van Beekvelt C A, Kooi E A, de Graaff-Vincent M, Riet J, Venema J, Raué H A
Department of Biochemistry and Molecular Biology, IMBW BioCentrum Amsterdam, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
J Mol Biol. 2000 Feb 11;296(1):7-17. doi: 10.1006/jmbi.1999.3432.
Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.
酿酒酵母25S rRNA的结构域III包含核糖体蛋白L25的主要rRNA结合识别位点，L25属于功能保守的核糖体蛋白EL23/L25家族。EL23/L25结合区域非常复杂，由几个不规则螺旋组成，这些螺旋通过长距离的二级和三级相互作用结合在一起。此外，它还包含真核生物特有的V9（D7a）扩展片段。通过详细的体外和体内突变分析对该位点的结构元件进行功能表征，结果表明存在两个直接参与L25结合的独立区域。特别是，螺旋49环中两个保守核苷酸中的任何一个发生突变，都会显著降低体外L25结合，从而有力地支持了它们作为核糖体蛋白附着位点的作用。另外两个螺旋似乎是结合位点正确折叠所必需的。消除L25体外结合的突变会阻止25S rRNA在体内的积累，因为它们会在其直接前体27S(B)前体rRNA水平上使前体rRNA加工停滞。令人惊讶的是，一些在体外不显著影响L25结合的突变在27S(B)前体rRNA加工中会导致相同的致死缺陷。V9扩展片段的缺失也会导致成熟25S rRNA积累不足和生长速率降低两倍。我们得出结论，完整的结构域III，包括V9扩展片段，对于25S rRNA的正常加工和组装至关重要。
