Olson H M, Grant P G, Cooperman B S, Glitz D G
J Biol Chem. 1982 Mar 10;257(5):2649-56.
Ribosomes from Escherichia coli strain Q13 have been photoaffinity labeled with [3H]puromycin in the presence of tetracycline. Puromycin-modified 50 S subunits appear to be identical with untreated subunits in electron micrographs and are precipitated by antibodies to the N6,N6'dimethyladenosine moiety of puromycin. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of subunits linked by an IgG molecule. Two regions of puromycin binding have been identified. The primary area, seen in 76% of the ribosome monomer complexes and 93% of the antibody-linked dimers, is beside (or on) the small central protuberance and on the side opposite the L7/L12 arm. A secondary area, maximally distant from the central protuberance, is seen in 22% of the monomeric complexes but only 7% of the antibody-linked dimers. In conjunction with our earlier localization of puromycin binding on the 30 S subunit (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894), we now define a puromycin-binding neighborhood of the 70 S ribosome. In addition to providing evidence for the localization of the peptidyl transferase center within the 50 S subunit, our results contribute to the formulation of a model for tRNA binding to both 30 S subunits and 70 S ribosomes.
来自大肠杆菌Q13菌株的核糖体已在四环素存在的情况下用[3H]嘌呤霉素进行了光亲和标记。在电子显微镜下，嘌呤霉素修饰的50 S亚基似乎与未处理的亚基相同，并且能被针对嘌呤霉素N6,N6'-二甲基腺苷部分的抗体沉淀。亚基-抗体复合物的电子显微镜图像显示，单个抗体分子结合在核糖体亚基上，并且IgG分子将亚基对连接起来。已确定了嘌呤霉素结合的两个区域。主要区域见于76%的核糖体单体复合物和93%的抗体连接的二聚体中，位于小的中央突起旁边（或之上）以及与L7/L12臂相对的一侧。次要区域距离中央突起最远，见于22%的单体复合物中，但仅见于7%的抗体连接的二聚体中。结合我们之前关于嘌呤霉素在30 S亚基上结合位置的定位研究（奥尔森，H.M.，格兰特，P.G.，格利茨，D.G.，和库珀曼，B.S.（1980年）《美国国家科学院院刊》77，890 - 894），我们现在定义了70 S核糖体的嘌呤霉素结合邻域。除了为肽基转移酶中心在50 S亚基内的定位提供证据外，我们的结果有助于构建tRNA与30 S亚基和70 S核糖体结合的模型。
