In vitro Synthesis and Characterization of AatR2

The DNA template for in vitro transcription-translation of ataR2 was prepared by PCR using the pBAD-ataRT2 vector template and T7-ataR2-F and T7-ataR2-R primers. The in vitro coupled transcription–translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding ataR2 or the dihydrofolate reductase (DHFR) (NEB, USA). The in vitro translation of firefly luciferase was carried our with in vitro transcribed mRNA (Luciferase T7 Control DNA Promega, USA). Before template addition, translation reactions were incubated for 10 min at 37°C in the presence of 0.2 mM of acetyl coenzyme A, with and without 2 μM AtaT2. DHFR- and ataR2- translation reaction mixtures were supplemented with FluoroTect™ GreenLys in vitro Translation Labeling System (Promega, USA). The fluorescent products of in vitro translation reactions were analyzed by 12% SDS-PAGE, and visualized by Typhoon fluorescence gel imager (GE, USA). The enzymatic activity of in vitro synthesized luciferase was measured by detection of chemiluminescence in the presence of 0.1 mM d-luciferin with VictorX5 multireader (Perkin-Elmer, USA).
The toeprinting assay was carried out using Rst1 and Rst2 mRNA as templates as described in (25 (link)), except that reactions were preincubated for 10 min with 0.4 mM Ac-CoA with or without the addition of 2 μM AtaT2.