1622 Nucleic Acids Research, 2005, Vol. 33, No. 5 appears that the experimental system used here recapitulates at the qualitative level the functional activity of the PTC as seen previously with reconstituted mutant ribosomes that contained full-length 23S rRNA. Modifications at the A2451 nucleobase have minor effects on peptide bond formation The possibility to introduce non-natural nucleotide analogs into a synthetic RNA oligonucleotide allowed us to study the effects of ﬁne changes at the nucleobase at position 2451 on peptidyl transfer activity. In total, we investigated eight different nucleoside analogs carrying base alterations. Modi- ﬁcations were introduced at the N1, C2, N3 or C6 position of adenine (Figure 2A). Particularly interesting was the 3-deaza A2451 where the nitrogen at position 3 was changed to a carbon, which renders this group unsuited for general acid/ base catalysis. In our assay, however, none of the introduced base modiﬁcations at A2451 had a severe impact on the initial rates of transpeptidation (Figure 2C). Neither do they affect signiﬁcantly the product yield at the endpoints (Figure 2B). Even attaching rather bulky groups to the base of A2451, such as N6,N6-dimethyl adenosine, had only minor consequences on the peptidyl transfer. Signiﬁcantly, even ribosomes in which the entire base was removed, thereby creating a ribose-abasic site at position 2451 of 23S rRNA, retained almost full peptide bond formation activity compared to gapped-cp-reconstituted 50S containing the wt A2451 (Figure 2B and C). These data suggest that neither the nature, nor the general presence of a nucleobase at position 2451 is critical to form a peptide bond. Removal of the A2451 ribose 20-hydroxyl inhibits peptide bond formation In addition to alterations in the nucleobase, the gapped- cp-reconstitution also allows to study transpeptidation in ribosomes that carry a chemically modiﬁed ribose. Compared to base modiﬁcations, alterations at the ribose at position 2451 more signiﬁcantly compromised peptide bond formation. Removing the 20-hydroxyl group of the abasic-site analog, thereby creating a deoxy-abasic site at 2451, almost com- pletely deprived the ribosome of peptidyl transferase activity. This was in clear contrast to ribosomes with the 2451 ribo- abasic site, which remained almost fully active (Figure 2B). The difference in measured peptidyl transferase rates between ribosomes containing the deoxy- and the ribo-abasic site at 2451 is at least 60-fold (Figure 2C). This is striking, given the fact that the chemical composition of the PTCs of these ribosomes differ only by a single oxygen atom. Introducing a deoxy adenosine nucleoside at position 2451, thereby adding back the entire adenine base to the deoxy-abasic analog, recovered some peptidyl transferase activity though it remained lower than with any of the tested ribo-nucleobase analogs (Figures 2 and 3A). This indicates that the ribose 20-hydroxyl at A2451 is not only in close proximity to the active site (11), but also hints at its potential functional import- ance for transpeptidation. In view of this result, it was not surprising that placing a C3-linker at A2451, where most of the ribose was deleted and three carbon atoms function as a placeholder for the nucleotide backbone, was not compatible with an active PTC (Figure 2). To investigate if the reduced activities seen with 20- deoxyribose-modiﬁed ribosomes are the result of a less efﬁcient P-site tRNA binding, we increased the peptidyl- tRNA concentration 10-fold. This change in peptidyl-tRNA concentration did not markedly affect the yield of product formation or the relative rates of the reaction catalyzed by the 2451 20-deoxyribose-modiﬁed ribosomes (data not shown) thus ruling out tRNA binding effects. It has been shown recently that the minimal A-site substrate puromycin may behave differently than aminoacyl-tRNA in peptide bond formation employing mutant ribosomes (10). In this study, base changes at active site nucleotides, including A2451, resulted in strong defects in catalysis with puromycin while essentially no effects were seen with aminoacyl-tRNA as acceptor. This indicates that puromycin is hypersensitive to base changes in the PTC. In our system however, we do not see strong effects on transpeptidation with any of the tested modiﬁed-bases at A2451 in the puromycin assay. To invest- igate whether the reduced peptidyl transferase activities of the 2451 20-deoxyribose-modiﬁed ribosomes (Figures 2 and 3) also result from a potential puromycin hypersensitivity, we re-tested the reactivity with a more natural A-site substrate. We used the donor N-acetyl-Phe-tRNA in combina- tion with [32P]pCpCp-puromycin, both present in excess to reconstituted ribosomes. pCpCp-puromycin mimics better than puromycin, the universally conserved CCA end of aminoacyl-tRNA with the cytidine residues making additional contacts with the PTC (1,11,22,23). This results in increased binding and acceptor activities of pCpCp-puromycin com- pared to puromycin (24). In spite of the extending contacts of this acceptor substrate with the ribosome, both 2451 20-deoxyribose-substituted ribosomes showed a decrease in peptidyl transferase activity similar to what we observed pre- viously in the standard puromycin assay (Figure 4). These results show that the reduced transpeptidation activities seen with ribosomes containing 20-deoxyribose modiﬁcations at position 2451 were apparent in two different peptidyl trans- ferase assays, and did not appear to critically depend on the nature of the A-site substrate used. Though the results of chemical engineering of A2451 pointed at the importance of the ribose 20-hydroxyl for peptide bond synthesis, the reduced activities of the modiﬁed 50S subunits could potentially stem from a less efﬁcient in vitro assembly. Sucrose gradient proﬁles did not reveal signiﬁcant differences in the overall reconstitution efﬁciency between 50S containing the wt A2451, the 20-deoxy-A, or the deoxy-abasic analog in the PTC (Figure 3B). The ribosomal PTC catalyzes two major reactions, namely peptide bond formation and peptide release. Importantly, ribosomes containing the 20-deoxy-A or the deoxy-abasic site analog at position 2451, substitutions that showed strong defects in peptide bond formation, had almost unimpaired peptide release activities. Both types of ribose 20-deoxy-modiﬁed ribosomes reached essentially the same endpoint of product formation compared to ribosomes with ribo-A at position 2451 (Table 1). Since both transpeptidation and peptide release are single turnover reactions under the applied conditions (see Materials and Methods), these ﬁndings indicate that comparable fractions of wild-type or modiﬁed gapped- cp-reconstituted 50S subunits have a functionally competent PTC. This re-emphasizes the notion that the negative effects