Grant P G, Olson H M, Glitz D G, Cooperman B S
J Biol Chem. 1983 Sep 25;258(18):11305-12.
Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.
在氯霉素存在的条件下，每摩尔核糖体掺入超过1摩尔抗生素，用[³H]嘌呤霉素对来自大肠杆菌TPR 201菌株的小（30 S）核糖体亚基进行光亲和标记。然后用3 M NH₄Cl洗涤亚基，得到核心颗粒和一个分裂蛋白组分；分裂蛋白再用硫酸铵进一步分级分离。接着，使用来自光亲和修饰亚基的一个组分（核心、分裂蛋白或硫酸铵上清液）和未修饰（对照）亚基的其他组分来重构亚基。通过一维聚丙烯酰胺凝胶电泳监测[³H]嘌呤霉素在核糖体蛋白中的分布，并通过免疫电子显微镜观察嘌呤霉素结合位点。确定了两个嘌呤霉素结合区域。一个高亲和力的嘌呤霉素位点位于亚基上部三分之一处，远离平台，与先前确定的主要位点相同（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）。在用高水平嘌呤霉素修饰的蛋白S14重构的亚基中，该位点的结合最大，而掺入未修饰的S14时结合会减少。由于主要位点的抗体结合百分比总是超过蛋白S14中嘌呤霉素标记的百分比，主要位点必定包含除S14之外 的其他组分。在亚基平台上发现了一个亲和力较低的次要嘌呤霉素位点。该位点在由嘌呤霉素修饰的核心颗粒重构的亚基中富集，可能包括蛋白S7。我们的结果证明了在重构的核糖体亚基中定位特异性修饰组分的可行性。
