Effect of Mutations on the Binding of Kanamycin‑B to RNA Hairpins Derived from the Mycobacterium tuberculosis Ribosomal A‑Site

• Published in: Biochemistry (Easton) 2015-12, Vol.54 (51), p.7425-7437
• Main Authors: Truitt, Amber R, Choi, Bok-Eum, Li, Jenny, Soto, Ana Maria
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - chemistry; Circular Dichroism; Escherichia coli; Kanamycin - analogs & derivatives; Kanamycin - chemistry; Molecular Structure; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - genetics; Nucleic Acid Conformation; Ribosomes - chemistry; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.5b00710

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.