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). Mesotetrakis(N-methyl-pyridyl)porphine followed linear competitive kinetics with pre-tRNA(Gly1) from E. coli as variable substrate (K-i 0.960 mu M). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA(Gly1) from E. coli (K-i 1.90 mu M). Inhibition by meso-tetrakis[4(trimethylammonio)phenyl]porphine versus pre-tRNA(Gly1) from E. coli followed non-competitive kinetics (K-i 4.1 mu M). 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 (mu M) 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 mesotetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong pi-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 pretRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M I RNA during catalysis, and part from the porphyrin binding to the M I RNA component. (C) 2005 Elsevier B.V. All rights reserved.