1620 Nucleic Acids Research, 2005, Vol. 33, No. 5 Synthetic RNA oligonucleotides Non-modiﬁed 26 nt RNAs as well as those containing 20-deoxyadenosine, a deoxy-abasic site analog, inosine, purine, 2-aminopurine, 2,6-diaminopurine or the C3-linker were purchased from Dharmacon. The phosphoramidite building blocks of 1-methylinosine, N 6-methyladenosine, N 6,N 6-dimethyladenosine were synthesized following pub- lished procedures (16). The phosphoramidite of the ribose- abasic site analog was obtained from Glen Research. The phosphoramidite of 3-deazaadenosine was synthesized in eleven steps starting from inosine via a 5-amino-4- imidazolecarboxamide (AICA) riboside derivative. Details of the synthesis will be described elsewhere (K. Lang and R. Micura, manuscript in preparation). The modiﬁed building blocks were incorporated into RNA by solid-phase syn- thesis using the 20-O-[(triisopropylsilyl)oxy]methyl (TOM) methodology (17–19). Deprotection and puriﬁcation of the oligoribonucleotides was performed as described previously (20). All oligoribonucleotides were analyzed and conﬁrmed by MALDI-TOF mass spectrometry. Peptide bond formation For the puromycin reaction, 10 pmol of 70S ribosomes were programmed with 60 pmol synthetic mRNA AAGGAG- AUAUAACAAUGUAAU (Dharmacon) containing a Shine– Dalgarno sequence (underlined) and a unique AUG codon (bold) and incubated with 0.3–3 pmol formyl-[3H]Met- tRNAfMet for 15 min at 37 C. The peptidyl transferase reaction was initiated by the addition of 2 mM puromycin and per- formed in 34 ml of the ﬁnal buffer containing 7 mM Tris–HCl, pH 7.5, 11.8 mM HEPES/KOH pH 7.5, 11.6 mM MgCl2, 167 mM NH4Cl, 2.5 mM spermidine, 0.03 mM spermine, 4.1 mM b-mercaptoethanol and 0.07 mM EDTA at 37 C for the indicated periods of time. Reaction products were extracted into ethyl acetate and quantiﬁed by liquid scintilla- tion counting as described in (12). In antibiotic inhibition experiments, 200 mM sparsomycin was added before puromy- cin addition. In all transpeptidation reactions, the background values (amount of product formed in samples containing only native T.aquaticus 30S subunits) were subtracted from all experimental data points. Under the applied conditions, peptide bond formation is a single turnover event. Chase experiments with deacylated tRNAPhe (in 33-fold excess over the donor N-acetyl-[H3]Phe-tRNA) added simultaneously with puromycin did not interfere with the measured reaction rates in poly(U) programmed 70S containing reconstituted 50S. However, addition of deacylated tRNAPhe during the N-acetyl-[H3]Phe-tRNA binding step completely inhibited transpeptidation (data not shown). The reaction with CC-puromycin was carried out with gapped-cp-reconstituted 50S assembled from 10 pmol cp-23S rRNA containing the wt RNA 26mer or its deoxy-adenosine or deoxy-abasic 2451 analogs. The assay employed 3 pmol of N-acetyl-Phe-tRNA and 4 pmol [32P]-end-labeled pCpCp- puromycin (Dharmacon), and was performed as described in (6). The radioactive species were separated by gel electro- phoresis and quantiﬁed using the Molecular Dynamics Storm PhosphorImager. Sucrose gradient analysis Gapped-cp-reconstituted 50S subunits assembled from 40 pmol cp-23S rRNA and the 26mer containing the wild- type sequence, dA or the deoxy-abasic analog at A2451 were layered over a 10–40% sucrose gradient prepared in recon- stitution buffer and centrifuged using a Beckmann SW-41 rotor as described (12). RF1-mediated peptidyl-tRNA hydrolysis Ten pmol 70S ribosomes containing gapped-cp-reconstituted 50S subunits were programmed with 250 pmol synthetic mRNA UUCAUGUAA (Dharmacon) and incubated with 0.3 pmol formyl-[3H]Met-tRNA for 15 min at 30 C in 17.9 ml of the P-site binding buffer described in (9). The peptidyl-tRNA hydrolysis reaction was initiated by the addi- tion of 15 pmol T.thermophilus RF1 (21) and performed at 25 C in a ﬁnal volume of 35.8 ml as described in (9). The incubation time, 30 min, corresponded to the endpoint of the reaction catalyzed by reconstituted wild-type ribosomes. The reaction was stopped and quantiﬁed by liquid scintillation counting as described in (9). Chase experiments with deacylated tRNAf Met (in 33-fold excess over f[3H]Met-tRNA) using 70S ribosomes contain- ing reconstituted 50S subunits revealed that peptidyl-tRNA hydrolysis is a single turnover reaction since the rate was not affected when excess of deacylated tRNAf Met was added simultaneously with RF1. In contrast, addition of excess deacylated tRNAf Met during f[3H]Met-tRNA binding com- peted efﬁciently with RF1-mediated f[3H]Met release (data not shown). RESULTS Use of circularly permuted 23S rRNA for 50S assembly In order to site-speciﬁcally position modiﬁed nucleosides within the 23S rRNA, we developed a ‘gapped-cp- reconstitution’ approach which expands the potential of the previously described in vitro reconstitution system of T.aquaticus 50S subunits (12). As the major rRNA component for the in vitro assembly, we employed a circularly permuted 23S rRNA (cp-23S rRNA) whose natural 50 and 30 ends are covalently connected and new ends have been introduced elsewhere in the molecule. For chemical engineering of the PTC, a cp-23S rRNA was designed so that its 50 end corres- ponds to position 2468 (E.coli nomenclature is used here and throughout the manuscript) and its 30 end to 2440 (Figure 1A). This cp-23S rRNA places a gap of 26 nt in the PTC, including the conspicuous residue A2451. The missing 23S rRNA seg- ment was chemically synthesized and used in combination with the cp-23S rRNA to assemble large ribosomal subunits in vitro. The reconstituted 50S subunits were reassociated with native 30S subunits from T.aquaticus and tested in a peptidyl transferase assay. Limiting amount of peptidyl-tRNA analog formyl-Met-tRNA was bound to the P-site and the reaction was initiated by the addition of saturating amounts of puromycin as an acceptor. Subunits assembled from cp-23S rRNA and the compensating RNA oligonucleotide showed