1730 Nucleic Acids Research, 2012, Vol. 40, No. 4 discontinuous glycerol gradients (10–70% glycerol, in a buffer containing 20 mM HEPES–KOH pH 7.9, 50 mM KCl, 1.5 mM MgCl2 and 1 mM DTT). 0.7 ml fractions were collected and analyzed for protein concentration (Bradford protein assay) and relative GFP ﬂuorescence intensity (excitation at 488 nm, light emission between 500 and 540 nm). Puriﬁcation of mt-RNase P activity To purify the native RNase P complex, we developed a preparative Blue Native Column Electrophoresis (NEC), which permits large-scale puriﬁcation of native protein complexes. It is based on the regular blue native gel elec- trophoresis (BN–PAGE) protocol (41–44), using instead of a slab gel a cylindrical running chamber ﬁlled with polyacrylamide gel (e.g. 7%). The different concentrations of native polyacrylamide gel were prepared as previously published (41–44). The mitochondrial extracts ( 250 mg) were separated at constant voltage (140 V and 9 mA, over- night, in a cold room 4 C), using the same electrophoresis buffers as in regular BN–PAGE. Samples were collected every 30 min from a 200 ml dialysis-cup placed at the outlet of the column. Collected fractions were assayed for mt-RNase P activity and analyzed for complexes compos- ition with BN–PAGE. In vitro preparation and radio-labeling of pre-tRNAproline The mitochondrial Reclinomonas americana pre-tRNAproline substrate for the RNase P assays was prepared by in vitro transcription and puriﬁed as described previously (45). The tRNAproline DNA ligated into pFBS/ EcoRV (2.9 kb) vector with T4 DNA ligase (Roche) and ampliﬁed by PCR using 50-GAAATTAATACGACTCAC TATAGGGTAACGTACTTAATGTAAAAGGTT-30, 50-TGGTCGGGATGACGTGATTTGAACA-30 and 50-TCACTAAAGGGAACAAAAGCTGGGT-30 primers produce respectively 50-leader pre-tRNAproline and, 50-leader and 30-termini pre-tRNAproline. The two ampliﬁed tRNAproline DNAs were used separately for two in vitro radio-labeled transcriptions. Two micrograms of each ampliﬁed tRNAproline DNA were used for tRNAproline transcription with Invitrogen T7 RNA poly- merase (2 u/ml) and a P32 ATP (10 mCi/ml) (Perkin Elmer) as previously described (45). After an overnight incuba- tion at 37 C, loading buffer was added and the samples were heated at 75 C for 2 min before loading on a 9% polyacrylamide/8 M urea gel (4 h, 200 V at room tempera- ture). The 117 nt (50-leader pre-tRNAproline) and 148 nt (50-leader and 30-termini pre-tRNAproline) RNA bands were cut out of the gel and incubated over night at 37 C in a 300 ml buffer extraction (30 ml of 1% SDS and 270 ml of H202). After phenol–chloroform extraction and ethanol precipitation, the precursor RNA was 50-labeled with a-ATP32. Activity assay of mt-RNase P To test the mt-RNase P activity we used the same proced- ure as described previously (46). Radio-labeled pre-tRNAproline (2000 cpm) was dissolved in 1 PA buffer (50 mM Tris–HCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2) and incubated 30 min at 37 C, in a 15 -ml mixture reaction. After incubation, 10 ml of loading buffer was added, and the sample was heated at 75 C for 2 min before loading on a 9% polyacrylamide/8 M urea gel (4 h, 200 V at room temperature). The gels were then either exposed to a Kodak ﬁlm ( 12 h) or a Biorad molecular imaging screen K (2 h). The M1 RNA ribozyme was used as positive control for 50-tRNA processing. The expression plasmid carrying the E. coli M1 RNA gene (provided by Sidney Altman) was used for in vitro transcription. For all experimental infor- mation for M1 RNA transcription and puriﬁcation see (45). The primers for ampliﬁcation of the rnpB gene were 50-GAAATTAATACGACTCACTATAGGGAAG CTGACCAGACAGTCGC-30 and 50AGGTGAAACTG ACCGATAAGCC 30. The M1 RNA was activated before use, by heating (65 C for 5 min), and slow cooling to room temperature in 1 PA buffer (50 mM Tris–HCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2). Radio-labeled pre-tRNAproline (2.000 cpm) was incubated at 37 C in 10 mM Tris–HCl, pH 7.5, 100 mM MgCl2, 100 mM NH4Cl, 4% PEG, in the presence of 10 nM M1 RNA. The total volume of the reaction is 15 ml. After 30 min, 10 ml of loading buffer was added, and the sample was heated at 75 C for 2 min before loading on a 9% polyacrylamide/8 M urea gel. Identiﬁcation of mt-RNase P RNA (RPM1) by RT–PCR Endogenous RNA from the identiﬁed MRT complex was puriﬁed using the ‘RNeasy plus’ kit by QIAGEN, and RT–PCR assays were performed on 10 ng RNA with AMV reverse transcriptase (cDNA synthesis). The cDNA was ampliﬁed with the Expand High Fidelity PCR system provided by Roche. The PCR primers for RPM1 RNA ampliﬁcation are 50-TAATAGGAAAGTC ATAAATAT-30, 50-GTAATATAT ATATATATATTG GAATAG-30 and 50-TTATATTATTATACAGAA ATA-30. For RT–PCR the same primers of the PCR were used in addition to 50-AGAAATAATATTATAAA TAAAATATAT-30, 50-GGATATTATATTATAAGC A-30 and 50-AAGCATATTTCTGTATAATAA-30. The expected PCR products have lengths of 51, 71, 52, 387 and 400 nt. Blue native gel electrophoresis Preparation of samples and of 4–14% BN–PAGE gel followed previously published procedures (41–44), except that detergents were omitted (their effect was only tested in supercomplex stability tests). Approximately 150 mg protein was loaded per well, and electrophoretic separ- ation was performed in a Ho¨ fer (18 16 cm) electrophor- esis chamber, at 140 V and 9 mA, overnight at 4 C. Analysis of supercomplex composition by mass spectrometry To identify protein complexes that form discrete bands in BN–PAGE, complex bands were cut from the gel and submitted to liquid chromatography tandem mass spec- trometry (LC–MS/MS) analysis (47,48). In-gel tryptic digestion and LC–MS/MS analysis was performed by a