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.cite journal | author = Tucker BJ, Breaker RR | title = Riboswitches as versatile gene control elements | journal = Curr Opin Struct Biol | volume = 15| issue = 3 | pages = 342–8 | year = 2005 | pmid = 15919195| doi = 10.1016/j.sbi.2005.05.003 ] cite journal | author = Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS | title = Riboswitches: the oldest mechanism for the regulation of gene expression? | journal = Trends Genet | volume = 20 | issue = 1 | pages = 44–50 | year = 2004 | pmid = 14698618 | doi = 10.1016/j.tig.2003.11.008 ] cite journal | author = Batey RT | title = Structures of regulatory elements in mRNAs | journal = Curr Opin Struct Biol | volume = 16 | issue = 3 | pages = 299–306 | year = 2006 | pmid = 16707260 | doi = 10.1016/j.sbi.2006.05.001] 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.cite journal | pmid=12323379 | author=Nahvi A, Sudarsan N, Ebert MS, Zou X, Brown KL, Breaker RR|title=Genetic control by a metabolite binding mRNA|journal=Chem Biol|volume=9|issue=9|year=2002|pages=1043|doi=10.1016/S1074-5521(02)00224-7] cite journal | pmid=12464185 | author=Mironov AS, Gusarov I, Rafikov R, Lopez LE, Shatalin K, Kreneva RA, Perumov DA, Nudler E | title = Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria | journal = Cell | year=2002|volume = 111 | issue = 5 | pages = 747–56 | doi=10.1016/S0092-8674(02)01134-0] cite journal|pmid=12410317 |author=Winkler W, Nahvi A, Breaker RR|title=Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression|journal=Nature|volume=419|issue=6910|year=2002|pages=890–1|doi=10.1038/nature01145] cite journal|pmid=12456892 |author=Winkler WC, Cohen-Chalamish S, Breaker RR|title=An mRNA structure that controls gene expression by binding FMN|journal=Proc Natl Acad Sci U S A|volume=99|issue=25|year=2002|pages=15908–13|doi=10.1073/pnas.212628899] 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.

Most known riboswitches occur in Bacteria, but functional riboswitches of one type (the TPP riboswitch) have been discovered in plants and certain fungi. TPP riboswitches have also been predicted in Archaea,cite journal | author = Sudarsan N, Barrick JE, Breaker RR | title = Metabolite-binding RNA domains are present in the genes of eukaryotes | journal = RNA | volume = 9 | issue =6 | pages = 644–7 | pmid = 12756322 | doi = 10.1261/rna.5090103 | year = 2003 ] but have not been experimentally tested.

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 genescite journal | author = Cheah MT, Wachter A, Sudarsan N, Breaker RR | title = Control of alternative RNA splicing and gene expression by eukaryotic riboswitches | journal = Nature | volume = 447 | issue = 7143 | pages = 497–500 | year = 2007 | pmid = 17468745 | doi = 10.1038/nature05769 ]
** A TPP riboswitch in plants modifies splicing and alternative 3'-end processing [cite journal |author=Wachter A, Tunc-Ozdemir M, Grove BC, Green PJ, Shintani DK, Breaker RR |title=Riboswitch control of gene expression in plants by splicing and alternative 3' end processing of mRNAs |journal=Plant Cell |volume=19 |issue=11 |pages=3437–50 |year=2007 |pmid=17993623 |doi=10.1105/tpc.107.053645] [cite journal |author=Bocobza S, Adato A, Mandel T, Shapira M, Nudler E, Aharoni A |title=Riboswitch-dependent gene regulation and its evolution in the plant kingdom |journal=Genes Dev. |volume=21 |issue=22 |pages=2874–9 |year=2007 |pmid=18006684 |doi=10.1101/gad.443907]

Types of riboswitches

The following riboswitch classes 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. 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.
* "PreQ1 riboswitches" bind pre-queuosine₁, to regulate genes involved in the synthesis or transport of this precursor to queuosine. Two entirely distinct classes of PreQ1 riboswitches are known: PreQ1-I riboswitches and PreQ1-II 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.
* "SAH riboswitches" bind S-adenosylhomocysteine to regulate genes involved in recycling this metabolite that is produced when S-adenosylmethionine is used in methylation reactions.
* "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.
* "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.
* "cyclic di-GMP riboswitches" bind the signaling molecule cyclic di-GMP in order to regulate a variety of genes controlled by this second messenger.

