studies were similar on a qualitative level: ribosomes with base changes at A2451 retain signiﬁcant peptidyl transferase activity. These data lead to a notion that the contribution of chemical catalysis involving the A2451 base, or other nearby 23S rRNA residues, to the overall rate of peptide bond forma- tion is relatively small, leaving substrate orientation as the main source of catalytic power provided by the ribosome. While mutational analysis did not appear to support direct implication of the A2451 base in catalysis of peptide bond formation, it also could not explicitly rule it out. Neither of the mutations at A2451 would entirely eliminate the hydrogen bonding capacity of a nucleotide base at this position: replace- ments of A by G, C or U would put a hydrogen bond acceptor (N3 for G and O2 for C or U) in approximately the same place as the N3 of A (11). Therefore, the role of substrate alignment and/or activation played by the base of A2451 could poten- tially be achieved by any of the three other bases as well. In addition to N3 of A2451, two other groups, the 20-hydroxyl of A2451 and the 20-hydroxyl of the terminal adenosine of P-site bound peptidyl-tRNA, were seen within hydrogen bonding distance to the attacking a-amino group of the acceptor sub- strate. These groups could therefore promote peptide bond formation by orienting or activating the a-amino group for its attack onto the carbonyl carbon of the peptidyl-tRNA ester bond (1,11). The level of chemical engineering that can be achieved by conventional mutational studies of 23S rRNA is fairly limited because at any particular position, it is restricted to only three possible mutations that can be incorporated by RNA poly- merase. At the same time, the complexity and size of 23S rRNA renders many of the molecular tools successfully used to study smaller ribozymes inapplicable. In particular, it is extremely difﬁcult to modify the chemical nature of a speciﬁc residue within a chemically homogeneous RNA molecule containing thousands of nucleotides. To overcome this obstacle, we have re-engineered the in vitro reconstitution system of Thermus aquaticus large ribosomal subunits (12) to make possible the assembly of ribosomal subunits from a combination of in vitro transcribed and chemically synthes- ized rRNA fragments. The centerpiece of this approach is the use of circularly permuted 23S rRNA (cp-23S rRNA) as the major rRNA component for the in vitro assembly. This new approach, which we named ‘gapped-cp-reconstitution’, allows placing a wide range of chemically altered rRNA residues at desired positions within the large ribosomal subunit. Investigation of the performance of the chemically engineered peptidyl transferase enzyme revealed the importance of the ribose 20-hydroxyl group at position 2451 of 23S rRNA for promoting peptide bond formation. MATERIALS AND METHODS tRNA Formyl-[3H]Met-tRNA (45 000 c.p.m./pmol) was prepared by aminoacylation of E.coli tRNAfMet in a buffer containing 20 mM HEPES/KOH (pH 7.6), 8 mM MgAc2, 150 mM NH4Cl, 4 mM b-mercaptoethanol, 2 mM spermidine, 0.05 mM sper- mine as described in (13). N-acetyl-[3H]Phe-tRNA (1000 or 15 000 c.p.m./pmol) was prepared as described previously (14). Formyl-[3H]Met-tRNA and N-acetyl-[3H]Phe-tRNA Nucleic Acids Research, 2005, Vol. 33, No. 5 1619 were puriﬁed using a reverse phase C4 HPLC column (Vydac) in buffer containing 50 mM MgAc2 and 50 mM NH4Ac (pH 6.0) with a linear gradient from 0 to 25% ethanol. Construction of circularly permuted 23S rRNA The circularly permuted gene product used for in vitro transcription of the cp-23S rRNA was produced by PCR using a plasmid (pCPTaq23S) carrying tandemly repeated 23S rRNA genes from T.aquaticus. This plasmid was created by the subsequent cloning of two 23S rRNA gene copies into pBR322. For the ﬁrst copy, the T7 RNA promoter, which is present in the parental plasmid (pT7TA), containing the cloned 23S rRNA gene (12), was removed and a BamH1 site at the 50 end of the gene was introduced by PCR. This 23S rRNA gene was then cloned as a BamH1/Bsp119l fragment into pBR322. For the second copy of the 23S rRNA gene, the parental plasmid pT7TA was altered so that the Bsp119l site at the 30 end of the gene was removed by Bsp119l digestion and subsequent ﬁlling up of the overhangs by Klenow poly- merase. After blunt-end re-ligation, PCR on this plasmid was employed to remove the T7 RNA promoter and to concom- itantly create a new Bsp119l site at the 50 end of the gene. This second 23S rRNA gene construct was excised by Bsp119l/ EcoRI digestion and cloned into the pBR322 vector that already contained the ﬁrst copy of the 23S rRNA gene. The resulting plasmid contained two copies of the 23S rRNA gene that were ligated tail to head via the Bsp119l site thereby covalently connecting the 30 and 50 ends of the two genes. This plasmid was subsequently used as PCR template using the forward primer TAATACGACTCACTATAGGGAG- CGTCCACAGCGGCG, which contained the T7 promoter sequence (underlined), and the reverse primer GTAACTTT- TATCCGTTGA. Two additional Gs following the T7 pro- moter were introduced during PCR to ensure efﬁcient transcription initiation. The position corresponding to the new 50 end of the cp-23S rRNA at G2468 (E.coli nomen- clature) is indicated in bold in the forward primer, while the position deﬁning the new 30 end (C2440) is bold in the reverse primer. The 2897 kb PCR product was puriﬁed and cp-23S rRNA was transcribed and puriﬁed as described previously (12). Reconstitution of 50S subunits 50S subunits were assembled by using 10 pmol (0.24 A260 units) in vitro transcribed cp-23S rRNA, 10 pmol (0.01 A260 units) 5S rRNA, total 50S subunit proteins (0.29 equivalent units) and 20 pmol synthetic RNA CCCGGGGAUAXCAGG- CUGAUCUCCCC in the presence of the macrolide antibiotic RU69874 (0.5 mM) in a total volume of 12.9 ml reconstitution buffer 20 mM Tris–HCl, pH 7.4, 4 mM MgCl2, 400 mM NH4Cl, 5 mM b-mercaptoethanol, 0.2 mM EDTA (12,15). The nucleotide corresponding to A2451 of 23S rRNA in the synthetic RNA oligonucleotide is indicated (X). The in vitro reconstitution procedure was performed essentially as described in (12), with the exception that the MgCl2 concentration during the last incubation step was adjusted to 30 mM. The in vitro reconstituted 50S subunits were reassociated with 4 pmol native T.aquaticus 30S subunits as previously described (6).