Characterizing the Stability of an Engineered Regulatory DNA Loop in Living E. coli Cells

Abstract

DNA looping has evolved as a regulatory mechanism in both eukaryotic and prokaryotic organisms. Cellular functions including replication, DNA packaging, recombination, and gene regulation utilize DNA looping. In prokaryotes, genes are typically available for expression unless switched off by repressor protein binding at or near the transcriptional start site. Repression may be further enhanced by formation of a DNA loop encompassing the transcriptional start site. The lac operon offers a classic example of a prokaryotic repression loop. The tetrameric LacI protein simultaneously binds two operators within relatively close proximity resulting in a DNA loop, repressing transcription of the downstream genes. Using the lac repression loop as a model, we have created a dimeric fusion protein capable of binding DNA in a sequence-specific manner to create tunable DNA loops that regulate gene expression in vivo. The fusion protein utilizes a transcription activator-like effector (TALE) protein as a programmable DNA binding domain fused to FK506 binding protein (FKBP) as a regulatable dimerization domain. The resulting TALE-FKBP fusion binds DNA in a bidentate manner, reminiscent to LacI, resulting in a closed loop. We utilize our established lac promoter looping system to assess a TALE-FKBP protein fusion targeted to the O2 operator. This system creates a single loop orientation. We optimize this engineered system to account for the directionality of operator binding by the designed protein, determining optimal operator spacings for transcriptional repression. Relative to binding of the fusion protein at the promoter without DNA looping, we report ∼5-fold enhancement of gene repression upon DNA looping. We find that repression enhancement is dependent on both the orientation and the spacing between the DNA operators. We will adapt this system to create an approach for artificial regulation of any promoter.