Riboprobe system t7

YEPR exponentially growing cell cultures of JKM139 derivative strains, carrying the HO-cut site at the MAT locus, were transferred to YEPRG at time zero. SspI-digested genomic DNA was run on an alkaline agarose gel and visualized after hybridization with an RNA probe that anneals with the unresected strand on one side of the HO-induced DSB as previously described (63 ). This probe was obtained by in vitro transcription using Promega Riboprobe System-T7 and plasmid pML514 as a template. Plasmid pML514 was constructed by inserting in the pGEM7Zf vector a 900-bp fragment containing part of the MAT locus (coordinates 200870 to 201587 on chromosome III). Quantitative analysis of DSB resection was performed by calculating the ratio of band intensities for ssDNA and the total amount of DSB products. The resection efficiency was normalized with respect to the HO cleavage efficiency for each time point. Densitometric analysis of band intensities was performed using Scion Image Beta 4.0.2.

The radioactive RNA probes were prepared by in vitro transcription in the presence of [α-³²P]CTP with Riboprobe System-T7 (Promega, Madison, WI, #P1446), using PCR products with a built-in T7 promoter as DNA templates. The following primers were used for MmuPV-1 DNA template preparation: oXYX-12 and oXYX-23 (S1 Table). The RNase protection assay (RPA) was performed with an RPA III kit (Ambion, Austin, TX, #1414) according to the manufacturer’s instructions with minor modifications. Briefly, 4 ng of each probe (specific activity, 35,000 cpm/ng) was hybridized overnight at 50°C with 30 μg of total tissue RNA in hybridization buffer and then digested with an RNase A-T1 mixture for 30 min at 37°C. Five micrograms of yeast RNA was used as a negative control (MmuPV1 -). Protected RNA fragments were separated in a denaturing 8% polyacrylamide gel containing 8 M urea. MmuPV-1 DNA sequencing ladders generated with the ³²P-labeled Primer Pr7237 (oXYX-28) (S1 Table) were used as size markers and run along with the RPA products as described [73 (link)]. Autoradiographic data were captured with a Typhoon Imaging System (GE Healthcare Life Sciences, Pittsburgh, PA) and analyzed with ImageQuant software (GE Healthcare Life Sciences).

Total RNA was extracted as reported previously (Ferrara et al., 2012 (link)). RNA for RNA/RNA interaction assays was prepared by T7 RNA polymerase transcription of gel-purified DNA fragments. DNA fragments for ErsA RNA and amrZ mRNAs (amrZ, amrZCIS1, amrZΔIS2, amrZCIS1ΔIS2) preparations were amplified from P.aeruginosa PAO1 genomic DNA with oligo pairs 4/5 or 4/6 and 7/8, respectively. The transcription reactions were performed using the Riboprobe^® System-T7 (Promega) with 300 ng of DNA template. DNA probe was 5′-end–labeled with (γ-³²P) ATP and T4 polynucleotide kinase (Promega) according to manufacturer’s instruction. Synthesized RNA was precipitated and resuspended in diethylpyrocarbonate-treated water. Purified RNA was checked by denaturing polyacrylamide gel electrophoresis and quantified using a Qubit Fluorometer.

The selected genes, PPE56, PE_PGRS41, PPE67, PE_PGRS39, PPE68 and PE5 (no rG4 control) were amplified from the H37Ra genomic DNA (PCR primers listed in File S2, Table S3) and were cloned into FLAG-HA-pcDNA3.1- (Addgene #52535). In vitro transcription was carried out with 500 ng of plasmids linearized by BglII, according to the manufacturer’s protocol for Riboprobe System-T7 (Promega) with 0 μM, 5 μM, and 10 μM of BRACO19. DNase I treatment was done upon completion of the reaction. RT-qPCRs were performed with QuantiNova SYBR Green RT-PCR Kit as per the manufacturer’s protocol. Each experiment was repeated at least thrice.

The in vitro transcription of sRNA was performed as previously described (22 (link)). The sRNA ReaL was synthesized using the Riboprobe System-T7 (Promega) from PCR product amplified from PA14 chromosomal DNA with the primers listed in Table S1 according to the manufacturer’s instructions. The RNA was purified by isopropanol precipitation and refolded by heating at 90°C for 10 min and then cooling down naturally at room temperature for 30 min. One hundred nanograms of the purified RNA was mixed with indicated amounts of purified YbeY or GFP in the binding buffer (10 mM Tris-HCl [pH 7.5], 5 mM MgCl₂, 50 mM KCl, 10% glycerol, 1 U recombinant RNase inhibitor [TaKaRa]) and incubated on ice for 30 min. Fifteen microliters of each sample was loaded onto a nondenaturing 8% polyacrylamide gel. The electrophoresis was performed at 100 V for 150 min in 0.5× TBE buffer on ice. The RNA bands were visualized after staining with Gel-red (Biotium) for 10 min.

The sRNA ReaL was synthesized using a Riboprobe System-T7 (Promega) from the PCR product amplified from PA14 chromosomal DNA with the primers listed in Table S1. The RNA was purified by isopropanol precipitation and refolded by heating at 90°C for 10 min and then cooling down naturally at room temperature for 30 min. One hundred nanograms of the purified RNA was mixed with indicated amounts of the purified YbeY or GFP protein in the activity buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 10% glycerol, and the indicated concentrations of MnCl₂) and incubated at 37°C for 30 min. Fifteen microliters of each sample was loaded onto a nondenaturing 8% polyacrylamide gel. Electrophoresis was performed at 100 V for 150 min in 0.5× TBE buffer on ice. The RNA was visualized after staining with Gel-red (Biotium) for 10 min.