More than 40 years of extensive research has revealed
that RNAs participate in a range of cellular func-
tions. Some RNAs, despite being composed of only
four chemically similar nucleotides, can fold into
distinct three-dimensional architectures. In many
cases, these constitute simple scaffolds that provide
binding sites for proteins that function together with
the RNAs. However, the discovery of the first catalytic
RNAs — later named RNA enzymes, or ribozymes
— in the 1980s demonstrated that RNAs can also have
important functional roles in their own right1,2. The list
of naturally occuring ribozymes is short, and additions
over the years have been rare, with each new discovery
eliciting considerable excitement in the field.
Studies of molecular evolution suggest that RNA
molecules significantly influenced the development
of modern organisms by mediating the genetic flow
from DNA to proteins, as well as through their own
contribution to catalytic functions. The ‘RNA world’
hypothesis implies that RNA molecules appeared
before DNA and proteins3. Nevertheless, even with a
head start, RNA catalysts do not prevail in the modern
world. Moreover, most ubiquitous ribozymes require
proteins for efficient catalysis in vivo, and most true
protein-devoid catalytic RNAs have been found in only
a few viral-like sources, suggesting that the contribu-
tion of protein-free RNAs to the functions of modern
cells has been limited.
The discovery of riboswitches4–6 and other RNA-based
sensors reignited interest in the roles of protein-devoid
RNA-based elements. These RNA sensors can be
broadly defined as mRNA regions that are capable of
modulating gene expression in response to internal
and external inputs, without the initial participation
of proteins. Unlike ribozymes, many of these sensors
direct gene expression purely through changes in
RNA conformation. For instance, riboswitches alter
their conformations in response to small metabolites.
However, the identification of the bacterial ribozyme
glmS, which specifically binds a metabolite and cleaves
the mRNA encoding the protein that controls the
metabolism of that metabolite, has established a closer
link between riboswitches and ribozymes7. Recent
findings of novel riboswitches, ribozymes and other
RNA-based regulatory elements further highlight the
essential contribution of such RNAs in gene expression
In this Review, we mainly focus on naturally
occurring RNAs that have distinct three-dimensional
structures and that can function without the help of
proteins. We first present an overview of ribozymes,
riboswitches and other related RNA sensors, and high-
light their impact on gene regulation. We then analyse
their structure–function relationships, dissecting the
features that are essential for gene expression control
and other cellular processes. Finally, we compare the
functions of protein-free RNAs with those of proteins
and RNA–protein complexes, providing a perspec-
tive on the evolution of the role of RNAs in the gene
regulatory processes of modern organisms.
Structural Biology Program,
Cancer Center, New York,
New York 10021, USA.
Correspondence to A.S. or D.J.P.
11 September 2007
Ribozymes, riboswitches and
beyond: regulation of gene
expression without proteins
Alexander Serganov and Dinshaw J. Patel
Abstract | Although various functions of RNA are carried out in conjunction with proteins,
some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require
little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing
riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that
cells use to regulate gene expression in response to internal and external changes. Structural
studies have shown how these RNAs can carry out a range of functions. In addition, the
contribution of ribozymes and riboswitches to gene expression is being revealed as far
more widespread than was previously appreciated. These findings have implications for
understanding how cellular functions might have evolved from RNA-based origins.
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Subviral agents whose
multiplication in a host cell
depends on coinfection with a
Although naturally occurring ribozymes, excluding
the ribosome, all catalyse the same reaction of RNA
strand scission and ligation, they can be divided into
two groups according to their main function: cleav-
ing ribozymes, which include self-cleaving RNAs and
trans-cleaving ribonuclease P (RNase P); and splic-
ing ribozymes, which are large RNAs involved in the
excision of introns from precursor RNAs (pre-RNAs)
Cleaving ribozymes. Self-cleaving ribozymes range from
~40–200 nucleotides in length, and have various sec-
ondary structures and three-dimensional folds (FIG. 1).
For the purpose of this Review, this group can be ten-
tatively split according to their function in either RNA
replication or mRNA cleavage.
The ribozymes of the first subgroup, which include
the hammerhead8, hairpin9, hepatitis δ virus (HDv)10,11
and varkud satellite (vS)12 ribozymes (FIG. 1a–d), are
predominantly found in satellite RNas of plant origin.
Table 1 | Major classes and distribution of ribozymes and RNA switches
65 Viroids, plant viral satellite RNA, eukaryotes
(plants, crickets, amphibians, schistosomes)
Plant viral satellite RNA Hairpin75
VS155 Satellite RNA of Neurospora spp.
Human satellite virus
190 mRNAs Transcription
CPEB3 70 Mammals
glmS GlcN6P170 Gram+ bacteria
140–500 Prokaryotes, eukaryotes
Group I Self-splicingGuanosine 200–1500Organelles (fungi, plants, protists), bacteria,
bacteriophages, mitochondria (animals)
Organelles (fungi, plants, protists), bacteria,
Phages, bacteria, eukaryotes
Mostly Gram+ bacteria
Bacteria, archaea, eukaryotes (fungi, plants)
Mostly Gram+ bacteria
α- and β-proteobacteria
γ-proteobacteria, Thermotogales, Firmicutes
AdoCbl, adenosylcobalamin; CoTC, co-transcriptional cleavage; FMN, flavin mononucleotide; GlcN6P, glucosamine-6-phosphate; Gram–, Gram-negative;
Gram+, Gram-positive; HDV, hepatitus δ virus; Hfq, host factor Q, interacting with some sRNAs; preQ1, pre-quenosine-1; SAM, S-adenosylmethionine; sRNA,
small RNA; TPP, thiamine pyrophosphate; VS,Varkud satellite. ? indicates an unconfirmed function.
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Nature Reviews | Genetics
Stem C Stem D
h RNase P
j Group I intron
i Group II intron
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a process of replication of
some circular genomes,
whereby one strand is
replicated first and the second
strand is replicated after
completion of the first one.
These RNAs are the product of a rolling-circle replication
mechanism involving ribozyme processing of long
multimeric RNAs into short monomers that, following
cyclization, participate in the next round of replication.
The second subgroup includes the recently discovered,
more diverse ribozymes that reside within eukaryotic
pre-mRNAs (the CPEB3 (ReF. 13) and co-transcriptional
cleavage (coTc)14 ribozymes) and a bacterial mRNA (the
glmS ribozyme)7. The CPEB3 ribozyme (FIG. 1e) lies within
the second intron of the mammalian CPEB3 gene, which
encodes cytoplasmic polyadenylation element binding
protein 3 (ReF. 13). The secondary structure and cleavage-
site organization of this catalytic RNA closely resemble
those of the HDv ribozyme (FIG. 1c). Although the exact
function of the CPEB3 ribozyme is unclear, it might func-
tion in the regulation of cPeb3 biosynthesis by interfering
with mRNA splicing and facilitating mRNA degradation
and/or the production of truncated protein forms.
The coTc element (FIG. 1f) has been identified down-
stream of the protein-coding sequences and poly(A) sites
of primate β-globin genes, suggesting that coTc has a
role in transcription termination and RNA processing14.
