Riboswitch

In molecular biology, a riboswitch is a part of an mRNA molecule that can directly bind a small target molecule, and whose binding of the target affects the gene's activity ^[1][2][3]. Thus, an mRNA that contains a riboswitch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule.

Although the metabolic pathways in which some riboswitches are involved have been studied for decades, the existence of riboswitches has only been relatively recently discovered, with the first experimental validations of riboswitches being published in 2002 ^[4][5][6][7]. This oversight may relate to an earlier assumption that genes are regulated by proteins, not by the mRNA transcript itself. Now that riboswitches are a known mechanism of genetic control, it is reasonable to speculate that more riboswitches will be found.

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Mechanics of riboswitches

Riboswitches are 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. Expression platforms include:

• The formation of rho-independent transcription termination hairpins
• Folding in such a way as to sequester the ribosome-binding site, thereby blocking translation
• Self-cleavage (i.e. the riboswitch contains a ribozyme that cleaves itself in the presence of sufficient concentrations of its metabolite)
• Folding in such a way as to affect the splicing of the pre-mRNA. A TPP riboswitch in Neurospora crassa (a fungus) controls alternative splicing to conditional produce a uORF, thereby affecting expressing of downstream genes^[8].

Prevalence of riboswitches

Most known riboswitches occur in eubacteria, but functional riboswitches of one type (the TPP riboswitch) have been discovered in eukaryotes, such as the fungus mentioned above. Sequences similar to known TPP riboswitches have also been found in archaea^[9], but have not been experimentally tested.

The following riboswitches are known:

• TPP riboswitch (also THI-box) binds thiamin pyrophosphate (TPP) to regulate thiamin biosynthesis and transport, as well as transport of similar metabolites
• FMN riboswitch (also RFN-element) binds flavin mononucleotide (FMN) to regulate riboflavin biosynthesis and transport.
• Cobalamin riboswitch (also B12-element), which binds adenosylcobalamin (the coenzyme form of vitamin B12) to regulate cobalamin biosynthesis and transport of cobalamin and similar metabolites, and other genes.
• 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 S_MK box riboswitch. SAM-I is widespread in bacteria, but SAM-II is found only in alpha-, beta- and a few gamma-proteobacteria. The S_MK box riboswitch is found only in the order Lactobacillales. These three varieties of riboswitch have no obvious similarities in terms of sequence or structure.
• Purine riboswitches binds purines to regulate purine metabolism and transport. Different forms of the purine riboswitch can bind either 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).
• Lysine riboswitch (also L-box) binds lysine to regulate lysine biosynthesis, catabolism and transport.
• glmS riboswitch, which is a ribozyme that cleaves itself when there is a sufficient concentration of glucosamine-6-phosphate.
• Glycine riboswitch binds glycine to regulate glycine metabolism genes, including the use of glycine as an energy source. As of 2007, this riboswitch is the only known natural RNA that exhibits cooperative binding, which is accomplished by two adjacent aptamer domains in the same mRNA.
• Magnesium riboswitch senses magnesium ions to regulate magnesium transport genes.
• PreQ1 riboswitch binds pre-queuosine₁, to regulate genes involved in the synthesis of this precursor to queuosine. The binding domain of this riboswitch is unusually small among naturally occurring riboswitches.

Riboswitches and 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 could be performed by RNA.

Identification of riboswitches

Before riboswitches were experimentally demonstrated, several groups had identified 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^[10] (now the SAM-I riboswitch), the THI-box ^[11] (now the TPP riboswitch) and the RFN element^[12] (now the FMN riboswitch), and in some cases experimental demonstrations that they were involved in gene regulation via an unknown mechanism. Some researchers, hypothesizing that riboswitches would exist, identified them in part by inspecting the scientific literature for pathways, such as cobalamin biosynthesis, whose regulation had long been studied without successful elucidation of a regulatory mechanism ^[4].

