Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies

Genes generally provide the information (in the form of RNA that is transcribed from the DNA) that ultimately leads to translation of the mRNA into protein, the gene product. Environmental signals act in numerous ways to compel transcription factors (TFs) to interact with modular regulatory regions of various genes, to turn them on or off (up- versus down-regulation). During the 1980s, the architecture of TFs was realized to be modular. Each TF broadly consists of a DNA-binding domain, and an activation domain, that recruits the cell’s transcription apparatus. These domains can be combined in a plug-and-play manner to build “synthetic-TFs” (synTFs). Such synTFs have been successfully used to program activation, or repression, of a gene of interest — a functionality that has proven essential for many studies in molecular biology as well as applications in biotechnology.

Authors [see attached article and editorial] present a successful approach for engineering cooperative synTF assemblies. These assemblies bring engineered control of gene expression closer to achieving the richness of behaviors exhibited by naturally cooperative TFs. When a synTF is designed to have one-to-one binding to its cognate DNA target (the promoter region of a particular gene), the dose-response of gene expression is largely a gradient (until one reaches saturation), typically fitted by a Michaelis-Menten saturation curve (i.e. similar to enzyme-substrate, transporter-substrate, or receptor-ligand interactions). As a result, the types of gene-expression regulation — that can be programmed with synTFs in this manner — represent only a fraction of the capability of natural TFs.

Cooperativity is a widespread phenomenon in biology by which coordinated behavior within a molecular system emerges from energetic coupling between its components. In eukaryotic gene networks, cooperative assembly occurs when core initiation machinery is recruited to basal promoter regions through multivalent, mutually reinforcing interactions between TFs and associated cofactors. The resulting nucleoprotein complexes play a critical signal-processing and decision-making role; they convert TF inputs into switch-like transcriptional outputs — or they incorporate multiple TFs — to carry out decision functions by activating transcription only in the presence of specific TF combinations.

To date, most synthetic gene circuits have been constructed by using TFs that bind to promoters in a one-to-one fashion constraining the

ability to tune circuit cooperativity and potentially imposing limits on engineerable behavior. Authors wondered whether circuits having expanded signal-processing function can be implemented by using engineered multivalent assembly. They established theoretical contexts and designed frameworks in yeast — for programming cooperative TF assembly on the basis of the configuration, and strength, of intra-complex interactions, and they constructed synthetic gene circuits composed of interconnected regulatory assemblies.

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