Indeed, coTc integrity was found to be crucial for effi-
cient termination of transcription of these genes by RNA
polymerase II15, and coTc RNA can be targeted by sev-
eral proteins that have been implicated in RNA processing
and degradation (A. Akoulitchev, personal communica-
tion). Auto-catalytic cleavage by the coTc ribozyme
requires the presence of GTP or its derivatives. because
the rate of coTc self-cleavage is low, the biological
significance of this ribozyme remains to be validated.
The glmS ribozyme (FIG. 1g), which was identified in
the 5′ region of the bacterial glmS gene, also requires
a specific cofactor for self-cleavage. This gene encodes
an amidotransferase7, which generates glucosamine-6-
phosphate (GlcN6P), a molecule that is used in cell-wall
biosynthesis. When sufficient GlcN6P is present, the
ribozyme uses GlcN6P as a coenzyme in the self-cleaving
reaction that is thought to cause degradation of the glmS
mRNA, thereby turning off the gene.
Almost all self-cleaving ribozymes break the RNA
backbone through the same reversible phosphodiester-
cleavage reaction (FIG. 2a). Nevertheless, they have
different catalytic pockets and use different cleavage
mechanisms, which are still being debated16. The coTc
ribozyme catalyses a different reaction14, producing
3′-hydroxyl (3′-oH) and 5′-phosphate (5′-P) ends, and
apparently carries out autolytic cleavage in the same way
as RNase P.
RNase P is the only naturally occurring ribozyme
identified so far that performs a multiple-turnover RNA
cleavage reaction in trans, involving multiple substrate
molecules2. This ubiquitous ribozyme removes extra
sequences from the 5′ ends of pre-tRNAs and some
other RNAs, by a different mechanism from the reaction
catalysed by self-cleaving ribozymes (FIG. 2b). RNase P
contains distinct protein subunits that are essential for
its in vivo function, but protein-free RNase P from both
bacteria2 and eukaryotes17 exhibits detectable activity
in vitro. Despite differences in sequence, length and
secondary structure, RNAse P RNAs contain five uni-
versally conserved regions that constitute the structural
core of the RNA18. These regions include the P4 helix, the
P10–P12 and the P2–P15.2 regions (FIG. 1h).
RNase P is structurally and evolutionarily related
to RNase MRP, which is found only in eukaryotes. both
RNases contain similar RNA components and share
several proteins19. Nevertheless, each RNase has its own
substrate preferences. In contrast to RNase P, RNase MRP
is implicated in 5.8S pre-ribosomal RNA (pre-rRNA)
processing and in RNA cleavage during mitochondrial
DNA synthesis, but not in pre-tRNA processing.
Splicing ribozymes. Typical nuclear mRNA splicing
requires a complex machinery that involves the assem-
bly of RNA–protein complexes. Splicing ribozymes
can perform the precise excision of an intron and the
covalent linkage of the boundary exons, with or without
assistance from specific protein factor(s). This group
encompasses two classes of heterogeneous self-splicing
introns (groups I and II), which can be found in many
tRNA, mRNA and rRNA precursors (Table 1).
Most large self-splicing introns fold into two types of
multidomain secondary structure that define their divi-
sion into groups I and II20,21 (FIG. 1i,j). In each group, helical
elements that form the catalytic core of the molecule
are relatively conserved, whereas the peripheral regions
vary, allowing classification into several subgroups.
Smaller introns that have a group II-like domain vI and
a streamlined domain I, but lack domains II–v, estab-
lish a separate family21. Self-splicing occurs in vitro for
most group I and for several group II introns; however,
Figure 1 | Domain organization and secondary and three-dimensional structures
of ribozymes. Secondary structures are depicted in thick lines and are connected by
thin black lines with arrows. Watson–Crick and non-canonical base pairs are shown
as solid lines and circles, respectively. Bulged-out nucleotides are represented as
triangles. Ribozymes with known three-dimensional structures are coloured according
to secondary structure elements and domains. Three-dimensional structures, if
available, are shown in a ribbon-and-stick representation below the secondary
structures. The two nucleotides that lie adjacent to the scissile phosphate are
indicated by red boxes. Nucleotides that are implicated in catalysis (panels a–f and i)
or that are essential for molecular recognition (panel j) are indicated by yellow boxes.
Black dashed squares and lines highlight important tertiary interactions. Coloured
dashed lines indicate elements that are missing in the structure or that are substituted
by non-natural sequences. a | Hammerhead ribozyme76. b | Hairpin ribozyme78.
c | Hepatitis δ virus ribozyme74. d | Varkud satellite (VS) ribozyme12. e | CPEB3
ribozyme13. f | Human CoTC ribozyme14. g | Bacillus antracis glmS ribozyme;
glucosamine-6-phosphate (GlcN6P) is represented as a red oval, with its interactions
with RNA indicated by dashed lines69. h | Bacillus subtilis RNase P, B-type73. i | Domain
organization of the group II intron, with IBS and EBS designating intron and exon
binding sequences, respectively21,22. The yellow-coloured A designates a conserved
unpaired adenosine that participates in splicing. j | Asoarcus spp. BH72 group I intron
in the state that precedes the second step of splicing79. The internal guide sequence
(IGS) aligns the 5′ and 3′ exons (ex), which are shown in grey. ωG and αG designate the
3′-terminal guanosine nucleotide of the intron and the external guanosine that is
linked to the intron after the first step of splicing, respectively. Secondary structures in
panels a, c, d, e, f, g, h and j are modified with permission from ReF. 76 (2006) Cell
Press, ReF. 117 (2007) Cell Press, ReF. 118 (1995) National Academy of Sciences
(USA), ReF. 13 (2006) American Association for the Advancement of Science, Nature
ReF. 14 (2004) Macmillan Publishers Ltd, ReF. 75 (2006) American Association for
the Advancement of Science and ReF. 69 (2007) Current Biology Ltd, ReF. 119
(2006) Elsevier Sciences and ReF. 120 (2005) Elsevier Sciences, respectively.
NATURe RevIeWS | GeNetiCS
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2′,3′ cyclic P
Exon Exon+ Intron
+ ‘Capped’ exon
c Group I introns
a Self-cleaving ribozymes
b RNase P
d Group I-like introns (GIR)
e Group II introns ‘branching’ reaction
f Group II introns ‘hydrolytic’ reaction
a protein machinery is required for efficient splicing
in vivo22. Introns can also encode RNa maturases, homing
endonucleases and reverse transcriptases (with only group
II introns), which help introns to splice and to spread
In contrast to the cleaving ribozymes, the splicing
ribozymes perform two consecutive reactions: cleav-
age and ligation of RNA. Sequence specificity and the
locations of splice sites are defined by the interactions
between the 5′ region of the intron (the internal guide
sequence, or IGS) and two exons (domains P1 and P10)
in group I introns (FIG. 1j), and by two or three pairs of
interactions between intron binding sites (IbSs) and exon
binding sites (ebSs) in group II introns22 (FIG. 1i). The first
step of self-splicing requires an exogenous nucleophile,
and is therefore similar to the RNase P-mediated reac-
tion. However, instead of water, group I and II introns
use external guanosine (αG) and internal adenosine as
nucleophiles, respectively (FIG. 2c,e). Growing evidence
suggests that some group II introns that lack the crucial
adenosine undergo an alternative hydrolytic pathway23
(FIG. 2f). Remarkably, the active sites of group II introns
can accommodate a diversity of nucleophiles (2′-oH
group, 3′-oH group and water) and substrates (DNA
and RNA) and, in addition to splicing, can catalyse
several other reactions, which are either adaptations of
the basic splicing activity or distinct reactions such as
terminal transferase activity24. Somewhat unexpectedly,
the group I-like ribozyme GIR1 from the slime mould
Didymium iridis forms a 2′,5′-lariat, similar to that of the
group II introns25 (FIG. 2d). This lariat structure appar-
ently serves as a cap, which protects the intron-encoded
endonuclease mRNA against degradation.
