Truitt Amber R, Choi Bok-Eum, Li Jenny, Soto Ana Maria
Department of Chemistry and ‡Molecular Biology, Biochemistry and Bioinformatics Program, Towson University , 8000 York Road, Towson, Maryland 21252, United States.
Biochemistry. 2015 Dec 29;54(51):7425-37. doi: 10.1021/acs.biochem.5b00710. Epub 2015 Dec 17.
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.
卡那霉素是一种氨基糖苷类抗生素，用于治疗耐药性结核病。结核分枝杆菌临床分离株中rRNA A位点的突变与卡那霉素耐药性有关。了解这些突变对结核分枝杆菌A位点构象的影响对于理解结核分枝杆菌的抗生素耐药机制至关重要。在这项工作中，我们研究了源自结核分枝杆菌A位点的RNA发夹结构，包括野生型以及以下位置的三个突变体（结核分枝杆菌/大肠杆菌编号）：A1400/1408→G、C1401/1409→U以及双突变体G1483/1491 C1401/1409→UA。具体而言，我们使用圆二色性、紫外光谱和荧光光谱来表征卡那霉素-B和其他氨基糖苷类抗生素对这些RNA发夹结构的构象、稳定性和结合亲和力。我们的结果表明，这些突变会影响解码位点的构象，1401/1409位点的突变会导致显著的去稳定化。有趣地是，这些突变体与巴龙霉素的结合亲和力比野生型弱，但它们与卡那霉素-B的结合亲和力与野生型相似。结果表明，突变的存在并不妨碍卡那霉素-B的结合。相反，与野生型相比，卡那霉素在突变体中可能与第三个伙伴促进不同的相互作用。此外，我们对更长和更短发夹结构的研究结果表明，生物体之间A位点不同的区域可能对A位点的结合和相互作用具有调节作用。
