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 (Ki 0.960 microM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA(Gly1) from E. coli (Ki 1.90 microM). Inhibition by meso-tetrakis[4-(trimethylammonio)phenyl]porphine versus pre-tRNA(Gly1) from E. coli followed non-competitive kinetics (Ki 4.1 microM). The porphyrins bound directly to E. coli tRNAVal, E. coli pre-tRNAGly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (microM) against E. coli tRNAVal and E. coli pre-tRNAGly 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 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 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.
Hori
Yoshiaki
Y
Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, 441-8580, Japan.
Rogert
Maria C
MC
Tanaka
Terumichi
T
Kikuchi
Yo
Y
Bichenkova
Elena V
EV
Wilton
Amanda N
AN
Gbaj
Abdul
A
Douglas
Kenneth T
KT
D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D007700:Kinetics; D011166:Porphyrins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D011166:Porphyrins; D012322:RNA Precursors; D012343:RNA, Transfer; D043262:Ribonuclease P; C470865:ribonuclease P, E coli