The term riboswitch is usually applied to metabolite-
sensing RNA switches, which share important
characteristics with other RNA sensors that are respon-
sive to cations, temperature and regulatory RNA mol-
ecules (Table 1). Indeed, all these mRNA-based control
systems, identified in many prokaryotes and some
eukaryotes, can sense various stimuli and transduce
them to the gene expression apparatus, with proteins
being recruited only at a later stage. by contrast, in
classical transcriptional attenuation control, ribosomes
or specialized proteins are directly involved in the ini-
tial step of regulation by helping to sense metabolite
Temperature sensing. RNA thermosensors26,27 are the
simplest RNA switches, yet they control adaptation to
an important physical parameter that affects multiple
cellular processes. because RNA secondary structure
is highly influenced by temperature, a thermosensor
can be as primitive as a stem–loop RNA structure that
bears a ribosome binding site (RbS) and an initiation
codon28 (FIG. 3a). At low temperatures, this RNA-based
thermometer adapts a conformation that masks the
RbS and prevents ribosome binding. At elevated tem-
peratures, secondary structure elements melt locally,
thereby allowing ribosome binding to the exposed RbS
Figure 2 | Reactions catalysed by ribozymes. a | The typical reaction of self-cleaving
ribozymes, initiated by 2′-hydroxyl (OH) attack, and yielding 2′,3′-cyclic phosphate (P)
and 5′-OH termini. b | The catalytic cleavage of pre-tRNA by RNase P (ReF. 2). A water
molecule serves as a nucleophile, and the reaction yields 2′,3′-diol and 5′-P termini.
c | Self-splicing by group I introns1. The reaction is initiated by nucleophilic attack by
the 3′-OH of external guanosine (αG) at the 5′ splice site. This results in covalent
linkage of αG to the 5′ end of the intron and release of the 3′-OH of the 5′ exon.
In the second step, the 3′-OH attacks the 3′ splice site located immediately after the
conserved guanosine (ωG), resulting in excision of the intron with αG at the 5′ end
and release of the ligated exons. d | The ‘capping’ reaction of the Didium iridis GIR1
ribozyme is similar to the first step of the ‘branching’ reaction of group II introns25.
The reaction joins nucleotides by a 2′,5′-phosphodiester linkage, thereby forming a
3-nucleotide ‘lariat’ that might be a protective 5′ cap of the mRNA. e | The self-splicing
of group II introns by a branching reaction22. In the first step, the 5′ splice site is
attacked by the 2′-OH of a conserved unpaired adenosine located in domain VI,
resulting in formation of a 2′,5′-phosphodiester linkage. In the next step, the free 3′-OH
group of the 5′ exon attacks the 3′ splice site, liberating the circular intron lariat and
ligated exons. f | Alternative ‘hydrolytic’ self-splicing of group II introns22. The reaction
involves a water molecule as a nucleophile and produces a linear intron.
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Nature Reviews | Genetics
12 base pairs
f TPP riboswitch
5′ Splice15′ Splice2
d T-box RNA
e Adenine riboswitch
Figure 3 | Gene regulation by RNA switches. RNA regions that are
involved in gene expression switching are shown in the same colour.
a | Translation activation of virulence genes in the pathogen Listeria
monocytogenes28. An increase in temperature melts the secondary
structure around the ribosome binding site (RBS) and start codon,
allowing ribosome binding and translation initiation. b | Upregulation of
Escherichia coli σs-factor gene by the DsrA antisense short RNA (sRNA).
DsrA RNA121 pairs with the translational operator of the rpoS gene122
using two sequences (coloured blue and light blue) located within helices
1 and 2 (ReFS 33,34,121). This base pairing exposes translation initiation
signals for ribosome binding and increases mRNA stability101.
c | Downregulation of transcription regulator HNS by DsrA sRNA. DsrA
RNA, transcribed in response to low temperature, pairs with 5′ and 3′
regions of hns mRNA and causes faster turnover of the mRNA101, possibly
by RNase E degradation123. d | Transcription termination of the
Bacillus subtilis glycyl-tRNA synthetase gene by aminoacylated tRNAGly
(ReF. 124). Non-aminoacylated tRNAGly interacts with the T-box region of
mRNA using an anticodon and an acceptor helix125, and promotes the
formation of the anti-terminator stem–loop structure. The aminoacylated
tRNA cannot contact mRNA using the acceptor stem, thus allowing the
formation of the transcription terminator. e | Transcription activation of
the purine efflux pump by the adenine riboswitch45. In the absence of
adenine, transcription of B. subtilis ydhL mRNA is aborted as a result
of formation of a transcription terminator. Adenine binding stabilizes the
metabolite-sensing domain and prevents the formation of the terminator.
f | Thiamine pyrophosphate (TPP)-riboswitch-mediated alternative
splicing of mRNA in Neurospora crassa43. In the absence of TPP, the mRNA
adopts a structure that occludes the 5′ splice site by base pairing with the
P4–P5 region of the riboswitch. Pre-mRNA splicing from 5′ splice site 1
leads to production of a short mRNA and expression of the NMT1 gene.
TPP binding causes a structural change in the RNA, opening the 5′ splice
site 2 and occluding the branch site. Therefore, splicing is inhibited (not
shown) and, were it to proceed, would result in the formation of a long
mRNA. The alternatively spliced and non-spliced mRNAs both carry a
short ORF (µORF) that begins from initiation codons 1–2 and competes
with translation of the main ORF, thereby repressing NMT1 expression.
Key splicing determinants are activated (indicated by green arrows) and
inhibited (indicated by red lines) during different occupancy states of the
TPP-sensing domain. Panels a, b and c, d and f are modified with
permission from ReF. 28 (2002) Cell Press, ReF. 126 (2000) National
Academy of Sciences (USA), ReF. 124 (2005) Academic Press and Nature
ReF. 43 (2007) Macmillan Publishers Ltd.
