Sapag A, Vartikar J V, Draper D E
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
Biochim Biophys Acta. 1990 Aug 27;1050(1-3):34-7. doi: 10.1016/0167-4781(90)90137-q.
The ribosomal protein S4 from Escherichia coli is essential for initiation of assembly of 30S ribosomal subunits. We have undertaken the identification of specific features required in the 16S rRNA for S4 recognition by synthesizing mutants bearing deletions within a 460 nucleotide region which contains the minimum S4 binding site. We made a set of large nested deletions in a subdomain of the molecule, as well as individual deletions of nine hairpins, and used a nitrocellulose filter binding assay to calculate association constants. Some small hairpins can be eliminated with only minor effects on S4 recognition, while three hairpins scattered throughout the domain (76-90, 376-389 and 456-476) are essential for specific interaction. The loop sequence of hairpin 456-476 is important for S4 binding, and may be directly recognized by the protein. Some of the essential features are in phylogenetically variable regions; consistent with this, Mycoplasma capricolum rRNA is only weakly recognized by S4, and no specific binding to Xenopus laevis rRNA can be detected.
来自大肠杆菌的核糖体蛋白S4对于30S核糖体亚基的组装起始至关重要。我们通过合成在包含最小S4结合位点的460个核苷酸区域内带有缺失的突变体，来鉴定16S rRNA中S4识别所需的特定特征。我们在分子的一个亚结构域中制作了一组大的嵌套缺失，以及九个发夹的单个缺失，并使用硝酸纤维素滤膜结合试验来计算结合常数。一些小发夹可以被去除，对S4识别只有轻微影响，而分布在整个结构域中的三个发夹（76 - 90、376 - 389和456 - 476）对于特异性相互作用至关重要。发夹456 - 476的环序列对于S4结合很重要，可能被该蛋白质直接识别。一些基本特征位于系统发育可变区域；与此一致的是，山羊支原体rRNA仅被S4微弱识别，并且未检测到与非洲爪蟾rRNA的特异性结合。
