 5 0 oxygen of G1; the last
is the leaving group of the transesterification reaction
catalyzed by the ribozyme. As in the TPP riboswitch,
the phosphate of the metabolite interacts with the RNA
through two solvated divalent metal ions ([19 ]; DJ Klein
and AR Ferré-D’Amaré, unpublished). TPP, SAM and
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276 Nucleic acids
Figure 2
GlcN6P all present negatively charged moieties to the
N1/N2 face of a guanine residue (G78, G11 and G1,
respectively). This face of guanine has previously been
shown to be an anion-binding site [31].
Aptamers and riboswitches
The discovery of RNA ‘aptamers’ that bind small
molecules through in vitro selection (reviewed in [32])
preceded the discovery of their naturally occurring counterparts,
riboswitches. We compared RNA and ligand solvent
accessibilities for several aptamer–ligand (reviewed in
[33]) and riboswitch–effector structures. Riboswitches
bury 90 6% (mean standard deviation) of the accessible
surface of their effectors. Aptamers, by contrast, bury
71 14% of the solvent-accessible surface of their cognate
ligand, consistent with an in vitro selection methodology in
which the ligand is tethered to beads. Furthermore, riboswitches
tend to have more compact structures than aptamers,
as indicated by solvent-accessible surface areas of
156 5Å 2 /nt for riboswitches and 171 8Å 2 /nt for
aptamers (end correction as in [34]). Despite these differences
and the different evolutionary origins of aptamers
and riboswitches, the binding energy correlates linearly
with the buried surface area of the ligand (Figure 3). Such a
relationship might be expected on theoretical grounds [35].
This relationship implies that the vitamin B 12 riboswitch
(K d 0.3 mM [36]) and the tetracycline aptamer
(K d 0.8 nM [37]) can achieve their known affinity by
burying 40% (of 1200 Å 2 ) and nearly all (600 Å 2 ),
respectively, of the surface area of their ligands. Exceptions
to the trend are RNAs that bind the smallest ligands, such
as the purine riboswitches and the theophylline aptamer,
which completely envelop the small molecule. For these
ligands, higher affinity must result from other factors, such
as RNA–RNA interactions. Our analysis implies that the
glycine riboswitch will also be an outlier, consistent with
the cooperative binding exhibited by this RNA [38].
Structural and biochemical analyses of several GTP aptamers
of varying degrees of complexity show that increased
RNA complexity correlates with higher affinity [39]. However,
higher affinity does not result in higher specificity
[40 ]. These studies suggest that higher affinity is derived
from additional RNA–RNA interactions that stabilize the
global RNA fold and that explicit selection is needed to
achieve higher specificity. Compared to aptamers, riboswitch
structures are stabilized by a variety of peripheral
RNA contacts (Figure 1) and these cellular regulators
presumably have undergone stringent natural selection,
thus accounting for their higher affinities and selectivity.
RNA–ligand interaction in the (a) purine, (b) thi-box, (c) SAM-I and
(d) glmS ribozyme riboswitches. Color scheme as in Figure 1, with the
addition of metal ions (green spheres) and water molecules (red
spheres). Dotted lines denote hydrogen bonds; solid lines denote
inner-sphere metal ion coordination. In (a), C74 is colored with
nitrogens in blue and oxygens in red to illustrate hydrogen bonding
with hypoxanthine (HX).
Current Opinion in Structural Biology 2007, 17:273–279
Riboswitch folding and regulation of gene
expression
Although chemical probing has been used to investigate
the small-molecule-dependent alternative folding of
some full-length riboswitches (reviewed in [1]), most
biochemical and biophysical studies of riboswitches have
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Metabolite recognition by riboswitches Edwards, Klein and Ferré-D’Amaré 277
Figure 3
thus far focused on isolated metabolite-binding domains.
Published studies include ensemble and single-molecule
fluorescence resonance energy transfer (FRET) characterization
of the adenine riboswitch (demonstrating the presence
of Mg 2+ -dependent folding intermediates [41 ]),
solution NMR experiments on the Mg 2+ and effector
dependence of the terminal loop–loop interaction in the
purine riboswitches [42 ], and small-angle X-ray scattering
analysis of the shape of the glycine riboswitch in the
unliganded, Mg 2+ -induced folded and effector-bound
states [43].