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a protein enzyme, intron-
specific, that acts as a cofactor
to facilitate splicing.
recognize large asymmetrical
DNa sequences, which are not
stringently defined, and which
mobilize these DNa elements
by facilitating their integration
into new genomic sites.
because these sites lack introns
and inteins (protein introns),
this form of mobility
is termed ‘homing’. Homing
endonucleases are encoded
by intervening sequences
embedded in either introns
an RNa-dependent DNa
polymerase that transcribes
ssRNa into dsDNa.
a regulatory mechanism
whereby gene expression is
controlled through the
formation of alternative
structures in the mRNa
sequence that inhibit or
facilitate the progression of
Derived from the latin aptus,
meaning ‘to fit’, aptamers are
oligonucleotide or peptide
molecules that bind specific
target molecules. The term is
usually applied to nucleic acid
molecules that are created
following selection from a large
random sequence pool, as well
as to natural metabolite-
sensing domains in
riboswitches, which possess
similar recognition properties
to artificially generated
and translation of the mRNA. Intrinsic thermometers
are well documented in two instances: during pathogenic
invasion and during heat-shock responses. When the
pathogen Listeria monocytogenes enters an animal host,
it encounters a warmer environment, and virulence-
associated genes that are normally silent at low
temperatures become activated. This occurs because
of a thermosensor located within the 5′ untranslated
region (5′-UTR) of the prfA mRNA28 (FIG. 3a). A simple
RNA thermometer, the repression of heat-shock gene
expression (RoSe) element, which was identified in
the hspA 5′-UTR, also upregulates a heat-shock gene
at high temperatures in the bacterium Bradyrhizobium
japonicum29. More complex RNA structures, including
large trans-acting RNAs, can participate in temperature
sensing, typically during heat and cold shocks, in various
organisms including bacteriophages26, bacteria27,30 and
RNA controls RNA. A similar strategy of either seques-
tering or exposing an RbS is utilized in gene expression
control by some regulatory antisense short RNAs
(sRNAs) in bacteria. These riboregulators are transcribed
in response to various changes in the environment, and
can in turn regulate genes by base pairing with target
mRNAs in the regions overlapping or adjacent to the
RbS. The sRNA–mRNA interactions can occur within
a single region, as for the replication control of plasmid
R1 by copA RNA32 and the control of biosynthesis of the
RNA polymerase σs-factor by DsrA RNA33,34, or within
two regions that are distant or close to each other, as
for DsrA- and oxyS-mediated control of transcrip-
tion factor production33,35,36 (FIG. 3b-c). Note that many
sRNAs require pairing with the RNA chaperone protein
Hfq (host factor Q) for their activity37,38. In addition to
translation, RNAs can also direct transcription of genes
by targeting transcription termination signals. The most
spectacular example of such regulation involves tRNA.
Non-aminoacylated tRNATyr (ReF. 39), tRNAGly (ReF. 40)
and others41 can selectively activate transcription of the
cognate aminoacyl-tRNA synthetase gene via specific
interaction of the anticodon and acceptor stem with the
so-called T-box region located in the 5′-UTR (FIG. 3d).
This binding promotes formation of the anti-terminator
stem and disruption of the terminator hairpin,
thereby producing the ‘on’ state of the gene. However,
aminoacylated tRNA does not bind mRNA using
its aminoacylated acceptor stem; therefore, the termina-
tor stem forms, rather than the anti-terminator, causing
Regulation by metabolites. Metabolite-binding ribos-
witches, numerous examples of which are found typically
embedded in the 5′-UTRs of bacterial metabolic genes,
represent genetic elements that are reminiscent of T-boxes
(Table 1). Riboswitches typically consist of evolutionar-
ily conserved ~35–200-nucleotide sensing or aptamer
domains — which specifically bind metabolites when
concentrations exceed a threshold — and expression
platforms that form or carry gene regulatory signals42.
Riboswitches can adapt alternative metabolite-free
and metabolite-bound conformations, which control
gene expression as on–off switches. In bacteria, this is
achieved by the formation of structures that form and
disrupt either transcription terminators or hairpins that
carry translation initiation signals. In fungi, a thiamine
pyrophosphate (TPP)-specific riboswitch located within
an intron was found to be involved in the regulation of
splicing43,44. Regulation generally depends on alternative
base pairing of the small region involved in the forma-
tion of helix P1, which locks the sensing domain in the
metabolite-bound conformation. Although most ribos-
witches are integrated into negative-feedback regulation
loops, some activate gene expression; for example, an
adenine-specific riboswitch achieves this by disrupting
a transcriptional terminator45 (FIG. 3e).
To date, metabolite-sensing riboswitches were
found to direct expression of the bacterial genes that
are implicated in the biosynthesis and transport of vari-
ous metabolites, including co-enzymes4–6,46–52, amino
acids53,54 and nucleobases45,55,56 (FIG. 4). A sugar deriva-
tive is also sensed by the glmS ribozyme7. Discovery of
the magnesium sensor that controls the magnesium
transporter MgtA of Salmonella enterica57 suggests that
riboswitches are involved in cellular processes that until
now were not thought to be subject to riboregulation.
The ligands of several conserved RNA elements are
still to be identifed, and these RNA elements might
represent novel riboswitch classes46,58. Remarkably,
riboswitches can discriminate between highly similar
compounds, such as S-adenosylhomocysteine (SAH)
and S-adenosylmethionine (SAM), which differ by only
a single methyl group47,49,52 (FIG. 4c). Recent studies have
identified several metabolite-like antimicrobial com-
pounds that function by targeting riboswitches. Among
them are the TPP analogue pyrithiamine pyrophosphate
(PTPP), which differs from TPP by the central ring59
(FIG. 4b), two analogues of lysine, l-aminoethylcysteine
(Aec) and Dl-4-oxalysine, which contain carbon
substitutions in the lysine side chain60 (FIG. 4i), and the
FMN analogue roseoflavin61 (FIG. 4f). Interestingly, high
specificity for a particular ligand can be conferred by
different sequences, and some metabolites such as SAM
interact with several types of sensing domain46–49,52
(FIG. 4c-e), suggesting that riboswitches have taken on
similar functions by following different evolutionary
The chemical simplicity of RNA molecules raises the
question of whether diverse RNA-based genetic elements
utilize a limited number of architectural components
and folding rules. The recently solved three-dimensional
structures of several riboswitches62–67 and the majority of
ribozyme types68–80 have shed light on this question.
Architectures that have an impact on function. The
structures of ribozymes and riboswitches highlight
the concept of modularity, a principle that is also used
by many other RNAs to generate and maintain specific
architectures81. All RNAs, ranging from simple ther-
mometers (FIG. 3) to complex ribozymes (FIG. 1), can be
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viewed as hierarchical assemblies that are composed of
helices as the major building blocks. The helices associate
into bundles by end-to-end stacking and edge-to-edge
docking, and are connected together by junctional
regions and tertiary interactions. Many oligonucleotide
elements that are present in secondary structures as
non-paired sequences can form compact and often
helix-like topologies. A comparison of riboswitch and
small ribozyme structures reveals that the key elements
defining these architectures are junctions composed of
three (FIGS 1a;4a,b) or more (FIGS 1b;4c) helical segments
that converge together. In large ribozymes68,73,80, the TPP
riboswitch63,65,67 and the hairpin77 ribozyme, these junc-
tions comprise structural elements that are necessary for
the correct orientation of the helical domains, in some
cases demonstrating a remarkable convergence in terms
of global architecture82. This feature is also observed
for the SAM-I riboswitch64 and the P4–P6 and P3–P7
domains of group I introns68,71,72.
