el-Baradi T T, de Regt V C, Einerhand S W, Teixido J, Planta R J, Ballesta J P, Raué H A
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands.
J Mol Biol. 1987 Jun 20;195(4):909-17. doi: 10.1016/0022-2836(87)90494-3.
The heterologous interaction of Escherichia coli ribosomal protein EL11 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the EL11 binding site present in these eukaryotic rRNAs. The fragments were obtained by SP6 polymerase-directed in-vitro run-off transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter. EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T1. In both cases, the position of this fragment in the L-rRNA sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E. coli 23 S rRNA. Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay. A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44', L44 and L45 protects exactly the same oligonucleotide fragment as does EL11 in both the yeast and mouse transcripts. Evidence is provided that L15, which is known to be structurally and functionally equivalent to EL11, is the rRNA-binding protein in this preparation. Thus the structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.
通过分析大肠杆菌核糖体蛋白EL11与各种合成rRNA片段形成特定复合物的能力,研究了该蛋白与酵母26S和小鼠28S rRNA的异源相互作用。这些合成rRNA片段跨越了这些真核rRNA中与EL11结合位点结构等效的区域。这些片段是通过SP6聚合酶指导的体外径流转录获得的,转录的是克隆在SP6启动子后面的酵母或小鼠大rRNA基因的部分片段。研究发现,EL11能保护酵母和小鼠转录本中一个63个核苷酸的寡核苷酸片段不被RNase T1消化。在这两种情况下,该片段在L-rRNA序列中的位置几乎与之前在大肠杆菌23S rRNA中发现的被EL11保护的片段位置完全一致。此外,通过硝酸纤维素滤膜结合试验表明,受保护的酵母片段能够重新与EL11结合。来自酿酒酵母的核糖体蛋白制剂,其中含有L15(YL23)以及酸性蛋白L44'、L44和L45,在酵母和小鼠转录本中保护的寡核苷酸片段与EL11完全相同。有证据表明,已知在结构和功能上与EL11等效的L15是该制剂中的rRNA结合蛋白。因此,酵母26S rRNA中存在的EL11结合位点的结构等效物构成了原核生物和真核生物rRNA上核糖体蛋白结合位点功能保守的第二个例子。