Effect of antibiotics on large ribosomal subunit assembly reveals possiblefunction of 5 S rRNA

Functional large ribosomal subunits of Thermus aquaticus can be reconstitut ed from ribosomal proteins and either natural or in vitro transcribed 23 S and 5 S rRNA. Omission of 5 S rRNA during subunit reconstitution results in dramatic decrease of the peptidyl transferase activity of the assembled su bunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstit ution can partly restore the activity of ribosomal subunits assembled witho ut 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most activ e: activity of the subunits assembled in the absence of 5 S rRNA was increa sed more than 30-fold if antibiotic was present during reconstitution proce dure. Activity of the subunits assembled with 5 S rRNA was also slightly st imulated by RU69874,but to a much lesser extent, approximately 1.5-fold. Ac tivity of the native T. aquaticus 50 S subunits incubated in the reconstitu tion conditions in the presence of RU69874 was, in contrast, slightly decre ased. The presence of antibiotics was essential during the last incubation step of the in vitro assembly, indicating that drugs affect one of the last assembly steps. The 5 S rRNA was previously shown to form contacts with segments of domains II and V of 23 S rRNA. All the antibiotics which can functionally compensa te for the lack of 5 S rRNA during subunit reconstitution interact simultan eously with the central loop in domain V (which is known to be a component of peptidyl transferase center) and a loop of the helix 35 in domain II of 23 S rRNA. It is proposed that simultaneous interaction of 5 S rRNA or of a ntibiotics with the two domains of 23 S rRNA is essential for the successfu l assembly of ribosomal peptidyl transferase center. Consequently, one of t he functions of 5 S rRNA in the ribosome can be that of assisting the assem bly of ribosomal peptidyl transferase by correctly positioning functionally important segments of domains II and V of 23 S rRNA. (C) 1999 Academic Pre ss.