Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.
Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.
复制标题
含有嘌呤霉素修饰的 L23 的大肠杆菌 50S 核糖体亚基的重建：功能后果。
DOI：
10.1021/bi00466a006
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Cooperman,BS
中科院分区：
文献类型：
--
作者：
Weitzmann,CJ;Cooperman,BS
宾夕法尼亚大学化学系，费城，宾夕法尼亚州 19104 1989 年 7 月 28 日收稿；修订稿 1989 年 11 月 29 日收稿 摘要；在之前的工作中，我们已经证明嘌呤霉素 [Weitzmann, CJ, & Cooperman, B. S.(1985) Biochemistry 24, 2268-2274] 和对叠氮嘌呤霉素 [Nicholson, AW, Hall, C. C., Strycharz, W. A., & Cooperman, B. S.(1982) Biochemistry 21, 3809-3817] 位点特异性光亲和标记蛋白 L23 的光亲和力是所有大肠杆菌核糖体蛋白中最高程度的。在这项工作中，我们证明了在 70S 核糖体中被嘌呤霉素 (嘌呤霉素-L23) 光亲和标记的 L23 可以通过反相高效液相色谱 (RP-HPLC) 与未修饰的 L23 分离，并且进一步证明，当将未修饰的 L23 添加到含有未修饰的其他 50S 成分的重构混合物中时，嘌呤霉素-L23 可以重构为 50S 亚基。形式。我们已实现每个重组 50S 亚基最多掺入 0.5 个嘌呤霉素-L23。与含有未修饰的L23或缺乏L23的重建的50S亚基相比，含有0.4-0.5嘌呤霉素-L23的重建的50S亚基保留了几乎所有（尽管较低）肽基转移酶活性，但仅保留了50-60%的mRNA依赖性tRNA结合刺激活性。我们得出的结论是，虽然 L23 并不直接位于肽基转移酶中心，但距离足够近，嘌呤霉素-L23 可以干扰 tRNA 结合。这一结论与将 L23 放置在肽基转移酶中心附近的许多其他实验一致，但很难与将 L23 放置在远离 30S 亚基一侧的 50S 亚基底部附近的免疫电子显微镜结果一致 [Hackl, W., & Stoffler-Meilicke, M.(1988) Eur. J.生物化学。 174, 431-435]，大肠杆菌核糖体是广泛研究的主题，旨在构建结构功能图谱，其中特定蛋白质和 RNA 区域位于核糖体结构内，并在蛋白质合成的整个过程中指定特定角色[有关最近的优秀文章集，请参阅 Hardesty 和 Kramer (1986) 以及 Noller 和 Moldave (1988)]。光亲和标记以其固有的能力来定义配体结合位点的组成部分，为实现这一目标做出了重要贡献（Cooperman，1987，1988）。然而，明确证明特定核糖体成分的标记发生在功能上存在固有的困难。
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received July 28, 1989; Revised Manuscript Received November 29, 1989 abstract; In previous work we have shown that both puromycin [Weitzmann, CJ, & Cooperman, B. S.(1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, AW, Hall, C. C., Strycharz, W. A., & Cooperman, B. S.(1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichiacoli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome bypuromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when addedin place of unmodified L23 to a reconstitution mixture containing the other 50S componentsin unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunitseither containing unmodified L23 or lacking L23, reconstituted 50S subunitscontaining 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopyresults placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & Stoffler-Meilicke, M.(1988) Eur. J. Biochem. 174, 431-435], The Escherichia coli ribosome is the subject of extensive studies directed toward the goal of constructing a structurefunction map in which specific proteinsand RNA regions are located within the ribosome structure and assigned specific roles in the overall process of protein synthesis [for excellent recent collections of articles, see Hardesty and Kramer (1986) and Noller and Moldave (1988)]. Photoaffinity labeling, with its intrinsic capability of defining the components of ligand binding sites, has contributed importantly toward this goal (Cooperman, 1987, 1988). However, the inherent difficulty of unambiguously demonstrating that labeling of a particular ribosomal component has taken place at a functionally im-