Presumed riboswitches:
* "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.

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. It has been suggested that some riboswitches might represent ancient regulatory systems, or even remnants of RNA-world ribozymes whose bindings domains are conserved. [cite journal |author=Corbino KA, Barrick JE, Lim J, "et al" |title=Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria |journal=Genome Biol. |volume=6 |issue=8 |pages=R70 |year=2005 |pmid=16086852 |doi=10.1186/gb-2005-6-8-r70 |url=] [cite journal |author=Cochrane JC, Strobel SA |title=Riboswitch effectors as protein enzyme cofactors |journal=RNA |volume= 14|issue= |pages= 993|year=2008 |month=April |pmid=18430893 |doi=10.1261/rna.908408 |url=]

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-boxcite journal|pmid=10094622|author=Grundy FJ, Henkin TM|title=The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria|journal=Mol Microbiol|year=1998|volume=30|issue=4|pages=737–49|doi=10.1046/j.1365-2958.1998.01105.x] (now the SAM-I riboswitch), the THI-box cite journal|pmid=11470904 |author=Miranda-Ríos J, Navarro M, Soberón M|title=A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria|journal=Proc Natl Acad Sci U S A|year=2001|volume=98|issue=17|pages=9736–41|doi=10.1073/pnas.161168098] (now the TPP riboswitch) and the RFN elementcite journal|pmid=10529804|author=Gelfand MS, Mironov AA, Jomantas J, Kozlov YI, Perumov DA|title=A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes|journal=Trends Genet|year=1999|volume=15|issue=11|pages=439–42|doi=10.1016/S0168-9525(99)01856-9] (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 q] .

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) cite journal|pmid=15096624 |author=Barrick JE, Corbino KA, Winkler WC, Nahvi A, Mandal M, Collins J, Lee M, Roth A, Sudarsan N, Jona I, Wickiser JK, Breaker RR|title=New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control|journal=Proc Natl Acad Sci USA|year=2004|volume=101|issue=17|pages=6421–6|doi=10.1073/pnas.0308014101] 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. Subsequent comparative genomics efforts using additional taxa of bacteria and improved computer algorithms have identified further riboswitches.cite journal|pmid=16086852|author=Corbino KA, Barrick JE, Lim J, Welz R, Tucker BJ, Puskarz I, Mandal M, Rudnick ND, Breaker RR|title=Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria|journal=Genome Biol|year=2005|volume=6|issue=8|pages=R70|doi=10.1186/gb-2005-6-8-r70] cite journal|pmid=17621584 |author=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|title=Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline|journal=Nucleic Acids Res|year=2007|doi=10.1093/nar/gkm487|volume=35|pages=4809]

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.cite journal|pmid=17160062|author=Blount KF, Breaker RR|title=Riboswitches as antibacterial drug targets|journal=Nat Biotechnol|year=2006|volume=24|issue=12|pages=1558–64|doi=10.1038/nbt1268] 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. However, many riboswitches have been shown to be non-essential, likely precluding them as effective antibiotic targets.

Engineering of synthetic riboswitches

Riboswitches have recently been engineered using a scaleable and modular approach cite journal|pmid=17709748|author=Win MN, Smolke CD|title=A modular and extensible RNA-based gene-regulatory platform for engineering cellular function.|journal=PNAS USA|year=2007|volume=104|issue=36|pages=14283–8|doi=10.1073/pnas.0703961104] . Engineered riboswitches allow for various forms of regulation of gene expression and other biological functions.

References