Fig. 2. Development of synthetic Ras/Erk target genes that recapitulate endogenous transcriptional kinetics.

a Schematic overview of synthetic target genes incorporating multi-step regulation. Signaling inputs can act at the transcriptional level through promoter/enhancer regulation (1), the mRNA level through UTR-based regulation (2), and at the protein level through signaling-responsive domains or linear motifs (3). b Design of synthetic immediate-early genes (SynIEGs). The Erk-responsive FOS promoter (pFOS) drives the expression of yellow fluorescent protein (YFP) followed by 24 MS2 stem-loops, which allow for the visualization of protein accumulation and transcription, respectively, in live cells. mRNA UTR elements and protein degrons can be added to modulate mRNA and protein stability. Representative images at different timepoints of a single cell expressing a SynIEG containing the FOS 5′ UTR and TUBULIN 3′ UTR before and after serum stimulation for both transcription and translation. Transcriptional focus of MCP-mCherry localization is denoted with a red arrow. Data from additional cells are shown in Supplementary Fig. 1. Scale bar is for 10 μm. c Images of an NIH3T3 nuclei after induction of transcription of SynIEG contain FOS 5′ UTR and BTG2 3′ UTR at multiple timepoints after serum stimulation. Transcriptional foci are denoted using red arrows. Scale bar is for 10 μm. d Quantification of the eight transcriptional foci in the cell shown in c, showing individual traces (left) their mean + SD (right). Transcriptional responses from 10 additional cells are shown in Supplementary Fig. 3.