To reinforce junctional conformations, spatial con-
straints are provided by diverse and complex tertiary
interactions, which most often involve loop–loop62,66
(FIGS 1a,h;4a), loop–helix63,65,67,76 (FIGS 1g;4b) and helix–
helix interlocking connections between peripheral
regions. In the absence of crucial tertiary loop–loop
contacts, the hammerhead ribozyme shows a 100-fold
loss of activity83,84, and purine riboswitches do not
interact with their ligands62. Ribozymes, riboswitches
and other RNAs share several conserved three-
dimensional nucleotide combinations called structural
motifs. These include the T-loop, a 5-bp hairpin that
is closed by a special non-canonical pair found in the
TPP riboswitch63,65,67 and RNase P85, and the kink-turn,
a sharp bend in the backbone of the RNA strand found
in the SAM-I riboswitch64 (FIG. 4c) and the Tetrahymena
thermophila group I intron72.
Folding of ribozymes and riboswitches. Not surprisingly,
splicing reactions and larger substrates require longer
ribozymes with intricate three-dimensional structures,
whereas smaller self-cleaving ribozymes and ribos-
witches adopt simpler folds. It is therefore notable that
ongoing in vitro studies indicate that large ribozymes
self-assemble into almost-active conformations in the
presence of magnesium86, and subsequently undergo
smaller substrate-induced conformational changes.
by contrast, riboswitches require their ligands for effi-
cient folding, thus demonstrating a typical induced-fit
binding mechanism. The name ‘riboswitch’ implies a
switch between two states in response to the presence
of metabolites; however, more than half of the putative
riboswitches that have been identified are predicted
to function through transcriptional attenuation, when
RNA polymerase must make a choice between mRNA
elongation and transcription termination soon after the
sensor domain of the riboswitch has been synthesized.
After that event, the energy barrier for re-folding of
the domain would be too great to overcome, suggest-
ing that, instead of switching between conformations, a
riboswitch makes a ‘once-in-a-lifetime’ choice between
‘on’ and ‘off’ conformations87.
because of the high speed of RNA polymerase, some
riboswitches such as the FMN riboswitch88 might not
have sufficient time for metabolite–RNA interactions to
reach thermodynamic equilibrium before the regulatory
decision is made; therefore, they might require higher
concentrations of metabolites (relative to the apparent
dissociation constants) to affect transcription4,50. other
riboswitches might need to attain thermodynamic
equilibrium with ligands to make a choice between
continuation or termination of transcription. However,
the adenine-sensing riboswitch utilizes kinetic or equi-
librium control depending on the speed of transcription
and external variables such as temperature89. Interestingly,
co-transcriptional folding seems to dictate the formation
of the tuning-fork-like architecture that is observed for
several riboswitches62,64–67. Such a mechanism, in which
two helical segments trap the metabolite between them,
thereby stabilizing the transient helix P1 and preventing
it from adopting an alternative base-pairing alignment,
might be superior to single-site recognition, especially for
bulky and extended metabolites.
An interesting situation is seen in the context of
phage and bacterial mRNAs that contain self-splicing
introns. In bacteria, transcription and translation are
coupled, and translating ribosomes can prevent the for-
mation of an elaborate intron structure that requires the
splice sites to be in close proximity. Therefore, efficient
splicing requires that RNA folding is coordinated with
transcription and translation90.
Catalysis and binding: similar trends? A direct compari-
son between molecular recognition mechanisms used by
ribozymes and riboswitches is difficult to undertake owing
to the fact that ribozymes target a specific sugar-phosphate
backbone position whereas riboswitches recognize vari-
ous small molecules. An analysis of available structures
also indicates that ribozymes and riboswitches exhibit
great structural diversity despite superficial similarities in
their global architectures. For example, a comparison of
RNAs with similar scaffolds, such as the three-way helical
junctions that are shared by the hammerhead ribozyme,
purine and TPP riboswitches (FIGS 1a;4a,b), shows that
they are used for conceptually different goals (bOX 1). In
the hammerhead ribozyme76, an open catalytic pocket
formed by a three-way junction functions to precisely
position a scissile phosphate. by contrast, the three-way
junction of the purine riboswitches62,66 consists of several
stacked layers of base triples, and nucleobase ligands such
as adenine, guanine or the guanine analogue hypoxan-
thine are sandwiched between layers so that ~98% of
the surface area is shielded from solvent62. In the TPP
riboswitch63,65,67, the junction adopts a simpler fold and
the ligand is recognized outside of the junctional region
by two helical segments (FIG. 4b). The same principle of
bridging two helical segments by the ligand is exploited by
the SAM-I riboswitch64 (FIG. 4c). Despite different ligand-
binding properties, the binding event in purine, TPP and
SAM-I riboswitches leads to the same result: stabilization
of the overall junctional region, which in turn contrib-
utes to the stabilization of the transient helix P1, which is
involved in the gene expression switch.
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Figure 4 | Secondary and tertiary structures of riboswitches. Secondary
structures are depicted by thick lines and are connected by black lines with arrows.
Watson–Crick and non-canonical base pairs are shown as solid lines and circles,
respectively. Natural riboswitch ligands and riboswitch-binding antibiotics are
shown next to the secondary structures. Grey shadings indicate areas in which the
ligands undergo changes. a | The Bacillus subtilis xpt gene guanine riboswitch bound
to guanine (represented as a red G)66. The discriminatory nucleotide C74 is coloured
yellow. b | The TPP riboswitch from the Escherichia coli thiM gene bound with TPP (in
red)65. A pair of hydrated Mg2+ cations is shown in magenta. G40 interacting with the
pyrimidine moiety of TPP is shown in yellow. The antibiotic pyrithiamine
pyrophosphate (PTPP) differs from TPP by the central ring. c | The class I SAM
Thermoanaerobacter tengcongensis riboswitch in complex with SAM (in red)64. U57
interacting with the purine moiety of SAM is shown in yellow. The grey area
highlights a methyl group that is missing in S-adenosylhomocysteine (SAH).
d | A class II SAM riboswitch from the Agrobacterium tumefaciens metA gene46.
e | An SMK (class III SAM) riboswitch from the Streptococcus gordonii metK gene48.
Helix P3 is formed by Shine–Dalgarno and anti-Shine–Dalgarno sequences. f | The
FMN riboswitch from the B. subtilis ribD gene50. g | The preQ1 riboswitch from the
B. subtilis queC gene56. h | The magnesium riboswitch from the Salmonella enterica
mgtA gene57. i | The lysine riboswitch from the B. subtilis lysC gene54. j | The AdoCbl
riboswitch from the E. coli btuB gene5. k | The glycine type II riboswitch from Vibrio
cholerae gcvT gene53. Secondary structures in panels c, d, e, f, g, h, i, j and k modified
with permission from Nature ReF. 64 (2006) Macmillan Publishers Ltd, ReF. 46
(2005) BioMed Central Ltd, Nature Structural & Molecular Biology ReF. 48 (2006)
Macmillan Publishers Ltd, ReF. 50 (2002) National Academy of Sciences (USA),
Nature Structural & Molecular Biology ReF. 56 (2007) Macmillan Publishers Ltd,
ReF. 57 (2006) Cell Press, Nature Biotechnology ReF. 61 (2006) Macmillan
Publishers Ltd, ReF. 5 (2002) Current Biology Ltd and ReF. 53 (2004) American
Association for the Advancement of Science, respectively.
