AN ELECTRON-MICROSCOPIC METHOD FOR LOCALIZATION OF RIBOSOMAL-PROTEINS DURING TRANSCRIPTION OF RIBOSOMAL DNA - A METHOD FOR STUDYING PROTEIN ASSEMBLY
AN ELECTRON-MICROSCOPIC METHOD FOR LOCALIZATION OF RIBOSOMAL-PROTEINS DURING TRANSCRIPTION OF RIBOSOMAL DNA - A METHOD FOR STUDYING PROTEIN ASSEMBLY
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DOI：
10.1073/pnas.78.8.4823
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
LEIBY, KR
中科院分区：
文献类型：
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作者：
CHOOI, WY;LEIBY, KR
描述了一种核糖体蛋白在活性rRNA基因的EM铺展上的定位方法。该方法包括提高针对果蝇黑腹果蝇蛋白的抗体，并允许这些抗体与果蝇卵室的裂解产物发生反应。用电子致密的聚甲基丙烯酸酯微球标记的羊抗兔抗体检测结合的抗体在活性转录本中的位置。通过统计分析，为特定蛋白质的蛋白质组装的起始点产生一个置信度区间。S14蛋白的第1个组装点位于18S rRNA前5‘端附近。相反，L4的蛋白质组装的第一个点在距起始点0.38个单位(一个单位是核糖体转录单位的长度)。S14和L4的结合模式与Pre-18S和Pre-28S rRNA的5‘’近端和5‘远端取向一致。本文描述的方法为阐明真核核糖体蛋白在体内的组装提供了一种途径。
A method is described for the localization of ribosomal proteins on EM spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates of D. melanogaster egg chambers. The locations of the bound IgG in the active transcripts are detected with goat antirabbit IgG that were labeled with electron-dense polymethacrylate spheres. By statistical analysis a confidence interval is generated for the initial point of protein assembly for a particular protein. The 1st point of protein assembly for S14 is located near the 5'' end of the pre-18S rRNA. In contrast, the 1st point of protein assembly for L4 is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and L4 are consistent with the 5'' proximal and the 5'' distal orientations of the pre-18S and the pre-28S rRNA. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.