Luciferase t7 control dna

For knockdown of DDX3, cells were transfected with psiDDX3-433 derived from pSUPER plasmid as described previously^{30 (link)}. Plasmid Flag-DDX3^R, which is a siDDX3-resistant Flag-DDX3 expressing construct, was generated as described previously^{58 (link)}. Plasmid pCMV-p53 WT and pCMV-p53 S15A mutant were gifts from Dr. Sheau-Yann Shieh (Institute of Biomedical Sciences, Academia Sinica, Taiwan). Plasmid pcDNA3-mCherry-α-tubulin was a gift from Dr. Tang K. Tang (Institute of Biomedical Sciences, Academia Sinica, Taiwan). Plasmid GFP-H2B was a gift from Dr. Jun-Yi Chien (Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan). Plasmid 5′ + 3′-pA, which directs the expression of reporter RNA, was generated by inserting HindIII/BamHI-treated PCR amplified p53 5′UTR (spanning from −134 to −1 related to translation start site)^{24 (link)} into HindIII/BamHI-treated Luciferase T7 Control DNA vector (Promega Corporation) and then the SacI-digested PCR amplified p53 3′UTR (spanning from 1183 to 2369 related to translation start site)^{22 (link)} was further ligated with SacI-digested p53 5′UTR-Luciferase T7 Control DNA plasmid.

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.

A 4.3-kbp plasmid DNA (Luciferase T7 Control DNA: 4331 bp) was purchased from Promega (Madison, WI, USA). A 1.7-kbp linear reporter DNA, a 4.3-kbp linear reporter DNA and a 2.6-kbp linear non-coding DNA were amplified by PCR using the following primers: for the 1.7-kbp linear DNA (forward 5′-TAATACGACTCACTATAGGGAGACC-3′; reverse 5′-CAATTTGGACTTTCCGCCCTTC-3′); for the 4.3-kbp linear DNA (forward 5′-TAATACGACTCACTATAGGGAGACC-3′; reverse 5′-ATTTCGATAAGCCAGCTGC-3′); and for the 2.6-kbp non-coding DNA (forward 5′-CTGTATTCAGCGATGACGAAATTC-3′; reverse 5′-ATTTCGATAAGCCAGCTGC-3′). 1 ng of the 4.3-kbp parental plasmid was used as a template for PCR. PCR was performed with KOD FX neo (Toyobo, Tokyo, Japan) using a Thermal Cycler Dice (TP600) (Takara Bio Inc, Shiga, Japan), according to the manufacturer’s protocol. The PCR products were precipitated with 0.3 M sodium acetate/ethanol, washed with 70% ethanol, and dried. The DNA pellets were dissolved in 50 μL of TE buffer (pH 8.0) and stored at − 20 °C until use. Each template DNA was analyzed using a DNA ladder marker (1 kbp DNA Ladder One, Nacalai Tesque, Kyoto, Japan) by standard 1% agarose gel electrophoresis (Fig. S1b,d in the Supplementary Information).