Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe

The protein subunit of Escherichia coli ribonuclease P (which has a cystein e residue at position 113) and its single cysteine-substituted mutant deriv atives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a s ulfhydryl-specific iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide (E PD-Fe). This reaction converts C5 protein, or its single cysteine-substitut ed mutant derivatives, into chemical nucleases which are capable of cleavin g the cognate RNA Ligand, M1 RNA, the catalytic RNA subunit of E. coli RNas e P, in the presence of ascorbate and hydrogen peroxide. Cleavages in M1 RN A are expected to occur at positions proximal to the site of contact betwee n the modified residue (in C5 protein) and the ribose units in M1 RNA. When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-me diated cleavages occurred predominantly in the P3 helix of M1 RNA present i n the reconstituted holoenzyme. C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C 5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot. However, hydr oxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA -Fe, perhaps indicative of Cys113 being distal from the RNA-protein interfa ce in the RNase P holoenzyme. Our directed hydroxyl radical-mediated footpr inting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide st ructural information essential for understanding the assembly of RNase P. ( C) 2000 Academic Press.