In
molecular biology, a riboswitch is a regulatory segment of a
messenger RNA molecule that binds a
small molecule, resulting in a change in
production of the
proteins encoded by the mRNA.[1][2][3][4] Thus, an mRNA that contains a riboswitch is directly involved in regulating its own activity, in response to the concentrations of its
effector molecule. The discovery that modern organisms use RNA to bind small molecules, and discriminate against closely related analogs, expanded the known natural capabilities of RNA beyond its ability to code for
proteins,
catalyze reactions, or to bind other RNA or protein
macromolecules.
The original definition of the term "riboswitch" specified that they directly sense small-molecule
metabolite concentrations.[5] Although this definition remains in common use, some biologists have used a broader definition that includes other
cis-regulatory RNAs. However, this article will discuss only metabolite-binding riboswitches.
Most known riboswitches occur in
bacteria, but functional riboswitches of one type (the
TPP riboswitch) have been discovered in archaea,
plants and certain
fungi. TPP riboswitches have also been predicted in
archaea,[6] but have not been experimentally tested.
History and discovery
Prior to the discovery of riboswitches, the mechanism by which some genes involved in multiple metabolic pathways were regulated remained mysterious. Accumulating evidence increasingly suggested the then-unprecedented idea that the mRNAs involved might bind metabolites directly, to affect their own regulation. These data included conserved RNA
secondary structures often found in the untranslated regions (
UTRs) of the relevant genes and the success of procedures to create artificial small molecule-binding RNAs called
aptamers.[7][8][9][10][11] In 2002, the first comprehensive proofs of multiple classes of riboswitches were published, including protein-free binding assays, and metabolite-binding riboswitches were established as a new mechanism of gene regulation.[5][12][13][14]
Many of the earliest riboswitches to be discovered corresponded to conserved sequence "motifs" (patterns) in
5' UTRs that appeared to correspond to a structured RNA. For example, comparative analysis of upstream regions of several genes expected to be co-regulated led to the description of the S-box[15] (now the SAM-I riboswitch), the THI-box[9] (a region within the TPP riboswitch), the RFN element[8] (now the FMN riboswitch) and the B12-box[16] (a part of the cobalamin riboswitch), and in some cases experimental demonstrations that they were involved in gene regulation via an unknown mechanism.
Bioinformatics has played a role in more recent discoveries, with increasing automation of the basic
comparative genomics strategy. Barrick et al. (2004)[17] used
BLAST to find UTRs
homologous to all UTRs in Bacillus subtilis. Some of these homologous sets were inspected for conserved structure, resulting in 10 RNA-like motifs. Three of these were later experimentally confirmed as the glmS, glycine and PreQ1-I riboswitches (see below). Subsequent comparative genomics efforts using additional taxa of bacteria and improved computer algorithms have identified further riboswitches that are experimentally confirmed, as well as conserved RNA structures that are hypothesized to function as riboswitches.[18][19][20]
Mechanisms
Riboswitches are often conceptually divided into two parts: an
aptamer and an expression platform. The aptamer directly binds the small molecule, and the expression platform undergoes structural changes in response to the changes in the aptamer. The expression platform is what regulates gene expression.
Expression platforms typically turn off gene expression in response to the small molecule, but some turn it on. The following riboswitch mechanisms have been experimentally demonstrated.
The riboswitch is a
ribozyme that cleaves itself in the presence of sufficient concentrations of its metabolite.
Riboswitch alternate structures affect the
splicing of the pre-mRNA.
A TPP riboswitch in Neurospora crassa (a fungus) controls alternative splicing to conditionally produce an
Upstream Open Reading Frame (uORF), thereby affecting the expression of downstream genes[21]
A TPP riboswitch in plants modifies splicing and alternative 3'-end processing[22][23]
A riboswitch in Clostridium acetobutylicum regulates an adjacent gene that is not part of the same mRNA transcript. In this regulation, the riboswitch interferes with transcription of the gene. The mechanism is uncertain but may be caused by clashes between two RNA polymerase units as they simultaneously transcribe the same DNA.[24]
A riboswitch in Listeria monocytogenes regulates the expression of its downstream gene. However, riboswitch transcripts subsequently modulate the expression of a gene located elsewhere in the genome.[25] This trans regulation occurs via base-pairing to the mRNA of the distal gene. As the temperature of the bacterium increases, the riboswitch melts, allowing transcription. Unpublished undergraduate research created a similar riboswitch or 'thermosensor' via random mutagenesis of the Listeria monocytogenes sequence.[26]
Types
The following is a list of experimentally validated riboswitches, organized by ligand.
cyclic di-GMP riboswitches bind the signaling molecule
cyclic di-GMP in order to regulate a variety of genes controlled by this second messenger. Two classes of cyclic di-GMP riboswitches are known:
cyclic di-GMP-I riboswitches and
cyclic di-GMP-II riboswitches. These classes do not appear to be structurally related.
