Cobalamin riboswitch

Riboswitches are RNA-based genetic regulatory elements present in the 5’ untranslated region (5'UTR) of primarily bacterial RNA.

In the alpha-axial position, the cobalt is coordinated to a dimethylbenzimidazole moiety attached to the corrin ring via a flexible aminopropanol linker.

Methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) are the biologically active forms of cobalamin, containing a methyl group and an adenosyl moiety in the beta-axial position, respectively.

[6] Hydroxocobalamin (HyCbl), with a hydroxyl group in the beta-axial position, is produced as a result of cobalamin photolysis, and is present in biological conditions but is not in an active form.

Cbl-I riboswitches are selective for AdoCbl, with a variable peripheral stem loop structure facilitating ligand specificity.

Cbl-IIa riboswitches are specific to cobalamin analogues with smaller β-axial ligands including MeCbl and HyCbl.

[6] Ethanolamine is abundant in the human intestinal tract as it is the product of the breakdown of the phosphatidylethanolamine from cell membranes and is also present in processed food.

[1][13] In addition, some cobalamin riboswitches exhibit promiscuous ligand binding, such as the B. subtilis yvrC riboswitch, which can adopt different structural conformations in order to bind cobalamin analogues with smaller β-axial ligands such as MeCbl and HyCbl in addition to AdoCbl, which has a much bulker β-axial moiety.

[5] Riboswitches are ideally suited to be engineered into biosensors due to their ability to undergo a conformational switch upon binding to specific ligands.

For example, the reporter gene can encode for green fluorescent protein (GFP) when fluorescence-based detection methods are desired.

[22] In the presence of ligand, the riboswitch undergoes a conformational change which blocks the ribosomal binding site, halting transcription of the reporter gene.

[4]  This sensor was also used to detect vitamin B12 biosynthetic precursors such as cobinamide and confirm the involvement of specific genes in cobalamin metabolism.

[4] More recently, this sensor was used to screen Ensifer meliloti mutants for their ability to synthesize large quantities of Vitamin B12.

For example, a biosensor developed from a Propionibacterium freudenreichii cobalamin riboswitch was used to determine the vitamin B12 concentration in fermented food with high sensitivity.

VB 12 -bound Riboswitches with PDB codes: a) 4FRN b) 6VMY c) 4FRG d) 4GMA
A basic schematic is shown here where the purple stars indicate the metabolite and the yellow arrow shows the reporter gene. The cells with a lower concentration of the metabolite have more riboswitches in the unbound state. The unbound conformation has an unstructured interaction between the ribosome binding site (RBS) and the blue and green segments. This unstructured interaction allows for the reporter gene to be translated efficiently downstream and produce a high signal output. At a higher metabolite concentration, the riboswitches form a bound conformation where the blue segment of the riboswitch interacts with the target RNA. This allows the green segment to interact with the RBS instead, and this allows the RBs to inhibit translation initiation of the reporter gene. Because of this, the signal output is lower than with the low concentration of the metabolite.