Part:BBa_K3743001
Cobalamin Riboswitch
Part description
Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA. It was proposed that cobalamin riboswitches can be classified into two classes , Cbl-I and Cbl-II, according to secondary and tertiary characteristics. They serve as precision sensors for their corresponding targets. Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression or translation of mRNA to yield a protein. Cobalamin in the form of adenosylcobalamin (Ado-CBL) is known to repress expression of proteins for vitamin B12 biosynthesis via a post-transcriptional regulatory mechanism that involves direct binding of Ado-CBL to 5' UTRs in relevant genes, preventing ribosome binding and translation of those genes.
Usage
For providing safety procedure for our vaccine, we thought about using cobalamin riboswitch that has the ability to regulate expression of downstream parts depending on the binding between vitamin B12 and the riboswitch. This could be applicable in our design in enable regulating vaccine transcripts whether increase or decrease them depending on the binding to perform as a safety switch.
Literature Characterization
A study made a plot showing the fold repression of the wild type of cobalamin riboswitches combared to variants of it that have been made by changes in the sequence of J1/13 as shown in figure(1).(1)
Characterization by Mathematical Modelling
In order to simulate the dynamics of the used riboswitches which acts as an essential safety switches in our vaccine design, Mathematical modeling is performed using ordinary differential equations (ODEs) and fitted parameters.
As shown in Figure(2), P2 which represents the binding state shown inhibition throughout the time which conclude inhibition of the circuit. On the other hand, P1 which represents removing the inhibitory effect of riboswitches showed increased transcription of the circuit which reach the steady state after about 200 seconds.
Structural Characterization
In order to model the structural stability of cobalamin riboswitch, we used RNAfold tool that predicts secondary structures of single stranded RNA.
The final result was that the free energy of the thermodynamic ensemble is -54.19 kcal/mol.
References
1.Polaski, J. T., Holmstrom, E. D., Nesbitt, D. J., & Batey, R. T. (2016). Mechanistic insights into cofactor-dependent coupling of RNA folding and mRNA transcription/translation by a cobalamin riboswitch. Cell reports, 15(5), 1100-1110.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 128
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 128
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 128
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 128
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 128
Illegal NgoMIV site found at 31
Illegal AgeI site found at 48 - 1000COMPATIBLE WITH RFC[1000]
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