11.2: Riboswitches

Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.

The aptamer has high specificity for a particular metabolite which allows riboswitches to specifically regulate transcription even in the presence of many other biomolecules. Aptamers bind a range of organic molecules including purines, coenzymes, and amino acids. They also bind inorganic molecules like magnesium cations and fluoride anions.  Most aptamers bind their ligands through hydrogen bonds or electrostatic interactions. There can be a single or multiple binding sites for the ligand on a riboswitch. In the lysine riboswitch, a single lysine binding site is present on the aptamer. In contrast, in the glycine riboswitch, two separate glycine-specific aptamers are present on the mRNA allowing the aptamer to sense only very high concentrations of glycine, as the riboswitch functions only when two molecules are bound.

The expression platform regulates transcription or translation by forming an anti-terminator or terminator structure. The formation of these structures is dependent on the metabolite binding to the aptamer. At low concentrations, metabolites will not bind to the aptamer. This will signal the expression platform to form an anti-terminator structure which will allow transcription or translation to continue. In contrast, when the metabolite is present at high concentrations, it will bind to the aptamer. In this case, the expression platform forms a terminator structure followed by a series of uracil residues, which forces RNA polymerase to dissociate from the transcript and the DNA strand, thereby terminating transcription. An expression platform can also inhibit ribosome binding to the transcript by forming a hairpin structure with the ribosome binding site, also known as Shine-Dalgarno sequence, preventing the initiation of the translation. Another mechanism by which riboswitches regulate transcription is by acting as RNA enzymes, or ribozymes, which is seen in the glmS riboswitch-ribozyme. These ribozymes cleave the riboswitch mRNA when the metabolite is bound, and then the remaining mRNA is degraded by RNase, leading to the inhibition of the translation.

Riboswitches were thought to be present only in bacteria and archaea, but recently they have also been observed in plants and fungi.  Only thiamine pyrophosphate (TPP) specific riboswitches have been found in eukaryotes so far. Unlike bacteria, eukaryotic genes contain introns that do not allow transcription and translation to occur in the same transcript simultaneously; therefore these riboswitches regulate transcription by alternative splicing. In some plants, a TPP riboswitch is present in the 3' untranslated intron region of the THIC gene. Low TPP levels mask the nearby 5’ splice site of the 3' untranslated region producing a stable mRNA. However, when high concentrations of TPP are present, TPP binds to the riboswitch and exposes the 5’ splice site of the 3' untranslated region. The removal of the intron produces unstable mRNA which cannot produce protein.