title: Effect of Mutations on the Binding of Kanamycin-B to RNA Hairpins Derived from the Mycobacterium tuberculosis Ribosomal A-Site.
abstract: Kanamycin is an aminoglycoside antibiotic used in the treatment of drug-resistant tuberculosis. Mutations at the rRNA A-site have been associated with kanamycin resistance in Mycobacterium tuberculosis clinical isolates. Understanding the effect of these mutations on the conformation of the M. tuberculosis A-site is critical for understanding the mechanisms of antibiotic resistance in M. tuberculosis. In this work, we have studied RNA hairpins derived from the M. tuberculosis A-site, the wild type and three mutants at the following positions (M. tuberculosis/Escherichia coli numbering): A1400/1408 → G, C1401/1409 → U, and the double mutant G1483/1491 C1401/1409 → UA. Specifically, we used circular dichroism, ultraviolet spectroscopy, and fluorescence spectroscopy to characterize the conformation, stability, and binding affinity of kanamycin-B and other aminoglycoside antibiotics for these RNA hairpins. Our results show that the mutations affect the conformation of the decoding site, with the mutations at position 1401/1409 resulting in significant destabilizations. Interestingly, the mutants bind paromomycin with weaker affinity than the wild type, but they bind kanamycin-B with similar affinity than the wild type. The results suggest that the presence of mutations does not prevent kanamycin-B from binding. Instead, kanamycin may promote different interactions with a third partner in the mutants compared to the wild type. Furthermore, our results with longer and shorter hairpins suggest that the region of the A-site that varies among organisms may have modulating effects on the binding and interactions of the A-site.
authors.affiliation: Department of Chemistry and ‡Molecular Biology, Biochemistry and Bioinformatics Program, Towson University , 8000 York Road, Towson, Maryland 21252, United States.
authors.forename: Amber R
authors.initials: AR
authors.lastname: Truitt
authors.affiliation: Department of Chemistry and ‡Molecular Biology, Biochemistry and Bioinformatics Program, Towson University , 8000 York Road, Towson, Maryland 21252, United States.
authors.forename: Bok-Eum
authors.initials: BE
authors.lastname: Choi
authors.affiliation: Department of Chemistry and ‡Molecular Biology, Biochemistry and Bioinformatics Program, Towson University , 8000 York Road, Towson, Maryland 21252, United States.
authors.forename: Jenny
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Chemistry and ‡Molecular Biology, Biochemistry and Bioinformatics Program, Towson University , 8000 York Road, Towson, Maryland 21252, United States.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Soto
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry*; D002942:Circular Dichroism; D007612:Kanamycin / Q000031:analogs & derivatives* / Q000737:chemistry; D015394:Molecular Structure; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; C001495:bekanamycin; D007612:Kanamycin