1624 Nucleic Acids Research, 2005, Vol. 33, No. 5 Figure 3. Peptidyl transferase activity of ribosomes containing 20-deoxyribose modifications at A2451. (A) Time course of peptide bond formation promoted by ribosomes containing 50S reconstituted from cp-23S rRNA and the com- pensating 26mer with either adenosine (wt), 20-deoxyadenosine (d-A2451), or a deoxy-abasic site analog (d-aba) at a position corresponding to A2451 of 23S rRNA. The curves represent the mean of 2–3 independent time course experiments, whereas the amount of product formed at the endpoint of the reaction catalyzed by ribosomes containing the wild-type 26mer was taken as 1.0. (B) Sucrose gradient analysis of gapped-cp-reconstituted large ribo- somal subunits carrying adenosine (wt), 20-deoxyadenosine (dA) or the deoxy- abasic analog (d-aba) at 2451. Vertical arrows indicate the mobility of native 50S subunits. Figure 4. Peptidyl transferase activities of gapped-cp-reconstituted subunits containing 20-deoxyribose modifications at A2451 using CC-puromycin as acceptor substrate. (A) The reaction between N-acetyl-Phe-tRNA and [32P]CC-puromycin was carried out for 120 min, a time point that corresponded to the endpoint of the reaction catalyzed by reconstituted wild-type large subunits. The product N-acetyl-Phe-CC-puromycin (CC-Pmn-AcPhe) was resolved from CC-puromycin (CC-Pmn) by gel electrophoresis (6). The control reaction (ctrl) contained the whole reaction mixture except ribosomal particles. The relative yields of product formation by gapped-cp-reconstituted subunits containing adenosine (wt), 20-deoxyadenosine (dA), or the deoxy-abasic analog (d-aba) at 2451 are shown below the gel. (B) The initial rates of peptide bond formation catalyzed by the wt or the deoxy-A2451-modified large ribosomal subunit were determined from experimental points within the first 45 min of incubation. During this incubation time, no product formation with ribosomal particles carrying the deoxy-abasic site analog at position 2451 could be mea- sured (n.d.). The rates were normalized to the rate of reconstituted subunits containing the synthetic wild-type RNA fragment (wt). Table 1. RF1-mediated peptide release activities of gapped-cp-reconstituted Table 2. Peptidyl transferase activities of gapped-cp-reconstituted T.aquaticus T.aquaticus ribosomes carrying nucleoside modifications at A2451 of 23S ribosomes carrying nucleoside modifications at A2450 or A2453 of 23S rRNA rRNA 23S rRNA position Modification Reaction ratesa RF1-mediated hydrolysisa Fraction formyl-Met releasedb A2451 (wt) d-A2451 d-aba 2451 100 79 – 4 92 – 6 0.27 – 0.01 0.22 – 0.01 0.25 – 0.02 aPeptide release activity of ribosomes containing 50S subunits carrying the wild-type (wt) 26mer was taken as 100%. The incubation time, 30 min, corre- sponded to the endpoint of the reaction catalyzed by reconstituted wild-type ribosomes. Values shown represent an average of two independent experiments using RNA fragments carrying either the wt, the deoxy (d-A2451) or the deoxy- abasic (d-aba 2451) nucleoside analog at a position corresponding to A2451 of 23S rRNA. bThe fraction of f-[3H]Met released from f-[3H]Met-tRNA after 30 min of incubation. wt A2450 A2453 none dA d-aba r-aba dA d-aba r-aba 1.00 1.29 0.36 0.61 1.23 n.d. 0.53 aThe peptidyl transfer rate of ribosomes containing 50S subunits carrying the wild-type (wt) 26mer was taken as 1.00 and compared to the rates of ribosomes carrying the deoxy adenine (dA), the deoxy-abasic site (d-aba) or the ribose- abasic site analog (r-aba) at positions A2450 or A2453. The initial rates were determined from experimental points in the linear range of the reactions within the first 30 min of incubation. The d-aba 2453 modification was not tested (n.d.). of removing the 20-hydroxyl at A2451 are speciﬁc for transpeptidation. To test whether the removal of a ribose 20-hydroxyl group or the complete nucleobase had similar negative effects at other conserved residues in the PTC, we tested the peptidyl trans- ferase activities of ribosomes containing a deoxy adenosine, a deoxy-abasic or a ribose-abasic site at positions A2450 or A2453. At both sites, the deoxy-adenosine modiﬁcations were tolerated and peptide bond formation proceeded with rates comparable to the wild-type control (Table 2). Furthermore, introduction of ribose-abasic analogs at residues 2450 or 2453 had only relatively small effects. In clear contrast to ribosomes with deoxy-abasic 2451, ribosomes with a deoxy-abasic site at 2450 were at least 40-fold more active in transpeptidation and readily formed the products of the peptidyl transferase reaction (compare Table 2 with Figure 2C). These results suggest that the inhibition of the peptidyl transferase activity by introducing a 20-deoxyribose at position A2451 of 23S rRNA are speciﬁc and re-emphasize a special role of this ribose 20-hydroxyl in functions of the PTC.