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FIGURE 5.

FIGURE 5.

Grc3 PNK active site motifs are critical in S. cerevisiae. (A) Schematic of tetracycline-off system. In the presence of doxycycline (DOX), the tetracycline-transcription activator (tTA) cannot bind the tetracycline response element (TRE); thus repressing transcription of endogenous GRC3. (B) S. cerevisiae tetO7-GRC3 and S. cerevisiae tetO7-GRC3–Las1 5X Flag strains were transformed with plasmids encoding wild-type Grc3 with and without an N-terminal Myc-tag and the ARS1-CEN4 YCplac vector. Serial dilutions were spotted on YPD agar plates in the absence and presence of DOX (20 µg/mL) and incubated at 30°C for 2–3 d. (C) S. cerevisiae tetO7-GRC3–Las1 5X Flag were transformed with plasmids encoding variants of the Grc3 PNK active site motifs (P-loop [red], Walker B [blue], Clasp [purple], Lid [orange]). Serial dilutions were spotted on YPD agar plates in the absence and presence of doxycycline (20 µg/mL) and incubated at 30°C for 2–3 d. (D) Growth curves from selected Grc3 PNK variants grown in the absence or presence of DOX (20 µg/mL) at 30°C. The absorbance at 595 nm was recorded over a 25-h time period. Each curve is the average of three independent replicates and errors bars mark the standard deviation.