Team:Oxford/CueR MOI

iGEM Oxford 2016 - Cure for Copper

CueR Mechanism

Gif shows CueR monomer (without copper in cyan, with copper in red cealinged in pymol) adapted from the PDB files 4wls and 4wlw.(1)

At weak promoters such as the promoter of the gene copA (pCopA), RNA polymerase (RNAP) can bind in two conformations. One form leads to the open complex and the start of transcription whilst the other form is a dead-end that does not lead to transcription.(2)

The E. coli copper regulator CueR does not, in fact, cause the inter-conversion of these two states but does affect the population distribution of RNAP between these two states by affecting the RNAP binding kinetics.

CueR without copper slightly increases the binding rate constant of the dead-end complex and slightly decreases the unbinding. It also slightly increases both the RNAP binding and unbinding rate constants for the open complex resulting in slight strengthening. Overall, however it causes a net reinforcement of the dead-end complex and thus represses transcription.

With copper bound however it does not change the dead-end complex RNAP’s binding rate constant significantly, but greatly increases its unbinding resulting in its weakening. Copper-CueR also significantly increases RNAP’s binding rate complex for the open complex, while slightly decreasing its unbinding, so has net strengthening. Overall therefore it causes a net favour for the open complex so activates transcription.

At the molecular level CueR operates by bending the DNA to which it binds, allowing better contact between the -35 and -10 sequences of the promoter and RNAP. With copper bound the DNA is stabilised by CueR in a conformation more resembling A-DNA than the standard B-DNA.(1)

References

  • (1) Philips, S.J., Canalizo-Hernandez M., Yildirim I., Schatz G., Mondragón A., O’Halloran T., (2015) "Allosteric transcriptional regulation via changes in the overall topology of the core promoter." Science 349: 877-881
  • (2) Danya J. Martell, Chandra P. Joshi, Ahmed Gaballa, Ace George Santiago, Tai-Yen Chen, Won Jung, John D. Helmann, and Peng Chen (2015) “Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription” Proc Natl Acad Sci U S A. 2015 Nov 3; 112(44): 13467–13472.
  • (3) Balogha R., Gyurcsika B., Hunyadi-Gulyásb E., Christensenc H., Jancsóa A., (2016) “Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein” Protein Expression and Purification Volume 123, July 2016, Pages 90–96.