Journal: The Journal of Biological Chemistry
Article Title: Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes
Figure Lengend Snippet: Characterization of MRPP3 ΔMTS alone and in complex with MRPP1 ΔMTS and MRPP2. A , domain organization for MRPP3 ΔMTS indicating the N extension (residues 50–206), PPR domain (residues 207–329, red ), core domain (residues 330–361 and 542–583, yellow ), NYN domain (residues 362–541, brown ), and structural zinc ion bound in a Cys-Cys-His-Cys motif in the core domain. B , top , analytical SEC profiles for the mixture of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS ( green line ) and the mixture of MRPP1 ΔMTS , MRPP2, MRPP3 ΔMTS , and pre-(mt)tRNA Ile ( black line ) applied to a Superdex S200 column. A replicate of this experiment is shown in Fig. S5 . The elution volumes for individual MRPP1 ΔMTS (from Fig. 3 A ), MRPP2 (from Fig. 3 A ), and MRPP3 ΔMTS (from Fig. S4 F ) proteins, as well as the mixture of MRPP1 ΔMTS and MRPP2 (from Fig. 3 A ), are indicated with arrows above the chromatogram ( blue , orange , red , and pink , respectively). Bottom , SDS-PAGE ( first two gel panels ) and urea-PAGE ( last two gel panels ) analysis of eluted fractions to visualize proteins and pre-(mt)tRNA Ile , respectively. The black dashed box indicates lanes where the complex containing MRPP1 ΔMTS , MRPP2, MRPP3, and pre-(mt)tRNA Ile would be found on the SDS-PAGE. C , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up, for mixtures of MRPP2 and MRPP3 ΔMTS with different MRPP1 truncated proteins (MRPP1 ΔMTS , MRPP1 MT+C(Δ202) , MRPP1 N ). All lanes shown are taken from one experimental gel only. A downward shift in band location of processed (mt)tRNA Ile relative to that of pre-(mt)tRNA Ile and the concomitant appearance of a band corresponding to the removed 5′-leader indicate RNase P activity. D , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up for the complex of MRPP1 ΔMTS and MRPP2 without MRPP3 ( lane 2 ) and in the presence of different truncated MRPP3 proteins ( lanes 3–6 ). A negative control of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS mixed with EDTA ( lane 1 ) is included. M r RNA markers are shown ( lane M ). All lanes shown are taken from one experimental gel only. Reactions in C and D were run for 30 min at protein concentrations of 300 n m MRPP1–MRPP2 and 150 n m MRPP3.
Article Snippet: RNase P activity assay RNase P cleavage was performed by mixing 300 nm MRPP1–MRPP2, 150 nm MRPP3, 10 units of RNase inhibitors (RNasin from Promega), and 400 nm in vitro transcribed pre-(mt) tRNAIle in a buffer of 30 mm Tris-HCl, pH 8, 40 mm NaCl, 4.5 mm MgCl2 , and 2 mm DTT to a total reaction volume of 8.25 μl.
Techniques: Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Activity Assay, Negative Control