Why architecture matters: Controlling gene expression through design

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Abstract Inducible gene expression systems are widely used in synthetic biology and gene therapy, yet their performance depends not only on regulator chemistry but also on circuit architecture. Here, we examine how promoter organization shapes TetR-based gene regulation in mammalian cells using a panel of single-vector constructs spanning a broad range of promoter strengths. Experiments and thermodynamic models show that bidirectional circuits impose a trade-off between output and control: increasing promoter strength elevates both induced and basal expression, compressing dynamic range. Incorporating transcriptional coupling explains the parallel scaling of these states in compact divergent designs. In contrast, autogenous regulation couples repressor production to transcription, introducing negative feedback that buffers promoter strength and preserves fold induction. Finally, adding ligand-responsive aptazymes as a post-transcriptional layer further suppresses basal expression while maintaining inducibility, albeit with reduced maximal output. Together, these results identify regulatory architecture as a primary determinant of circuit performance and establish design principles for constructing more predictable gene expression systems in eukaryotic cells.