As noted in the introduction, in 2002, several reports demonstrated that identified motifs, or pathways with stubbornly unknown means of regulation, were controlled by riboswitches. Proof that an RNA element is a riboswitch most often includes in vitro evidence that the RNA can bind the putative small molecule ligand, and in vivo genetic evidence that the riboswitch controls gene expression in the cell.

In vitro binding assays include structural probing assays, most often in-line probing, size-exclusion assays (where the radiolabeled metabolite ligand is observed to not travel through a membrane when it binds to a much larger riboswitch RNA) and equilibrium dialysis (where radiolabeled ligand is observed to be more concentrated in an RNA-containing chamber, than in an RNA-free chamber connect by a membrane).

Bioinformatics has played a role in more recent discoveries, with increasing automation of the basic comparative genomics strategy. Barrick et al. (2004) ^[13] 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 riboswitches. Subsequent comparative genomics efforts using additional taxa of bacteria and improved computer algorithms have identified further riboswitches^[14][15]

Riboswitches 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 ^[16]. 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.

One potential advantage that riboswitches have as an antibiotic target is that many of the riboswitches have multiple instances per genome, where each instance controls an operon containing many genes, many of which are essential. Therefore, in order for bacteria to evolve resistance to the antibiotic by mutations in the riboswitch, all riboswitches must be mutated. However, other mechanisms for resistance may exist, and some — such as altering the specificity of an exporter to export the drug — may require fewer mutations.

References

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2. ^ Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS (2004). "Riboswitches: the oldest mechanism for the regulation of gene expression?". Trends Genet 20 (1): 44-50. doi:10.1016/j.tig.2003.11.008. PMID 14698618.
3. ^ Batey RT (2006). "Structures of regulatory elements in mRNAs". Curr Opin Struct Biol 16 (3): 299-306. doi:10.1016/j.sbi.2006.05.001. PMID 16707260.
4. ^ ^{a b} Nahvi A, Sudarsan N, Ebert MS, Zou X, Brown KL, Breaker RR (2002). "Genetic control by a metabolite binding mRNA". Chem Biol 9 (9): 1043. doi:10.1016/S1074-5521(02)00224-7. PMID 12323379.
5. ^ Mironov AS, Gusarov I, Rafikov R, Lopez LE, Shatalin K, Kreneva RA, Perumov DA, Nudler E (2002). "Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria". Cell 111 (5): 747-56. doi:10.1016/S0092-8674(02)01134-0. PMID 12464185.
6. ^ Winkler W, Nahvi A, Breaker RR (2002). "Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression". Nature 419 (6910): 890-1. doi:10.1038/nature01145. PMID 12410317.
7. ^ Winkler WC, Cohen-Chalamish S, Breaker RR (2002). "An mRNA structure that controls gene expression by binding FMN". Proc Natl Acad Sci U S A 99 (25): 15908-13. doi:10.1073/pnas.212628899. PMID 12456892.
8. ^ Cheah MT, Wachter A, Sudarsan N, Breaker RR (2007). "Control of alternative RNA splicing and gene expression by eukaryotic riboswitches". Nature 447 (7143): 497-500. doi:10.1038/nature05769. PMID 17468745.
9. ^ Sudarsan N, Barrick JE, Breaker RR. "Metabolite-binding RNA domains are present in the genes of eukaryotes". RNA 9 (6): 644-7. PMID 12756322.
10. ^ Grundy FJ, Henkin TM (1998). "The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria". Mol Microbiol 30 (4): 737-49. doi:10.1046/j.1365-2958.1998.01105.x. PMID 10094622.
11. ^ Miranda-Ríos J, Navarro M, Soberón M (2001). "A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria". Proc Natl Acad Sci U S A 98 (17): 9736-41. doi:10.1073/pnas.161168098. PMID 11470904.
12. ^ Gelfand MS, Mironov AA, Jomantas J, Kozlov YI, Perumov DA (1999). "A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes". Trends Genet 15 (11): 439-42. doi:10.1016/S0168-9525(99)01856-9. PMID 10529804.
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