Effect of antibiotics on large ribosomal subunit assembly reveals possible function of 5 S rRNA.
Functional large ribosomal subunits of Thermus aquaticus can be reconstituted 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 subunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstitution can partly restore the activity of ribosomal subunits assembled without 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most active: activity of the subunits assembled in the absence of 5 S rRNA was increased more than 30-fold if antibiotic was present during reconstitution procedure. Activity of the subunits assembled with 5 S rRNA was also slightly stimulated by RU69874, but to a much lesser extent, approximately 1.5-fold. Activity of the native T. aquaticus 50 S subunits incubated in the reconstitution conditions in the presence of RU69874 was, in contrast, slightly decreased. 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 compensate for the lack of 5 S rRNA during subunit reconstitution interact simultaneously 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 antibiotics with the two domains of 23 S rRNA is essential for the successful assembly of ribosomal peptidyl transferase center. Consequently, one of the functions of 5 S rRNA in the ribosome can be that of assisting the assembly of ribosomal peptidyl transferase by correctly positioning functionally important segments of domains II and V of 23 S rRNA.
Khaitovich
P
P
Center for Pharmaceutical Biotechnology, University of Illinois, Chicago 60607, USA.
Mankin
A S
AS
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D004352:Drug Resistance, Microbial; D015180:Electrophoresis, Gel, Two-Dimensional; D018942:Macrolides / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D009690:Nucleic Acid Conformation; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D018942:Macrolides; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins; D010458:Peptidyl Transferases