Spickler C, Brunelle M N, Brakier-Gingras L
Département de Biochimie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada.
J Mol Biol. 1997 Oct 31;273(3):586-99. doi: 10.1006/jmbi.1997.1323.
Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.
链霉素是一种可诱导错误的氨基糖苷类抗生素，它与细菌小核糖体亚基上的一个位点结合，但该位点尚未被精确界定。在此，我们证明在没有核糖体蛋白的情况下，链霉素能与大肠杆菌16S rRNA结合，并保护解码区域中的一组碱基免受硫酸二甲酯的攻击。结合研究是在含有20 mM Mg2+的高离子强度缓冲液中进行的。解码区域的保护模式与链霉素结合到30S亚基时观察到的模式相似。然而，链霉素还能保护30S亚基内16S rRNA的915区域，而它不能保护裸露的16S rRNA的915区域。通过使用对应于16S rRNA 3'小结构域和解码类似物（该结构域包含解码中心的一部分）的两个片段，进一步确定了链霉素与16S rRNA的相互作用。在链霉素存在的情况下，这两个片段对硫酸二甲酯攻击的保护模式与全长16S rRNA的相似。这表明3'小结构域以及解码类似物都包含链霉素结合的识别信号。然而，在没有Mg2+时，链霉素不能与解码类似物结合。这与新霉素（另一种可诱导错误的氨基糖苷类抗生素）形成对比，新霉素在没有Mg2+时能与解码类似物结合，但在20 mM Mg2+时不能。我们的结果表明，新霉素和链霉素都与解码中心相互作用，但识别该区域的不同构象。