Interesting parallels are observed when riboswitch
structures are compared with hairpin ribozyme and
group I intron structures. Formation of the catalytic
pocket in the hairpin ribozyme77 via docking of two
helical domains might be considered to be reminiscent
of TPP and SAM-I riboswitches (FIG. 1b). In group I
introns, the guanosine-binding pocket, formed by
nucleotides at the junction of J6–J7 and P7 and occu-
pied by ωG (the 3′ splice site)68,71,72, is surrounded
by several stacked base triples (FIG. 1j), reminiscent
of the purine-binding site in purine riboswitches.
Unlike other ribozymes and riboswitches, the glmS
ribozyme contains pre-formed catalytic and ligand-
binding sites and, in contrast to riboswitches, binds
its ligand GlcN6P in an open, solvent-accessible
pocket69,75 (bOX 1; FIG. 1g).
Regulation of gene expression
Given that RNase P has a universal catalytic function
in both prokaryotes and eukaryotes, there has been a
long-standing appreciation of the important role that
ribozymes have in cells. The discovery of riboswitches
and novel ribozymes, however, points to a different
trend for protein-devoid RNAs; namely, a direct
involvement in a range of gene-control mechanisms
in both prokaryotes and eukaryotes.
The role of ribozymes in gene expression. Self-splicing
introns continually modulate the genomic organiza-
tion of various organisms by mediating the mobility of
genetic material within the genome and by spreading
between species. How important are these ribozymes
for the regulation of gene expression? Self-splicing
introns can interrupt genes, but the ability of introns
to self-excise from mRNA potentially renders them
neutral to the host91. Nevertheless, in the roaA gene of
the Euglena gracilis chloroplast, self-splicing introns
cause alternative splicing of pre-mRNAs to produce
two distinct transcripts92, highlighting one way in
which these RNAs can affect gene expression. Splicing
might also be an important mechanism for post-
transcriptional regulation. For example, the addition
of a 3′ ccA sequence, which is required for amino-
acylation, to a plastid tRNA is less efficient when
intron removal is blocked93. Importantly, in addition
to homing, which occurs when introns spread into
similar genes, self-splicing introns can also invade
ectopic or non-homologous sites22, and these new
integration sites might not necessarily be neutral for
other self-cleaving ribozymes, such as the CPEB3
and coTc ribozymes, which seem to be involved in
splicing13 and transcription14, respectively, might have
a direct impact on gene expression. These ribozymes
have been identified in only some mammalian species,
and further study is needed to determine whether
similar RNA elements exist, providing a more gen-
erally applicable mechanism for their function. by
contrast, small self-cleaving ribozymes (hammerhead,
hairpin, vS and HDv ribozymes) have specialized
functions in replication, and are therefore unlikely to
influence expression of the host genes specifically and
A few more catalytically active self-cleaving
sequences have been found in humans13 and plants94.
Homology searches in various genomes have also iden-
tified several hammerhead-like motifs, the activities
of which have not been demonstrated95,96. Potentially,
cleavages produced by the catalytically active elements
might provide specific entry sites for distinct exo-
nucleases, with direct implications for transcriptional
termination, intron degradation and mRNA turnover.
Future research should reveal whether these ribozymes
are an integral part of gene expression mechanisms
or are involved in specific mechanisms of gene
expression regulation. So far, a regulatory function
has been clearly shown for only the glmS ribozyme,
which cleaves off the 5′ mRNA region and, probably
through mRNA degradation, downregulates expres-
sion of the amidotransferase gene. Unexpectedly, the
pre-rRNA-processing enzyme RNase MRP also par-
ticipates in mRNA degradation by targeting specific
mRNAs that are involved in cell-cycle regulation97,
suggesting that RNase MRP might be involved in gene
The complexity of riboswitch-dependent regulation
in bacteria. The number of genes controlled by
metabolite-sensing riboswitches (~2% in some
bacteria)55 is comparable with the number of genes
regulated by metabolite-sensing proteins. Indeed, a
bacterial genome can contain riboswitches that are
specific to different metabolites, as well as multiple
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Box 1 | The architecture of binding and catalytic pockets
The functioning of ribozymes and riboswitches depends on the precise formation of
catalytic domains and binding pockets, respectively. How do these RNA molecules achieve an
exceptionally high specificity for recognition and catalysis despite a limited arsenal of functional
groups, and are there common principles that underlie ligand–substrate recognition?
In the hammerhead ribozyme76, the scissile phosphate (indicated by a yellow sphere in panel A)
is recognized in an ‘open’ pocket. Nucleotides C17 and C1.1, which are neighbours to the scissile
phosphate, are splayed apart. C1.1 forms hydrogen bonds (indicated by black dashed lines)
with G2.1, whereas C17 is intercalated into the core of the junction. The nucleophilic
2′-hydroxyl (OH) of C17 is positioned in line with the scissile phosphate and the 5′-oxygen of the
leaving group (indicated by a blue dashed line coming from the phosphate group). The conserved
G8 and G12 are located nearby to facilitate acid–base catalysis. Red dashed lines indicate
hydrogen bonds that are potentially active for the catalysis.
Purine riboswitches contain a ‘closed’ ligand-binding pocket
that is located between several layers of triples that constitute
the three-way junction62,66. The bound guanine (G) and adenine (A)
are surrounded by uridines along their periphery66 ( shown in panels
Ba and Bb, respectively). Specific recognition of the ligands is
achieved by Watson–Crick pairing with a discriminatory nucleotide
at position 74 (either cytosine or uridine in guanine and adenine
Similarly to purine riboswitches, the guanosine-binding pocket
of group I introns68,71,72 is built by several stacked triples, and the
3′ splice site (ωG) is bound by extensive stacking and the hydrogen
bond interactions of its nucleobase. However, ωG is not fully
enveloped and is likely to be inserted or removed without a pronounced conformational change in the
pocket. ωG interacts with a guanosine residue that is engaged in a G130–C177 base pair, forming a
nucleotide triple (shown in panel C). Specific recognition of guanosine also has an essential role in the
selection of the 5′ splice site by the ribozyme68. In this case, a conserved wobble pair is formed between the
terminal nucleotide of the 5′ exon, U-1 and a G within the intron’s internal guide sequence (IGS) (FIG. 1j).
Such pairing exposes the exocyclic amine and 2′-OHs of the G for readout by a wobble receptor element,
containing two non-canonical A•A pairs, in J5–4.
