Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro transcribed rRNA
Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro transcribed rRNA
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DOI：
10.1021/bi9822473
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发表时间：
1999-02-09
期刊：
影响因子：
2.9
通讯作者：
Mankin, AS
中科院分区：
文献类型：
--
作者：
Khaitovich, P;Tenson, T;Mankin, AS
利用天然或体外转录的23S rRNA和5S tRNA，利用核糖体蛋白在体外组装出具有功能活性的大核糖体亚基，重组亚基的沉降性与天然核糖体50S亚基相似。用体外转录的rRNA重组的亚基在肽基转移酶实验和依赖聚(U)的无细胞翻译系统中表现出很高的活性(分别比天然50S亚基的活性高22%和30%)，重组亚基的催化活性关键取决于5S rRNA的存在，已知的影响天然核糖体功能的rRNA突变在重组水蚤50S亚基中产生了类似的影响。在体外转录的含有G2267A突变的水曲霉菌23S rRNA(在大肠杆菌中为G2252a)组装的亚基，干扰了核糖体P-位点上的肽基-tRNA的结合，显示出显著的肽转移酶活性降低，而克林霉素抗性突变A2084G(在大肠杆菌中为A2058G)使组装的亚基对克林霉素抑制具有耐受性。因此，功能亚基与体外转录的rRNA的重组使利用体外遗传学对翻译中的23S rRNA功能进行突变分析成为可能。此外，从没有任何转录后修饰的rRNA转录本组装具有催化活性的50S亚基的能力清楚地表明，23S rRNA中的修饰核苷酸对于核糖体的主要活性是必不可少的。
Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S TRNA, Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits), Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA, rRNA mutations known to affect functions of The native ribosome produced similar effects in reconstituted T. aquaticus 50S subunits. Subunits assembled with in vitro-transcribed T. aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E. coli) rendered assembled subunits tolerant to clindamycin inhibition. Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome.