Colca Jerry R, McDonald William G, Waldon Daniel J, Thomasco Lisa M, Gadwood Robert C, Lund Eric T, Cavey Gregory S, Mathews W Rodney, Adams Lonnie D, Cecil Eric T, Pearson James D, Bock Jeffrey H, Mott John E, Shinabarger Dean L, Xiong Liqun, Mankin Alexander S
Pharmacia Corporation, 301 Henrietta Street, Kalamazoo, MI 49001, USA.
J Biol Chem. 2003 Jun 13;278(24):21972-9. doi: 10.1074/jbc.M302109200. Epub 2003 Apr 10.
Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.
恶唑烷酮类抗生素是一类重要的新型合成抗菌药物，通过干扰核糖体功能来抑制蛋白质合成。恶唑烷酮作用的确切位点和机制尚未阐明。尽管遗传学数据表明核糖体肽基转移酶是药物作用的主要位点，但一些体外生化研究表明其与核糖体的不同区域相互作用。这些体内和体外获得的不一致观察结果使得对恶唑烷酮作用的理解变得复杂。为了在活细胞中定位恶唑烷酮的作用位点，我们将一种光活性药物类似物与完整的、活跃生长的金黄色葡萄球菌中的靶点进行了交联。恶唑烷酮特异性地交联到23S rRNA、tRNA和两种多肽上。与23S rRNA的交联位点被定位到普遍保守的A-2602。交联的多肽是核糖体蛋白L27，其N端可能到达肽基转移酶中心，以及LepA，一种与翻译因子同源的蛋白质。只有与核糖体相关的LepA，而不是游离蛋白，被交联，这表明LepA是被与核糖体结合的抗生素交联的。证据表明，在肽基转移酶中心紧邻区域的正在翻译的核糖体中形成了一个特定的恶唑烷酮结合位点。
