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bioRxiv · 10.1101/2020.07.14.202762

Characterization of gene repression by designed transcription activator-like effector dimer proteins

Becker, N.·Peters, J.·Schwab, T.·Phillips, W.·Wallace, J.·Clark, K.·Maher, L. J.
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Abstract

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the E. coli lac operon switch, where DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed Transcription Activator-Like Effector Dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system. Statement of SignificanceWe are designing and testing in living bacteria artificial DNA looping proteins engineered based on principles learned from studies of the E. coli lac repressor. The engineered proteins are based on artificial dimers of Transcription Activator-Like Effector (TALE) proteins that have programmable DNA binding specificities. The current work is the first to create unique DNA repression loops using this approach. Systematic study of repression as a function of loop size, with data fitting to a thermodynamic model, now allows this system to be compared in detail with lac repressor loops, and relevant biophysical parameters to be estimated. This approach has implications for the artificial regulation of gene expression.

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Becker, N., Peters, J., Schwab, T., Phillips, W., Wallace, J., Clark, K., Maher, L. J.. 2020-07-15. Characterization of gene repression by designed transcription activator-like effector dimer proteins. https://doi.org/10.1101/2020.07.14.202762

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