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. 2021 Jun 2;10:e66739. doi: 10.7554/eLife.66739

Figure 1. The N-terminal domain of TPL contains two independent repression domains.

(A) Schematic of the ARCSc. The auxin-responsive promoter driving the fluorescent protein Venus carries binding sites for the auxin-responsive transcription factor (ARF). In the absence of auxin, the IAA-TPL-N fusion protein is bound to the ARF and maintains the circuit in a repressed state. Upon addition of auxin, the IAA-TPL protein is targeted for ubiquitination and subsequent protein degradation, activating transcription of the fluorescent reporter. (B) TPL domains are LisH (LIS1 homology motif, blue), CTLH (C-terminal LisH motif, orange), CRA (CT11-RanBPM; red, dimerization; green, foldback), and two WD40, beta-propeller motifs (purple). N-terminal domains are indicated on the solved structure of the first 202 amino acids (Martin-Arevalillo et al., 2017, 5NQS). The termini of the TPLN100 truncation used in the original ARCSc studies is indicated. (C) Diagram indicating the structure of constructs analyzed in experiments shown in subsequent panels. For constructs with identical behavior (H1-H3, H1-H5, H1-H6, H1-H7), we included only a representative member (H1-H7) for simplicity. Repression Index (Rep.) is a scaled measure of repression strength with 0 set to the level of repression observed with IAA3 and 10 set to the level of repression by TPLN188. Auxin induction level (Aux. ind.) indicates the fold change difference between reporter expression before auxin addition (time zero) and at the end of an experiment (~500 min). (D-F) Helix 1 and the CRA domain (Helix 3–Helix 8) can act independently to repress transcription. Each panel represents two independent time-course flow cytometry experiments of the TPL helices indicated, all fused to IAA3. Every point represents the average fluorescence of 5–10,000 individually measured yeast cells (a.u.: arbitrary units). Auxin (IAA-10 µM) was added at the indicated time (gray bar, +Aux).

Figure 1.

Figure 1—figure supplement 1. Helix 1 and the CRA domain (Helix 3-Helix 8) can act independently to repress transcription.

Figure 1—figure supplement 1.

Each color represents two independent time-course flow cytometry experiments of the TPL helices indicated, all fused to IAA3 or IAA3 alone (black line). Every point represents the average fluorescence of 5–10,000 individually measured yeast cells (a.u.: arbitrary units). Auxin (IAA-10 µM) was added at the indicated time (gray bar, +Aux).