In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function

In vitro transcripts of Escherichia coli 23S rRNA are compromised severely (at least five orders of magnitude below natural 23S rRNA) in their ability to reconstitute into catalytically active, correctly assembled 50S subunits in a standard reconstitution procedure. Denaturation experiments suggest that this deficiency is the result of missing posttranscriptional modifications present in natural 23S rRNA. An in vitro complementation analysis was performed where partial natural 23S rRNA fragments prepared by RNase H digestion or hammerhead ribozyme cleavage were combined with the remaining RNA as a partial in vitro transcript in a standard reconstitution reaction and the peptidyl transferase activity was measured. This approach has identified a ca. 80-nt region in 23S rRNA (extending from position 2445 to 2523) containing the natural RNA element essential for E. coli 50S subunit assembly and has excluded the requirement for all but six of the known posttranscriptional modifications in 23S rRNA for 50S subunit assembly or peptidyl transferase activity. Importantly, this chimeric reconstitution approach provides a system for the analysis of pure mutant populations of 23S rRNA reconstituted into 50S subunits.