(418b) Physical Structure of Autoregulatory Gene Circuits

Complex arrays of regulatory gene circuits enable cells to efficiently utilize resources and respond to changes in their environments. Analysis of these circuits in a variety of organisms has identified a number of recurring regulatory mechanisms or so-called network motifs. In addition to the specific regulatory mechanism utilized, the behavior of a circuit may also be determined by how it is physically encoded within the genome. By physical encoding, we mean the orientation and relative proximity of a transcription factor to its target structural genes. In particular, a given regulatory mechanism involving multiple genes can be physically encoded in any number of ways. The specific location of the genes may, therefore, represent an additional factor determining circuit behavior. In other words, two circuits involving the same genes and regulatory mechanism, nonetheless, can potentially behave differently if they are physically encoded in alternate configurations. One specific example in bacteria of where the physical encoding may affect behavior is autoregulatory gene circuits involving a divergent promoter.

Divergent promoters are characterized by having closely spaced or overlapping binding sites for RNA polymerase. A number of hypotheses have been proposed to explain why divergent promoters are so prevalent in E. coli and other bacteria. The most common is that close proximity of the binding sites for RNA polymerase couple transcription on both sides of the promoter, either directly through steric interactions or indirectly through alterations in local DNA topology. Such interactions may affect transcription in one or both directions. Given that, in Escherichia coli, approximately 40% of all operons expressed from divergent configurations and that 60% of these promoters are linked to the expression of transcription factors, we hypothesized that this configuration may represent one example where the physical topology of the circuit affects its behavior. More specifically, we hypothesized that autoregulatory gene circuits may be arranged in a divergent configuration to achieve a degree of control not possible in other transcriptional configurations.

In this work, we investigated how the transcriptional organization of a negatively autoregulated gene circuit affects its behavior. Using the tetRA divergent promoter from Tn10 as our basis, we compared dose-response behavior of circuits utilizing divergent, tandem, or decoupled configurations. Mathematical modeling predicted that the divergent configuration had a lower cost than the other two in terms of the amount of regulator needed to fix the gain of the circuit and also a higher sensitivity in a sense that it could be induced by the inducer at a concentration over 10 times lower. We experimentally validated these predictions by measuring the response to inducer of TetR-autoregulated circuits utilizing these three configurations. These results suggest that cells may utilize divergent promoters to minimize the metabolic cost associated with regulating gene expression in response to an inducer more effectively. Furthermore, they illustrate how the physical topology of a gene circuit can influence its performance in addition to the general feedback mechanisms employed.