The non coding regions of mRNA host some important domains which can bind specifically to metabolites and regulate gene expression. These domains are called Riboswitches. They are highly specific in binding and undergo allosteric conformational changes on binding to the target. These domains are found in bacteria and recently in humans.
These riboswitches are essentially aptamer sequences that can bind to small effector molecules specifically. These domains usually switch off/on the protein translation process and thus regulate the mRNA activity. Bacterial species such as E.coli, B. anthracis, Streptococcus etc are found to contain riboswitches.
Riboswitches were discovered by Ronald Breaker in 2002. Earlier instances of riboregulation were discovered in regulation of tryptophan biosynthesis by Charles Yanofsky. This type of regulation was attributed to the coupling of translation and transcription at the mRNA level.
Many other RNA based regulatory mechanisms have been discovered after that. One prominent example is the RNA based regulation of interference and epigenetics in eukaryotes.
RNA aptamers have been developed in vitro also for many target molecules. Examples include theophylline aptamer. These aptamers are found to undergo conformational changes on binding to the ligands just like the induced fit mechanism of enzymatic binding.
Aptamers can be either synthesized or naturally be present in the non coding regions of mRNAs. The natural aptamers are capable of interacting with the metabolites for gene regulation without any intermediate proteins. The usual mechanism of regulation is feedback based.
The riboswitches comprise mainly two regions- an aptamer and an expression platform. The aptamer portion binds to the target molecule and this causes changes in the structure of expression platform which in turn alters the gene expression.
Methods of riboregulation with riboswitches
There are several methods through which riboswitches carry out the process of riboregulation.
1. Translation control
a. The most common is the induced fit mechanism described above. A change in the structure of expression platform switches on/off the genes involved.
b. Induced self cleavage
In this method, the ribozyme within the riboswitches cleaves itself upon binding of expression platform to the target. This induces conformational changes in riboswitches causing them to block the ribosomal binding site/ Shine Dalgarno sequence and making them inaccessible to translation process.
2. Intrinsic termination / Transcription control
a. This is mediated through dissociation of transcription complex terminating the process. The process involves the action of RNA polymerases. These enzymes bind to the stem-loop structure of riboswitches which are often enriched with Cytosine- Guanine base pairs. These loop structures are often followed by a trail of Uracil residues. The binding of RNA polymerase inhibits the transcription. The interaction between DNA and mRNA at the Uracil rich chain is often weak and inadequate to support the formation of transcription complexes. Hence they dissociate easily.
b. A newer variant of transcription control involves alternative splicing discovered in TPP riboswitches in Neurospora. In this, the transcription complex is formed, but the riboswitch modifies the transcript by shifting the 5'splice site resulting in a larger transcript than the usual one. This transcript is unable to undergo translation efficiently since the ribosomes tend to dissociate once they read the upstream open reading frames which are incorporated in the transcript due to the action of riboswitch.
3. RNA processing
Riboswithces are known to regulate gene expression by splicing or degradation of mRNA.
Examples of riboswitches
• Coenzymne B12 aptamer consensus
• TPP aptamer consensus- The action of riboswitch is mediated by metal ions (Mg2+) and contact with the nucleobases. This enable the recognition of phosphate group of the ligand molecule. The antibiotic pyrithiamine pyrophosphate (PTPP) induces a conformational change to the guanosine 60 of the sequence in conjugation with the riboswitch.
• FMN aptamer
• S-Adenosyl Methionine aptamer
• Purine aptmer sequence (Guanine specific)
• Purine aptamer consensus ( Adenine specific)
• Lysine aptamer
• Glycine aptamer - uses cooperativity between two aptamer regions which results in metabolite induced conformational transition to regulate gene expression.
• Glucosamine 6-phosphate specific aptamer- riboswitch has a ribozyme which regulates through induced self cleavage mechanism. This riboswitch was recently found to have the ability to bind glucose-6-phosphate also which causes opposite results. When the riboswitch was bound to glucosamine-6-phosphate, it induced self-destruction turning the glmS gene off. Binding to glucose-6-phosphate prevented self-destruction keeping the glmS gene turned on.
• I-A aptamer (specific to 2-deoxyguanosine)
• PreQuinosine specific aptamer
These riboswitches have been the basis of RNA world hypothesis since they have been found in numerous bacterial and archaeal species. Riboswitches were thought to be 'vestigial' sequences lost in the process of evolution. Now, scientists have discovered a riboswitch in humans in the non coding regions of mRNA for Vascular Endothelial Growth Factor (VEGF). This riboswitch has two complexes. One of them induces conformational change in the other when bound to the target. The factor which binds is dependent on the environmental signals. This is a class of protein dependent RNA which analyze the individual inputs precisely to produce a response.
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