Glutamine riboswitches bind
glutamine to regulate genes involved in glutamine and
nitrogen metabolism, as well as short peptides of unknown function. Two classes of glutamine riboswitches are known: the glnA RNA motif and Downstream-peptide motif. These classes are believed to be structurally related (see discussions in those articles).
Glycine riboswitch binds glycine to regulate glycine metabolism genes, including the use of glycine as an energy source. Previous to 2012, this riboswitch was thought to be the only that exhibits
cooperative binding, as it contains contiguous dual aptamers. Though no longer shown to be cooperative, the cause of dual aptamers still remains ambiguous.[27]
PreQ1 riboswitches bind pre-queuosine1, to regulate genes involved in the synthesis or transport of this precursor to
queuosine. Three entirely distinct classes of PreQ1 riboswitches are known:
PreQ1-I riboswitches,
PreQ1-II riboswitches and
PreQ1-III riboswitches. The binding domain of PreQ1-I riboswitches are unusually small among naturally occurring riboswitches. PreQ1-II riboswitches, which are only found in certain species in the genera Streptococcus and Lactococcus, have a completely different structure, and are larger, as are PreQ1-III riboswitches.
Purine riboswitches binds
purines to regulate purine metabolism and transport. Different forms of the purine riboswitch bind
guanine (a form originally known as the G-box) or
adenine. The specificity for either guanine or adenine depends completely upon Watson-Crick interactions with a single
pyrimidine in the riboswitch at position Y74. In the guanine riboswitch this residue is always a
cytosine (i.e. C74), in the adenine residue it is always a
uracil (i.e. U74). Homologous types of purine riboswitches bind
deoxyguanosine, but have more significant differences than a single nucleotide mutation.
SAM riboswitches bind
S-adenosyl methionine (SAM) to regulate
methionine and SAM biosynthesis and transport. Three distinct SAM riboswitches are known: SAM-I (originally called S-box), SAM-II and the SMK box riboswitch. SAM-I is widespread in bacteria, but SAM-II is found only in
Alpha-,
Beta-, and a few
Gammaproteobacteria. The SMK box riboswitch is found only in the order
Lactobacillales. These three varieties of riboswitch have no obvious similarities in terms of sequence or structure. A fourth variety,
SAM-IV riboswitches, appears to have a similar ligand-binding core to that of SAM-I riboswitches, but in the context of a distinct scaffold.
SAM-SAH riboswitches bind both SAM and SAH with similar affinities. Since they are always found in a position to regulate genes encoding
methionine adenosyltransferase, it was proposed that only their binding to SAM is physiologically relevant.
TPP riboswitches (also THI-box) binds
thiamin pyrophosphate (TPP) to regulate
thiamin biosynthesis and transport, as well as transport of similar metabolites. It is the only riboswitch found so far in eukaryotes.[28]
Moco RNA motif is presumed to bind
molybdenum cofactor, to regulate genes involved in biosynthesis and transport of this coenzyme, as well as enzymes that use it or its derivatives as a cofactor.
Gallery of secondary structure images
Cobalamin riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00174.
Cyclic di-GMP-I riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF01051.
Cyclic di-GMP-II riboswitch: Secondary structure for the riboswitch marked up by sequence conservation.
FMN riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00050.
GlmS ribozyme: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00234.
Glycine riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00504.
Lysine riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00168.
PreQ1 riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00522.
PreQ1-II riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF01054.
Purine riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00167.
SAM riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00162.
SAM-II riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00521.
SAM-III riboswitch (SMK): Secondary structure for the riboswitch marked up by sequence conservation.
SAM-IV riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00634.
SAM-V riboswitch: Secondary structure for the riboswitch marked up by sequence conservation.
SAM-SAH riboswitch: Secondary structure for the riboswitch marked up by sequence conservation.
SAH riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF01057.
TPP riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00059.
Tetrahydrofolate riboswitch: Secondary structure for the riboswitch marked up by sequence conservation.
