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. 2019 Apr 23;38(11):e100368. doi: 10.15252/embj.2018100368

Figure 3. The transcription factor‐like Euc1 shows transactivation in reporter gene assays.

Figure 3

  1. Euc1 domain structure is reminiscent of transcription factors. Top: Schematic representation of Euc1, with a predicted coiled‐coil domain (CC) and GCR1 domain indicated. Predicted DNA binding of the C‐terminal part was confirmed in Y1H experiments (Appendix Fig S3A–C). Bottom: Euc1‐like protein sequences from closely related Saccharomyces species were aligned, and a phylogenetic tree was generated using Clustal Omega. Jalview was used to graphically display the degree of sequence conservation (see also Appendix Fig S4A).
  2. The ub‐HS‐motif is conserved in closely related yeast species. The 7 yeast Multiz Alignment & Conservation tool of the UCSC Genome Browser was used to retrieve alignments of sequences corresponding to ub‐HS‐motifs from other Saccharomyces species. Dot (.) indicates a conserved base.
  3. Euc1 can induce transactivation via its N‐terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig 2D, and serial dilutions were spotted on control or selective media to test HIS3 activation. Cells were grown at 30°C for 3 days.
  4. Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC‐Leu), harvested in logarithmic growth phase, and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT–qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). P = 2.43 × 10−5 (Student's t‐test).