Regulatory

Part:BBa_K3286202

Designed by: Ben Kuipers   Group: iGEM19_Wageningen_UR   (2019-10-20)


Vc2 Riboswitch

This is the regulatory sequence of the Vc2 riboswitch. The Vc2 riboswitch is a class I c-di-GMP riboswitch which acts on the translational level. Upon binding of c-di-GMP, the downstream gene will be repressed from getting translated. When c-di-GMP is getting degraded in the cell, the alterations in structure will alleviate the repression and translation of the gene will occur [1,2].


Vc2 riboswitches are regulatory elements naturally occurring in Vibrio cholerea. Although, class I riboswitches, in general, are phylogenetically widespread among the bacterial kingdom [3].


  1. S. Inuzuka et al., “Mutational analysis of structural elements in a class-I cyclic di-GMP riboswitch to elucidate its regulatory mechanism,” J. Biochem., vol. 160, no. 3, pp. 153–162, 2016.
  2. S. Inuzuka et al., “Recognition of cyclic-di-GMP by a riboswitch conducts translational repression through masking the ribosome-binding site distant from the aptamer domain,” Genes to Cells, vol. 23, no. 6, pp. 435–447, 2018.
  3. N. Sudarsan et al., “Riboswitches in Eubacteria Sense the Second Messenger Cyclic Di-GMP,” Science (80-. )., vol. 321, no. 5887, pp. 411–413, Jul. 2008.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution from iGEM 2021 RDFZ-China

Group: iGEM 2021 RDFZ-China
Author: Wenjing Zhang

Vc2 Riboswitch resides upstream of the gene (VC1722) homologous to tfoX of V. Cholerae. Fig1 shows the crystal structure of three-helix Vc2 Riboswitch. Two terminal ends of base-paired regions(P2 and P3, respectively shown in light blue and green) form a third helix(P1, shown in black-blue). c-di-GMP is shown in orange.


   < img src="T--RDFZ-CHINA--Contribution-Vc2-riboswitch-structure2.png">
Figure 1. Crytsalline structure of vc2 riboswitch with c-di-GMP

Vc2 RNA tends to form a one-to-one saturable complex with a dissociation constant (KD) of ~1 nM for c-di-GMP. As shown by Fig2, Various analogs of c-di-GMP are tested, including the linear breakdown products pGpG, GpG, and GpGpG etc. and results have shown that they are strongly discriminated against by Vc2 aptamer. Moreover, it has an affinity of ~10 pM, making it the tightest binding c-di-GMP receptor and the highest-affinity RNA-ligand interaction known. 

   < img src="T--RDFZ-CHINA--Contribution-Vc2-riboswitch.jpg">
Figure 2. Comparison of KD values exhibited by Vc2 aptamer for c-di-GMP and various aptamers


Reference

<p>[1]Smith, K., & Strobel, S. (2011). Interactions of the c-di-GMP riboswitch with its second messenger ligand. Biochemical Society Transactions, 39(2), 647–651. https://doi.org/10.1042/bst0390647

[2] Nelson, J. W., Sudarsan, N., Furukawa, K., Weinberg, Z., Wang, J. X., & Breaker, R. R. (2013). Riboswitches in eubacteria sense the second messenger c-di-AMP. Nature Chemical Biology, 9(12), 834–839. https://doi.org/10.1038/nchembio.1363

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