Riboswitch

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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.

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:

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

  1. Tucker BJ, Breaker RR (2005). "Riboswitches as versatile gene control elements". Curr Opin Struct Biol. 15 (3): 342–8. doi:10.1016/j.sbi.2005.05.003. PMID 15919195.
  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. 4.0 4.1 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.
  13. Barrick JE, Corbino KA, Winkler WC, Nahvi A, Mandal M, Collins J, Lee M, Roth A, Sudarsan N, Jona I, Wickiser JK, Breaker RR (2004). "New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control". Proc Natl Acad Sci USA. 101 (17): 6421–6. doi:10.1073/pnas.0308014101. PMID 15096624.
  14. Corbino KA, Barrick JE, Lim J, Welz R, Tucker BJ, Puskarz I, Mandal M, Rudnick ND, Breaker RR (2005). "Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria". Genome Biol. 6 (8): R70. doi:10.1186/gb-2005-6-8-r70. PMID 16086852.
  15. Weinberg Z, Barrick JE, Yao Z, Roth A, Kim JN, Gore J, Wang JX, Lee ER, Block KF, Sudarsan N, Neph S, Tompa M, Ruzzo WL, Breaker RR (2007). "Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline". Nucleic Acids Res. doi:doi:10.1093/nar/gkm487 Check |doi= value (help). PMID 17621584.
  16. Blount KF, Breaker RR (2006). "Riboswitches as antibacterial drug targets". Nat Biotechnol. 24 (12): 1558–64. doi:10.1038/nbt1268. PMID 17160062.

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