YkoK leader: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF00380.
Moco riboswitch: Secondary structure for the riboswitch marked up by sequence conservation. Family
RF01055.
Candidate metabolite-binding riboswitches have been identified using bioinformatics, and have moderately complex
secondary structures and several highly conserved
nucleotide positions, as these features are typical of riboswitches that must specifically bind a small molecule. Riboswitch candidates are also consistently located in the 5' UTRs of protein-coding genes, and these genes are suggestive of metabolite binding, as these are also features of most known riboswitches. Hypothesized riboswitch candidates highly consistent with the preceding criteria are as follows:
crcB RNA Motif,
manA RNA motif,
pfl RNA motif,
ydaO/yuaA leader,
yjdF RNA motif,
ykkC-yxkD leader (and related ykkC-III RNA motif) and the
yybP-ykoY leader. The functions of these hypothetical riboswitches remain unknown.
Computational models
Riboswitches have been also investigated using in-silico approaches.[29][30][31] In particular, solutions for riboswitch prediction can be divided into two wide categories:
riboswitch gene finders, i.e. systems aimed at uncovering riboswitches by genomic inspections, mostly based on motif-searching mechanisms. This group contains Infernal, the founding component of the
Rfam database,[32] and more specific tools such as RibEx[33] or RiboSW.[34]
conformational switch predictors, i.e. methods based on a structural classification of alternative structures, such as paRNAss,[35] RNAshapes[36] and RNAbor.[37] Moreover, family-specific approaches for ON/OFF structure prediction have been proposed as well.[38]
The SwiSpot tool[39] somehow covers both the groups, as it uses conformational predictions to assess the presence of riboswitches.
The RNA world hypothesis
Riboswitches demonstrate that naturally occurring
RNA can bind small molecules specifically, a capability that many previously believed was the domain of
proteins or artificially constructed RNAs called
aptamers. The existence of riboswitches in all domains of life therefore adds some support to the
RNA world hypothesis, which holds that life originally existed using only RNA, and proteins came later; this hypothesis requires that all critical functions performed by proteins (including small molecule binding) could be performed by RNA. It has been suggested that some riboswitches might represent ancient regulatory systems, or even remnants of RNA-world
ribozymes whose bindings domains are conserved.[13][18][40]
As antibiotic targets
Riboswitches could be a target for novel
antibiotics. Indeed, some antibiotics whose mechanism of action was unknown for decades have been shown to operate by targeting riboswitches.[41] For example, when the antibiotic
pyrithiamine enters the cell, it is metabolized into pyrithiamine pyrophosphate. Pyrithiamine pyrophosphate has been shown to bind and activate the TPP riboswitch, causing the cell to cease the synthesis and import of TPP. Because pyrithiamine pyrophosphate does not substitute for TPP as a coenzyme, the cell dies.
Since riboswitches are an effective method of controlling gene expression in natural organisms, there has been interest in engineering artificial riboswitches[42][43][44]
for industrial and medical applications such as
gene therapy.[45][46]
See also
RNA thermometer - Another class of mRNA regulatory segments which change conformation in response to temperature fluctuations, thereby exposing or occluding the ribosome binding site.
References
^Nudler E, Mironov AS (January 2004). "The riboswitch control of bacterial metabolism". Trends in Biochemical Sciences. 29 (1): 11–17.
doi:
10.1016/j.tibs.2003.11.004.
PMID14729327.
^Tucker BJ, Breaker RR (June 2005). "Riboswitches as versatile gene control elements". Current Opinion in Structural Biology. 15 (3): 342–348.
doi:
10.1016/j.sbi.2005.05.003.
PMID15919195.
^Batey RT (June 2006). "Structures of regulatory elements in mRNAs". Current Opinion in Structural Biology. 16 (3): 299–306.
doi:
10.1016/j.sbi.2006.05.001.
PMID16707260.
Ferré-D'Amaré, Adrian R.; Winkler, Wade C. (2011). "Chapter 5. The Roles of Metal Ions in Regulation by Riboswitches". In Astrid Sigel, Helmut Sigel and Roland K. O. Sigel (ed.). Structural and catalytic roles of metal ions in RNA. Metal Ions in Life Sciences. Vol. 9. Cambridge, U.K.: RSC Publishing. pp. 141–173.
doi:
10.1039/9781849732512-00141.
ISBN978-1-84973-094-5.
PMC3454353.
PMID22010271.