With the exception of the glmS ribozyme, riboswitchmodulated
gene regulation results from the sequestration
of a switch segment (red in Figure 1) into either a
metabolite-bound or a metabolite-free conformational
fold. For riboswitches that exert genetic control at the
transcriptional level, co-transcriptional mRNA folding is
an essential component of the mechanism of action [44]
and must be considered alongside the thermodynamics
and kinetics of ligand binding [24 ,45], as well as the
relative speed of transcription [46 ]. In a study of the
flavin mononucleotide (FMN) riboswitch, it was shown
that the speed of transcription relative to the kinetics of
FMN binding precludes this RNA from reaching thermodynamic
equilibrium before the point at which a genetic
decision must be made [46 ]. Consequently, metabolite
concentrations needed to trigger this riboswitch in vivo
must be considerably higher than the in vitro dissociation
constant measured with previously transcribed RNA.
These studies also implicate transcriptional pause sites
as critical components of riboswitch-mediated regulation.
Several ribosensors have been described that function by
means other than direct metabolite binding. These include
non-coding mRNA elements that respond to the presence
of uncharged tRNAs [47], the concentration of Mg 2+ [48]
and temperature [49]. A series of ‘RNA thermometers’ in
the 5 0 -UTRs of heat shock response genes unfold at
elevated temperatures, releasing a Shine–Dalgarno sequence
sequestered at lower temperatures (Figure 4a). A
solution NMR structure of the lower temperature state
has been determined, in which several residues form noncanonical
base pairs (Figure 4b) [49]. The imino proton
resonances of these residues disappear at elevated temperatures,
indicating that these pairs melt. The coupling of
folding with the control of gene expression is a feature
shared by these ribosensors and riboswitches.
Small-molecule binding by aptamers and riboswitches. (a) The
solvent-accessible surface area of the ligand that is buried when in
complex with aptamers or riboswitches correlates linearly with affinity,
with a binding energy of 19 cal/mol per Å 2 of buried area. Red and
blue represent riboswitches and aptamers, respectively; squares and
circles denote structures solved by X-ray crystallography or NMR,
respectively; 95% prediction intervals are shown as dashed lines.
The purine and theophylline complexes were excluded from the
analysis. (b) Solvent-accessible surfaces of riboswitch- or aptamerbound
ligands. Panels show the absolute solvent-accessible area of
each ligand atom (dark blue 0–1 Å 2 , light blue 1–10 Å 2 , green
10–20 Å 2 , yellow 20–30 Å 2 , orange 30–40 Å 2 , red 40–60 Å 2 ) mapped
onto the molecular surface overlaying it. Buried surface areas were
calculated using a 1.4 Å radius probe [50]. Aptamer structures are
reviewed in [32].
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278 Nucleic acids
Figure 4
Foundation. TEE and DJK contributed equally to the writing of this
review.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
of special interest
of outstanding interest
Secondary structure (a) and NMR structure (PDB code 2GIO) (b) of the
ROSE (repressor of heat-shock gene expression) ribosensor, which
functions as an RNA thermometer. Upon increase in temperature (D), a
helix primarily consisting of non-canonical Watson–Crick pairings melts
to liberate a Shine–Dalgarno sequence (depicted in gray boxes). Except
for line drawings, figures were prepared with PyMol [51].
Conclusions
The recent determination of the structures of the metabolite-binding
domains of riboswitches opens the way to
multiple research avenues that take advantage of highresolution
information. These include analysis of the
molecular recognition mechanisms employed by riboswitches
and design of artificial effectors, elucidation of
the mechanism of action of the coenzyme GlcN6P in the
glmS ribozyme and, most generally, coupling of ligandinduced
folding of RNA to the cellular processes of
transcription, post-transcriptional RNA processing and
translation.
Acknowledgements
TEE and DJK are Damon Runyon Fellows, and ARF is a Distinguished
Young Scholar in Medical Research of the WM Keck Foundation.
Supported by grants from the Damon Runyon Cancer Research
Foundation (DRG-1844-04 to TEE and DRG-1863-05 to DJK), the
National Institutes of Health (GM63576 to ARF) and the WM Keck
Current Opinion in Structural Biology 2007, 17:273–279
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