1732 Nucleic Acids Research, 2012, Vol. 40, No. 4 Figure 1. Density-based fractionation of mitochondrial GFP-Rpm2p. After fractionation on a discontinuous 10–70% glycerol gradient, relative GFP ﬂuorescence absorbance/fraction identify most Rpm2-GFP with high-molecular-weight complexes. Figure 2. Puriﬁcation of mt-RNase P from the mitochondrial matrix extract. Puriﬁcation and assays conditions are given in ‘Materials and Methods’ section. Mt-RNase P activities in (A) correspond to a separation on a 5.5% NEC column and (B) on 7.5% NEC slab gel. Aliquots from each fraction were assayed for RNase P activity. The negative control (C–) is without and the positive control (+M1) with E. coli M1 RNA. The RNA corresponding to 50-leader pre-tRNAproline (117 nt), tRNAproline (78 nt) and the removed 50-leader sequence (39 nt) are indicated. Mt-RNase P activity was found in fractions 7, 8 and 9 in preparative NEC (A), and in fraction 38 only in a subsequent regular NEC separation (B). Note that the activity test of fraction 38 (B) was performed with more material, leading to almost complete processing of the tRNA precursor, but also to partial, unspeciﬁc cleavage of the mature tRNA into smaller fragments. With less material, the processing pattern is similar to that in (A) (Supplementary Figure S2).