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首页> 外文期刊>Biochemistry >Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.
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Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.

机译：用体外转录的rRNA重建功能活跃的水生栖热菌大核糖体亚基。

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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 rRNA. 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.
机译：嗜热细菌水生栖热菌的功能活跃的大核糖体亚基已从核糖体蛋白，天然或体外转录的23S rRNA和5S rRNA体外组装而成。重建的亚基的沉积特性与天然核糖体50S亚基的相似。用体外转录的rRNA重组的亚基在肽基转移酶测定法和poly（U）依赖性无细胞翻译系统中表现出高活性（与天然50S亚基相比分别为22％和30％）。重建的亚基的催化活性主要取决于5S rRNA的存在。已知会影响天然核糖体功能的rRNA突变在重构的水生水母50S亚基中产生了相似的作用。与含有G2267A突变（在大肠杆菌中为G2252A）的体外转录的水生T. aquaticus 23S rRNA组装的亚基，其干扰肽核糖体P位点中的肽基tRNA结合，显示出肽基转移酶活性大大降低，而克林霉素抗性突变A2084G （在大肠杆菌中为A2058G）使组装的亚基耐受克林霉素抑制。因此，用体外转录的rRNA重建功能性亚基使得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。另外，从没有任何转录后修饰的rRNA转录物中组装催化活性的50S亚基的能力清楚地表明，23S rRNA中修饰的核苷酸对于核糖体的主要活性是可有可无的。

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