α 32p utp

Mitochondria were isolated from fresh heart and liver tissue by differential centrifugation (1 × 1,000g, 1 × 10,000g). For in organello transcription experiments, freshly purified mitochondria (500 μg) were washed three times in incubation buffer (25 mM sucrose, 75 mM sorbitol, 10 mM Tris–HCl, 10 mM K₂HPO₄, 100 mM KCl, 0.05 mM EDTA, 1 mM ADP, 5 mM MgCl₂, 10 mM glutamate, 2.5 mM malate, and 1 mg/ml BSA, pH 7.4). Washed mitochondria were resuspended in 500 μl of incubation buffer and supplemented with 50 μCi of α-³²P-UTP (Hartmann Analytic). Samples were incubated at 37°C for 1 h. Afterwards, mitochondria were pelleted, resuspended in incubation buffer containing 0.2 mM UTP and incubated for 10 min at 37°C. Mitochondria were subsequently washed twice in cold wash buffer (10% glycerol, 0.15 mM MgCl₂, and 10 mM Tris–HCl, pH 6.8). An aliquot of the mitochondria (10 μl) was taken as a loading control. Then, RNA was extracted using TRIzol (Ambion) following the manufacturer’s recommendations and precipitated overnight at −20°C. The purified RNA was loaded onto a formaldehyde–agarose gel and blotted as a northern blot. The membrane (Hybond-N+; GE Healthcare) was exposed to a phosphorimager screen. Loading controls were run on 10% NuPage gels using MOPS buffer and equal loading was assessed by immunoblotting against HSP60.

Transcription from promoter DNA fragments was performed essentially as described (20 (link)). Briefly, reactions were performed in 10 μl of transcription buffer TB (20 mM Tris–HCl pH 7.9, 40 mM KCl, 10 mM MgCl₂). 1 pmol of E. coli RNAP core with 3 pmol of σ⁷⁰ or 1 pmol of T. aquaticus RNAP core with 3 pmol of T. aquaticus σ^A or 1 pmol of S. aureus RNAP core with 3 pmol of S. aureus σ^A or 1 pmol of M. smegmatis or S. epidermidis RNAP holoenzymes were incubated in TB with 1 μl of DMSO containing or not containing Urd at 37°C (or 60°C in case of T. aquaticus RNAP) for 5 min. Transcription was initiated by the addition of 2 μl mixture of nucleotides and promoter DNA in TB, containing (final concentrations): 10 nM promoter DNA, 25 μM CpA (for T7A1 and GalP1 promoters) or 100 μM ApA (for lacUV promoter), 0.2 μl α-[³²P]UTP (10mCi/ml) (Hartmann Analytic), 10 μM UTP with (run off transcription) or without (abortive transcription) 100 μM ATP, CTP and GTP. Reactions were stopped after 10 min incubation at 37°C (or 60°C in case of T. aquaticus RNAP) for run off transcription or 5 min for abortive transcription by the addition of equal volume of formamide-containing loading buffer. Products were resolved in denaturing polyacrylamide gels, revealed by PhosphorImaging (GE Healthcare), and analyzed using ImageQuant software (GE Healthcare)