In the thiamine pyrophosphate (TPP) riboswitch63,65,67 (shown in panel D), the ligand is bound in an extended
conformation by two helical segments that lie outside of the junctional region65 . The pyrimidine-like ring pairs
with G40 (coloured yellow) and is stacked between G42 and A43, which constitute part of a T-loop motif
(coloured orange). Two Mg2+ ions (the coordinated water molecules are shown in magenta) are bound to the
pyrophosphate moiety, which interacts with the RNA through direct contact of phosphate oxygens with
the base edges of C77 and G78. In addition, Mg12+ makes direct contacts with G60
and G78, and also makes water-mediated contacts with several other nucleotides.
The ligand also bridges two helical segments in the SAM-I riboswitch (shown in
panel e)64. However, unlike TPP, the bound S-adenosylmethionine (SAM) adopts a
compact conformation. The purine moiety of SAM specifically binds U57 and A45
(coloured yellow), and is sandwiched between a methionine moiety and C47. Main-
chain atoms of methionine interact with the (G58–C44)•G11 base triple. SAM forms
van der Waals interactions with U7 and U88, positioning P1 close to P3. These
multiple interactions zip up helices P1 and P3, and stabilize the P1 helix and the
overall four-way junctional fold, with the former constituting the integral part of
the gene expression switch.
The preformed open glucosamine-6-phosphate (GlcN6P)-binding pocket of the
glmS ribozyme (shown in panel F)69,75 consists of nucleotides from the P2 loop (FIG. 1g)
that form a double pseudoknot conformation, which is observed in the structures
of the hepatitis δ virus (HDV)70 and Diels–Alder ribozymes116. Like the HDV ribozyme,
the double pseudoknot in the glmS ribozyme
forms an active-site cleft, with the scissile
phosphate located at the bottom. GlcN6P
is locked in place by stacking with the G+1
nucleotide and multiple direct and
magnesium-mediated interactions involving
ring and phosphate moieties. The reaction
mechanism might include deprotonation of
the 2′-OH of A-1 by guanine (not shown),
and protonation of the 5′-O leaving group
by GlcN6P, consistent with the observation
that GlcN6P is essential for catalysis. Note that
Mg2+ does not directly participate in catalysis.
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Boolean NOR logic gate
boolean logic, named after
George boole, is a complete
system for logical operations.
a logic gate performs a logical
operation on one or more
logical inputs and produces
a single logical output. The
logical NOR, or joint denial, is
a logical operator, meaning
that the output is true if none
of the inputs are true.
consequently, if one or both
inputs are true, then the
output is not true.
riboswitches of the same type, each regulating a dif-
ferent operon. Moreover, two sensing domains or
even two complete switches can lie adjacent to each
other, resulting in a more complex mechanism of
gene expression regulation53,98. For example, tandem
glycine-specific aptamers bind glycine cooperatively
and accomplish regulation through a single expres-
sion platform, thus providing a greater dynamic
response to small changes in glycine concentration53.
Another adjoining arrangement constitutes two com-
plete riboswitches that independently sense different
metabolites, such as SAM and adenosylcobalamin
(Adocbl), and function as a two-input boolean NOR
logic gate, wherein binding of either ligand causes
repression98. Tandem riboswitches can also be com-
posed of two complete riboswitches of the same type.
Such composite arrangements, exhibited by some TPP
riboswitches98,99, candidate riboswitches98 and T-box
RNAs98,100, probably enable a greater dynamic range
for gene control and greater responsiveness to changes
in metabolite concentration98.
Remarkably, depending on the expression platform,
riboswitches of the same type are capable of either
repression or activation of gene expression, greatly
increasing their regulatory potential. A similar trend
is seen in regulation by bacterial sRNAs, although
this involves an increased level of sophistication. The
DsrA sRNA can pair with two different mRNAs, hns
and rpoS, down- and upregulating their translation,
respectively33,34,101 (FIG. 3b,c). To add further complex-
ity, rpoS mRNA can, in turn, be repressed by another
sRNA, oxyS, possibly by titration of Hfq37.
Riboswitches as mediators of eukaryotic gene expression.
To date, virtually all riboswitch-like riboregulators
have been found in bacterial species. because of
differences in transcription, translation and mRNA
structure, eukaryotes rely on post-transcriptional
gene-control mechanisms that differ to some extent
from their prokaryotic counterparts. Nevertheless,
functional TPP riboswitches have been identified in
the 5′-UTR introns of fungal genes44 and the 3′-UTRs
of plant genes involved in thiamine biosynthesis102,
suggesting a regulatory role in splicing and mRNA
processing, respectively. Indeed, a recent study has
shown that fungal TPP riboswitches can either down-
regulate or upregulate gene expression using an elegant
alternative splicing mechanism43 (FIG. 3f). computer
searches have also identified TPP riboswitch-
like motifs in two other eukaryotic species and in
archaea, and SAM-II, preQ1 and Adocbl riboswitch-
like sequences have been found in eukaryotes103,
although some of them such as preQ1 and Adocbl
cannot be functional riboswitches. Furthermore, a
novel candidate riboswitch that senses arginine has
been recently suggested to control the arginase gene
in fungi104. These examples highlight the plasticity of
riboswitch-mediated genetic control and point out the
need for experimental studies that might reveal
the distinct features of eukaryotic versus prokaryotic
RNAs versus proteins
As they are composed of many more variable building
blocks, proteins are generally considered to be more
versatile than RNAs, so why did nature also implement
RNA-based catalysts and regulatory elements? In fact,
the versatility of RNA might have been underestimated.
Studies of ribozymes and RNA sensors have shown
that, despite having a much simpler composition, RNA
molecules have a number of features that are typical
of protein molecules. like their protein counterparts,
ribozymes and especially riboswitches interact with
small-molecule cofactors, and some of these regula-
tory and catalytic RNAs show allosterically controlled
activity. Similarly to proteins, RNAs demonstrate high
affinity and specificity towards their ligands and can
recognize a wide range of molecules, from simple gly-
cine to bulky Adocbl. Also in common with proteins,
some RNA regulators, such as tandem riboswitches,
have a modular organization that seems to be crucial
for cooperative and other types of complex control.
In addition to their similarities with proteins, RNAs
might have functional advantages over proteins in some
situations. Direct sensing of metabolites and other mol-
ecules by mRNA provides a significant advantage for
cells, which can save energy and resources by eliminat-
ing the need for special regulatory proteins. Another
important feature of RNA regulators that are positioned
in cis is a synchronized response to changing surround-
ings. In contrast to the long-lived trans-acting protein
factors, regulatory elements that are embedded within
an mRNA exclusively control that mRNA, providing
precise regulation of gene expression.
both RNA and protein enzymes use binding interac-
tions and energy to facilitate catalysis105. Remarkably,
despite a limited arsenal of functional groups and a sim-
ple composition, some ribozymes turn out to be efficient
catalysts. The cleavage-rate constants for the HDv106
and hammerhead ribozymes (~1 sec–1 and 15 sec–1,
respectively)107 approach the corresponding values
for the protein enzyme ribonuclease A (160 sec–1 and
69 sec–1 for UpG and cpc substrates, respectively)108.
