Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro transcribed rRNA

Functionally active large ribosomal subunits of thermophilic bacterium Ther mus aquaticus have been assembled in vitro from ribosomal proteins and eith er natural or in vitro-transcribed 23S rRNA and 5S TRNA, Sedimentation prop erties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibi ted high activity in the peptidyl transferase assay and in a poly(U)-depend ent cell-free translation system (22 and 30%, respectively, compared to tha t of native 50S subunits), Catalytic activity of reconstituted subunits cri tically 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 c oli), which interferes with binding of peptidyl-tRNA in the ribosomal P-sit e, showed drastically reduced peptidyl transferase activity, whereas clinda mycin resistance mutation A2084G (A2058G in E. coli) rendered assembled sub units tolerant to clindamycin inhibition. Thus, reconstitution of functiona l subunits with in vitro-transcribed rRNA makes possible the use of in vitr o genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from t he rRNA transcript lacking any posttranscriptional modifications clearly de monstrates that modified nucleotides in 23S rRNA are dispensable for the pr incipal activities of the ribosome.