Porphyrins and porphines bind strongly and specifically to tRNA, precursor tRNA and to M1 RNA and inhibit the ribonuclease P ribozyme reaction

Porphyrins and porphines strongly inhibit the action of the RNA subunit of the Escherichia coli ribonuclease P (M1 RNA). Meso-tetrakis(N-methyl-pyridyl) porphine followed linear competitive kinetics with pre-tRNA^Gly1 from E. coli as variable substrate (K_i 0.960 μM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA^Gly1 from E. coli (K_i 1.90 μM). Inhibition by meso-tetrakis[4-(trimethylammonio) phenyl]porphine versus pre-tRNA^Gly1 from E. coli followed non-competitive kinetics (K_i 4.1 μM). The porphyrins bound directly to E. coli tRNA^Val, E. coli pre-tRNA^Gly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (μM) against E. coli tRNA^Val and E. coli pre-tRNA^Gly were: meso-tetrakis(N- methyl-pyridyl)porphine 1.21 and 0.170; meso-tetrakis[4-(trimethylammonio) phenyl]porphine, 0.107 and 0.293; protoporphyrin IX, 0.138 and 0.0819. For M1 RNA, dissociation constants were 32.8 nM for meso-tetrakis(N-methyl-pyridyl) porphine and 59.8 nM for meso-tetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong π-stacking of the porphine nucleus and base pairs in a rigid low-polarity environment. Part of the inhibition of ribonuclease P is from interaction with the pre-tRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M1 RNA during catalysis, and part from the porphyrin binding to the M1 RNA component.