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Antibiotic inhibition of catalytic RNA function

University of British Columbia

A number of compounds inhibit group I intron splicing. Competitive inhibitors include deoxyguanosine, dideoxyguanosine, arginine and streptomycin; the non-competitive inhibitors include members of the aminoglycoside family of antibiotics. Further screening of a collection of antibiotics for their ability to inhibit group I intron splicing identified several novel compounds. In particular, the pseudodisaccharide antibiotic lysinomicin, the peptide antibiotics netropsin and distamycin, and the tetracycline analog chelocardin, were found to inhibit group I intron splicing at concentrations of 250 μM or lower. Inhibition of group I intron splicing by pseudodisaccharide antibiotics was studied in detail. Lysinomicin and three closely related compounds were found to inhibit the self splicing reaction of the Tetrahymena, Bacillus phage SP01 and T4 phage td and sun Y group I introns at concentrations less than 50 μM. Lysinomicin competitively inhibited sunY intron splicing with a Ki of 8.5 μM (+/- 5 μM). The pseudodisaccharides were also shown to interact at the A-site on the ribosome, as Escherichia coli strains resistant to neomycin, which binds to the ribosomal Asite, were also resistant to the pseudodisaccharides. To further examine antibiotic/ribozyme interactions, antibiotic inhibition of a second ribozyme system, the human hepatitis delta virus (HDV) ribozyme, was examined. The small size (150 nucleotides) of this ribozyme and the fact that it lacks a guanosine binding site (the proposed site of interaction of inhibitors of group I intron splicing) made it a good candidate for detailed studies of antibiotic/ribozyme interactions. The antibiotics that have been shown to inhibit group I intron splicing were found to inhibit the HDV genomic and antigenomic ribozymes. Kinetic analysis showed that neomycin competes with magnesium binding to the ribozyme with a Ki of 28 μM (+/- 10 μM). Lead cleavage also suggested that neomycin inhibits the self-cleavage reaction of the HDV ribozyme by competing with divalent cation binding. Footprint analysis also supported this hypothesis as neomycin binds HDV RNA near the cleavage site. I propose that the binding of neomycin to several different RNAs (Rev Responsive Element, 16S rRNA, and the hammerhead, group I intron and HDV ribozymes) may be due to neomycin recognition of divalent cation binding site(s) in these RNAs.

Thesis/Dissertation

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2009-03-17

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http://hdl.handle.net/2429/6141

Microbiology and Immunology

University of British Columbia

UBCV

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