DISCUSSION In this study, we used circularly permuted 23S rRNA (cp-23S rRNA) for engineering the chemical make-up of the ribosomal PTC. The generation of the cp-23S rRNA is possible due to the proximity of the natural 50 and 30 ends of mature 23S rRNA in the tertiary structure of the ribosome. The 50 and 30 ends of natural 23S rRNA are base-paired to each other forming helix 1 of 23S rRNA (25). Helix 1 is located on the solvent side of the 50S ribosomal subunit, far from known functional centers. Therefore, covalent connection of the 23S rRNA ends is unlikely to cause major structural changes in the large ribosomal subunit. The ‘gapped-cp-reconstitution’ of the large ribosomal subunit used here for engineering the chemical environment of one of the key nucleotides in the PTC, A2451, employed a cp-23S rRNA that lacked a 26 nt long RNA fragment encom- passing A2451. The missing rRNA segment was chemically synthesized to either contain the wild-type sequence, natural mutations or non-natural nucleoside analogs at position A2451 of 23S rRNA. The synthetic RNA fragment was provided in trans to the in vitro reconstitution of 50S ribosomal sub- units. Base-pairing with the complementary sites of the cp-23S rRNA transcript locks the synthetic rRNA piece in its native place thus restoring the structure of 23S rRNA (Figure 1A). The main beneﬁt of using cp-23S rRNA in these experiments is that it allows the reconstitution of the large ribosomal sub- unit from two rather than three pieces of rRNA. Using three or more rRNA fragments markedly reduces the efﬁciency of the in vitro assembly procedure [(26), N. Polacek and A. Mankin, unpublished data, 2005]. The obvious advantage of the gapped-cp-reconstitution system is that it allows to incorporate non-natural nucleosides at desired sites within 23S rRNA. This technique signiﬁcantly expands the chemical repertoire of nucleotide residues that can be placed at speciﬁc 23S rRNA sites. In this work, we substituted functional groups of the nitro- gen base or the ribose of A2451 and tested the effects on the ability of the ribosome to catalyze peptide bond formation. Our major ﬁnding is that the removal of the ribose 20-hydroxyl group at A2451 reproducibly reduces the ribo- somes’ ability to promote peptide bond formation in a standard puromycin assay. Subsequent removal of the base, thereby creating a deoxy-abasic site, almost completely depleted the ribosome of its ability to catalyze peptidyl transfer (Figures 2B and 3A). Notably, placing a ribose-abasic instead of the deoxy- abasic site at 2451, thereby reintroducing a single hydroxyl group to the ribose 20 position, stimulates the reaction rate at least 60-fold, thereby restoring near wild-type levels of pep- tidyl transferase activity (Figure 2B). This indicates that the base at A2451 does not contribute signiﬁcantly to transpeptidation in our assay system. In support of this, all the tested base modiﬁcations at A2451 had relatively minor impact on the performance of the PTC (Figure 2). This was the case for placing bulky groups at the N1 (1-methyl inosine), the C2 (e.g. 2,6-diaminopurine) or the C6 position (e.g. N6,N6-dimethyl adenosine) as well as for changing an allegedly functional nitrogen atom at position 3 to a carbon (3- deaza adenosine). To conﬁrm these ﬁndings in a peptidyl transfer assay employing a more natural A-site substrate, we replaced puromycin by CC-puromycin. The two cytidine residues enable this acceptor substrate to make additional Nucleic Acids Research, 2005, Vol. 33, No. 5 1625 important hydrogen bonding interactions with the A-loop of the PTC (1,11,22,23) thus enhancing its resemblance to the natural acceptor substrate (10,24). Using this assay, the neg- ative effects of removing the ribose 20-hydroxyl group at A2451 remained apparent and similar rate reductions com- pared to the puromycin reaction were observed (Figure 4). Taken together, these results are compatible with the notion that this 20-hydroxyl is of functional or structural signiﬁcance for transpeptidation. Although the theoretical possibility remains that the observed differences in activity is the result of a less efﬁcient subunit reconstitution with the 20-deoxy- adenosine or the deoxy-abasic site analog at 2451, this scen- ario seems unlikely. Our observation that ‘wild-type’ or ‘modiﬁed’ ribosomes showed indistinguishable sucrose gradient proﬁles argue against major reconstitution defects (Figure 3B). This conclusion is further supported by the results of the peptide release assay where the deoxy-A2451 and the 2451 deoxy-abasic versions of the reconstituted ribosomes showed similar product yields at the reaction endpoint (Table 1) compared to the wild-type control. In a single turn- over reaction with incomplete conversion of the substrate into the product, the portion of the reacted substrate shows the active fraction of the enzyme. Therefore, the results of the release assay demonstrate that comparable fractions of cata- lytically active 50S subunits were assembled with the wild- type RNA 26mer, or its deoxy-A or deoxy-abasic versions. Our ﬁndings reveal a possible functional or structural importance of the 20-hydroxyl group of A2451 for peptide bond formation. This interpretation is compatible with recent in vivo data showing that snoRNA-guided methylation of the 20-hydroxyl group at A2451 resulted in a lethal growth pheno- type in yeast (27). In the crystallographic structure of the PTC, this hydroxyl group was found to be in hydrogen bonding distance to the reactive amino group of the A-site substrate (11). The ribose 20-hydroxyl of 2451 may therefore potentially help to orient the attacking amino group of the acceptor sub- strate. Earlier theoretical studies as well as more recent experi- mental work suggest that peptide bond formation is mainly driven by positioning of the two reactants, the a-amino group from the A-site aminoacyl-tRNA and the carbonyl carbon from the P-site peptidyl-tRNA (1,6–8,10,28,29). The role and contribution of chemical catalysis in the overall scheme of events that take place during the formation of a new peptide bond is less clear. Recently, a model was proposed in which the 20-hydroxyl of the terminal nucleotide of the P-tRNA is directly involved in substrate-assisted catalysis of the peptide bond (30). This model is compatible with the notion that the main role of the ribosome to promote efﬁcient peptide bond formation is to provide the molecular template where the two reactants can be properly aligned. The 20-hydroxyl of A2451 of 23S rRNA might be one of the critical groups to fulﬁll this task. The unperturbed pKa of the 20-hydroxyl of RNA is far from neutral, 13 (31). Therefore, its direct participation in general acid–base catalysis is unlikely. However, by analogy with the model proposed for the ribose 20-hydroxyl of the terminal A of P-tRNA, it may possibly serve as a hydrogen bond acceptor in the transition state intermediate (30,32,33) or be involved in metal ion coordination similar to catalytically important nucleotides in other ribozymes (34). We cannot exclude, however, that the removal of the 20-hydroxyl at A2451 may somehow disturb the functionally competent