Chemical engineering of the peptidyl transferase center reveals an important role of the 2'-hydroxyl group of A2451

Abstract

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2'-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2'-hydroxyl was present. This implicates a functional or structural role of the 2'-hydroxyl group at A2451 for transpeptidation.

Figure 1

Structural and functional characteristics of reconstituted 50S subunits containing circularly permuted (cp) 23S rRNA (gapped-cp-reconstitution). (A) Schematic representation of the secondary structure of the cp-23S rRNA. The natural 5′ and 3′ ends of 23S rRNA, which form helix 1 (H1) in the native 50S subunit, were covalently connected (bold black line) and new endpoints at positions 2468 and 2440 (5′, 3′) in helices 89 (H89) and 74 (H74) have been introduced to generate the cp-23S rRNA used in this study. The sequence of helix 1 of the cp-23S rRNA is shown whereas the natural ends of native 23S rRNA are indicated by numbers. The additional nucleotides that were added during clone construction are in bold lower case letters. The chemically synthesized 26 nt long RNA fragment, which compensates for the missing 23S rRNA segment in the peptidyl transferase center (boxed), is shown in red. (B) The peptidyl transferase activity of ribosomes composed of gapped-cp-reconstituted 50S subunits and native T.aquaticus 30S subunits. Product yields in the absence of the compensating 26mer (−) or in the presence of fragments containing the wild-type sequence (wt; red bar) or carrying base changes (C, G, U; blue bars) at the position corresponding to A2451 of 23S rRNA are indicated. Yield of product formation of 50S carrying the wild-type sequence in the presence of 200 μM sparsomycin (+ Spa) are shown. In all cases, the mean and the SD values of the fraction of f[³H]Met-tRNA that reacted with puromycin after 240 min of incubation are shown.

Figure 2

Peptidyl transferase activity of ribosomes containing non-natural nucleoside analogs at A2451. (A) Chemical structures of all the tested nucleoside analogs. The introduced chemical modifications are depicted in red. (B) Product yields in the absence of the compensating 26mer (no oligo) or in the presence of fragments containing the wild-type sequence (wt; black bar), ribose sugar modifications (red bars) or carrying base modifications (green bars) at the position corresponding to A2451 of 23S rRNA are indicated. The yield of f-Met-puromycin formed with gapped-cp-reconstituted ribosomes carrying the wt 26mer after 240 min of incubation was taken as 1.0. Values shown represent the mean of at least three independent experiments. (C) Initial rates of peptide bond formation catalyzed by A2451-modified ribosomes. The initial rates were determined from experimental points in the linear range of the reactions within the first 30 min of incubation (with the exception of the deoxy-abasic ribosome, where the first time point that could be measured was at 240 min). The rates were normalized to the rate of reconstituted ribosomes containing the synthetic wild-type (wt) RNA fragment (A). In all cases, the background values (amount of product formed in reactions containing only native 30S subunits) were subtracted from all experimental data points.

Figure 3

Peptidyl transferase activity of ribosomes containing 2′-deoxyribose modifications at A2451. (A) Time course of peptide bond formation promoted by ribosomes containing 50S reconstituted from cp-23S rRNA and the compensating 26mer with either adenosine (wt), 2′-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 ribosomal subunits carrying adenosine (wt), 2′-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 2′-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), 2′-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 measured (n.d.). The rates were normalized to the rate of reconstituted subunits containing the synthetic wild-type RNA fragment (wt).