Nevertheless, many ribozymes have much slower rate
constants (~1 min–1)109, with these values being sufficient
for typical ribozyme reactions involving single cleavage
and ligation in cis. The cleavage and ligation reactions
can be carried out by specific host endoribonucleases
and ligases. However, virus-like RNAs, the sources of
many ribozymes, could improve their chances for sur-
vival by encoding cleavage and ligation activity within
their sequences, thereby eliminating a dependence on
specialized host enzymes.
Finally, cells undoubtedly benefit from RNA-dependent
gene expression control under stress conditions.
Relatively small sRNAs can efficiently and precisely
regulate protein synthesis by base pairing with target
mRNAs, thereby preventing a waste of resources for
Despite the benefits described above, some features
of RNA-based regulatory systems can be limiting.
For instance, because RNA typically degrades more
quickly than proteins in vivo, RNA regulators that work
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a non-protein component
of a conjugated protein that
is usually essential for the
protein’s function. Prosthetic
groups are also called
coenzymes and cofactors.
a process of transferring
genetic material from one
organism to another without
reproduction. also called
horizontal gene transfer.
an RNa-containing reverse
transcriptase that adds DNa
sequence repeats (telomeric
repeats) to the 3′ end of
DNa strands in eukaryotic
chromosomes using its RNa
component as a synthesis
in trans, such as sRNAs, are probably not well suited
to functioning under conditions in which control is
required over an extended period of time. current
research suggests that many RNA catalysts and regula-
tors might be considered as relics of a pre-protein era,
either perfectly adapted to particular functions or still
subject to evolutionary pressure for replacement by pro-
teins. Indeed, functions that are carried out by certain
ribozymes and riboswitches have been taken over by
protein-based systems in other species110.
Descendants of the RNA world?
If RNAs constituted the predominant species in the early
biological world as suggested by the RNA world hypoth-
esis, are modern RNA modules direct descendants of
those primordial molecules? Given the key participa-
tion of RNA in fundamental cellular processes such as
protein synthesis, it is tempting to consider a positive
answer, especially as it is assumed that proteins, at first,
largely assisted functional RNAs. Therefore, the exist-
ing ribonucleoprotein enzymes, including the ribosome,
RNase P and the spliceosome, could be considered as
remnants of the RNA world, although their origins
cannot be unambiguously traced owing to the many
missing links in their evolutionary history. A similar
problem is encountered in the evolutionary analysis of
modern small ribozymes. The complexity and narrow
distribution of vS, HDv and hairpin ribozymes argues
against their descent from the RNA world. However,
the distribution of the hammerhead ribozyme in highly
divergent organisms, suggesting an ancient origin,
might be misleading: this ribozyme might instead have
arisen independently multiple times111. The evolution-
ary relationships between similar ribozymes in different
species might be even more intricate than initially
thought, given the similarity between the HDv and
CPEB3 ribozymes, which, along with the limited distri-
bution of the HDv ribozyme, suggests a human origin
for this viral ribozyme13.
Knowledge of the origin and evolution of self-
splicing introns, although uncertain, is important for
understanding the origins of spliceosomal introns112.
Indeed, terminal structures of group II self-splicing
introns, which contain the parts that carry out the
splicing reaction, show a remarkable similarity to
the structures of the spliceosomal small nuclear RNAs
(snRNAs)22. This prompted speculation that self-splicing
introns are ancestors of spliceosomal introns. Growing
evidence suggests that group II introns, which are present
in many bacteria, are among the most ancient genetic
entities, and probably moved into the evolving eukary-
otic genome from the α-proteobacterial progenitor
of mitochondria, later giving rise to the spliceosomal
introns and splicing machinery112.
Several aspects of riboswitches are suggestive of their
RNA world origin. Riboswitch scaffolds have probably
stayed unaltered over long periods of time, because
riboswitches recognize coenzymes and nucleobases that
have remained unchanged for billions of years113. These
nucleotide-like molecules could initially have been teth-
ered to RNAs and used as prosthetic groups for catalytic
reactions. With the emergence of proteins and their
take-over of catalytic functions, the ancient cofactor-
dependent ribozymes could have lost their catalytic
ability and instead evolved to create RNA switches42. In
addition, the presence of some riboswitches in diverse
organisms supports their ancient origin, although even
this criterion is not absolute because of possible lateral
Perspectives and future challenges
Modern genomic and biochemical techniques provide
unique opportunities to identify novel roles for RNAs
that function independently of proteins, as shown by the
recent discoveries of riboswitches and several ribozymes
that are encoded by cellular genomes. Some of these
studies, such as the discovery of riboswitches4–6, have
taken advantage of well-established biological observa-
tions, suggesting the existence of metabolite-dependent
gene expression control. other examples, such as the
metabolite-dependent glmS and CPEB3 ribozymes, were
discovered serendipitously during the characterization
of a potential riboswitch7 or by careful labour-intensive
experimentation involving in vitro selection to identify
self-cleaving RNAs13. Many of these exciting findings,
such as the identification of glmS7, coTc13 and CPEB314
RNAs, have highlighted our limited understanding of
key biological processes including the termination
of mRNA transcription, and mRNA processing and
A priority for future studies is to characterize the
self-cleavage activities of recently identified genome-
encoded ribozymes13,94, as well as candidate ribozyme
and RNA sensor sequences46,58,95,96,103, that might reveal
new aspects of genetic regulation by RNA elements. As
a pivotal example, we cite the recent and long-awaited
dissection of the functional role of eukaryotic ribos-
witches, which participate in gene expression control
by alternative splicing of their pre-mRNAs43. This
outstanding work will encourage experimentation on
archaeal and other eukaryotic riboswitches, especially
those that are found in the 3′ region of mRNAs and
are potentially involved in novel types of regulation.
These biochemical and genetic studies should be sup-
plemented by searches for novel RNA sensors and
ribozymes using new computer-based approaches
that can identify candidate RNA sequences with low
Despite considerable progress in studies of
ribozymes and riboswitches at the atomic level, future
research must resolve many uncertainties in functional
mechanisms, especially for some classical (vS ribozyme,
group II introns and RNase P) and recently identified
(coTc and CPEB3) ribozymes, for which structures
are either not yet available or are restricted to a subset
of functional states. This need also applies to many
riboswitches for which three-dimensional structures
await determination. comprehensive research in this
area would enhance our mechanistic understanding
of the biological function of these molecules and more
complex RNA-containing cellular machineries such as
the spliceosome, the ribosome and telomerase.
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This work was supported by the US National Institutes of
Health grant GM073618.
Competing interests statement
The authors declare no competing financial interests.
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
β-globin | CPEB3
Protein Data Bank Identification (PDB ID): http://www.rcsb.
Asoarcus spp. BH72 group I intron | Bacillus antracis glmS
ribozyme | Bacillus subtilis RNase P, B-type | Bacillus subtilis xpt
gene | Escherichia coli thiM | Hairpin ribozyme | Hammerhead
ribozyme | Hepatitis δ virus ribozyme | Thermoanaerobacter
Dinshaw J. Patel’s homepage:
All liNkS ARe ACtive iN the oNliNe pDF.
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