Nucleic Acids Research, 2005, Vol. 33, No. 5 1621 Figure 1. Structural and functional characteristics of reconstituted 50S subunits containing circularly permuted (cp) 23S rRNA (gapped-cp-reconstitution). (A) Schematic representation of the secondary structure of the cp-23S rRNA. The natural 50 and 30 ends of 23S rRNA, which form helix 1 (H1) in the native 50S subunit, were covalently connected (bold black line) and new endpoints at positions 2468 and 2440 (50, 30) in helices 89 (H89) and 74 (H74) have been introduced to generate the cp-23S rRNA used in this study. The sequence of helix 1 of the cp-23S rRNA is shown whereas the natural ends of native 23S rRNA are indicated by numbers. The additional nucleotides that were added during clone construction are in bold lower case letters. The chemically synthesized 26 nt long RNA fragment, which compensates for the missing 23S rRNA segment in the peptidyl transferase center (boxed), is shown in red. (B) The peptidyl transferase activity of ribosomes composed of gapped-cp-reconstituted 50S subunits and native T.aquaticus 30S subunits. Product yields in the absence of the compensating 26mer ( ) or in the presence of fragments containing the wild-type sequence (wt; red bar) or carrying base changes (C, G, U; blue bars) at the position corresponding to A2451 of 23S rRNA are indicated. Yield of product formation of 50S carrying the wild-type sequence in the presence of 200 mM sparsomycin (+ Spa) are shown. In all cases, the mean and the SD values of the fraction of f[3H]Met-tRNA that reacted with puromycin after 240 min of incubation are shown. considerable peptidyl transferase activity and at the endpoint of the reaction (240 min) 30% of peptidyl-tRNA was con- verted into the reaction product, f Met-puromycin. Control experiments with excess of deacylated tRNA added simultan- eously with puromycin demonstrated that the peptidyl transfer reaction catalyzed by gapped-cp-reconstituted ribosomes is a single turnover event (data not shown). Therefore, the incom- plete conversion of the substrate indicates that only a fraction of the reconstituted ribosomes was catalytically active. Nev- ertheless, in these ribosomes, product formation could be read- ily inhibited by the peptidyl transferase inhibitor sparsomycin (Figure 1B). This reveals the general integrity of the PTC and suggests that the short RNA fragment probably binds to its natural site within the PTC restoring its functionally competent structure. Importantly, it was necessary for the supplementing RNA fragment to be present from the very beginning of the reconstitution procedure. Addition of the RNA fragment at a later point did not yield active 50S particles (data not shown). Ribosomes composed of the cp-23S rRNA missing the 26 residues long RNA fragment of the PTC showed essentially no peptidyl transfer activity (Figure 1B). ‘Natural’ mutations at A2451 do not eliminate peptidyl transferase activity Previous studies showed that mutations at A2451 only mod- erately affect peptidyl transferase activity (6,7). In accordance with these ﬁndings, ribosomes carrying the 26mer with all three base changes at 2451 showed <3-fold reduced product yields after 240 min of incubation (Figure 1B). It, therefore,