Ribosome-Catalyzed Peptide-Bond Formation with an A-Site Substrate Covalently Linked to 23S Ribosomal RNA

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Science  10 Apr 1998:
Vol. 280, Issue 5361, pp. 286-289
DOI: 10.1126/science.280.5361.286


In the ribosome, the aminoacyl–transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA). This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase–catalyzed reaction. This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA. Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.

Catalysis of peptide bond formation requires precise juxtaposition of the acceptor ends of P (peptidyl)- and A (aminoacyl)- site–bound tRNAs in the active site of the ribosome. Accumulating evidence points to a role for the 23S rRNA in the function of peptidyl transferase (1-3). Identification of a peptidyl transferase–reactive crosslink between a benzophenone-derivatized peptidyl-tRNA and A2451-C2452 localized the central loop of domain V of 23S rRNA to the peptidyl transferase site (2). More recently, identification of a base-pairing interaction between C74 of P-site–bound tRNA and G2252 of domain V established a direct role for 23S rRNA in the function of peptidyl transferase (3). Here we describe an aminoacyl-tRNA analog, 4-thio-dT-p-C-p-puromycin (s4TCPm), which crosslinks to 23S rRNA with high efficiency and specificity. This substrate remains fully active as an acceptor in the peptidyl transferase reaction while covalently bound to 23S rRNA, imposing constraints on the proposed concerted events of tRNA movement and peptide-bond formation and unambiguously placing the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site.

The aminoacyl-tRNA analog, s4TCPm, was chemically synthesized and purified (4); the Michaelis constant (K m) of this compound for peptidyl transferase is about 10 μM (5). The phosphorylated compound, [32P]s4TCPm, was then bound toEscherichia coli 70S ribosomes programmed with gene 32 mRNA (6) in which the P site was filled with deacylated tRNAPhe. After irradiation with ultraviolet (UV) light (366 nm), total RNA was prepared from the ribosomal complexes and analyzed (Fig. 1A) (7). Exclusive labeling of 23S rRNA is consistent with crosslinking to the peptidyl transferase center of the ribosome. Based on incorporation of 32P into 23S rRNA, about 30% of ribosomes were crosslinked in the presence of 20 μM s4TCPm. The absence of substantial crosslinking to ribosomal proteins (8) provides evidence for the specificity of the crosslinked A-site ribosomal complex and the RNA-rich nature of the peptidyl transferase site (9). This contrasts with the recent proposal of a proteinaceous A-site environment (10).

Figure 1

Identification and localization of a crosslink between [32P]s4TCPm and 23S rRNA. (A) Ribosomal complexes (7) were exposed for various lengths of time to long wavelength (366 nm) UV light. Ribosomal RNAs were extracted with phenol and analyzed on a 3.8% polyacrylamide gel containing 7 M urea. (B) 32P-radiolabeled A-site substrate [32P]s4TCPm (lane 1) and an RNase T1 digestion of crosslinked 23S rRNA resolved on a 24% polyacrylamide gel containing 6 M urea (lane 2) (11). (C) Primer extension analysis of crosslinked E. coli 23S rRNA. Lanes: G and A, sequencing lanes; 1 to 3, absence of s4TCPm for 0, 5, and 15 min of exposure, respectively, to UV light; 4 to 6, presence of s4TCPm for 0, 5 and 15 min of exposure, respectively, to UV light (15). The strong reverse transcriptase stop correlated with the presence of crosslinking reagent is indicated at G2553.

Digestion of the crosslinked E. coli 23S rRNA species with ribonuclease (RNase) T1 (11) revealed a single crosslinked product (Fig. 1B). The efficiency and specificity of crosslinking was high, which indicates an extremely close juxtaposition of s4TCPm and rRNA, comparable to that between position 34 of tRNA and position C1400 of 16S rRNA (12). Typically, even highly efficient crosslinks to the ribosome, such as those obtained with benzophenone-derivatized Phe-tRNAPhe, target multiple rRNA sites (2).

Assignment of the site of crosslinking to position G2553 was achieved with RNase H digestion (13) followed by primer-extension analysis (Fig. 1C) (14). The strong stop induced by crosslinking with s4TCPm is consistent with the estimated crosslinking efficiency of 30%. Crosslinking of s4TCPm toBacillus stearothermophilus ribosomes was similarly efficient and also yielded a single RNase T1 product; primer extension again localized the crosslinked position to nucleotide G2553 (E. coli numbering) (8).

The crosslink between s4TCPm and 23S rRNA places the conserved 2555 loop at the site of interaction between the ribosome and the conserved CCA end of A-site tRNA. This conclusion is consistent with protection of G2553 from chemical modification by A-site tRNA, which depends on the presence of the terminal adenosine (A76) of tRNA (15) and with directed cleavage of this region of the RNA backbone by hydroxyl radicals generated from Fe(II) tethered to the 5′ end of A-site–bound tRNA (16). Other biochemical and genetic experiments are similarly consistent (17). Both G2553 and Um2552 are universally conserved nucleotides, suggesting possible interactions between these nucleotides of 23S rRNA and the similarly conserved CCA end of A-site–bound tRNA.

Crosslinked E. coli 50Ssubunit–s4TCPm complexes were isolated and tested for the ability of the covalently bound A-site substrate to participate in peptide bond formation (18). The minimal P-site oligonucleotide substrate CACCA-(N-Ac-Phe) was supplied and its reaction with the crosslinked puromycin complex was followed by a shift in the electrophoretic mobility of the RNase T1 fragment of 23S rRNA resulting from acquisition of N-Ac-Phe [resulting product,N-Ac-Phe-(s4TCPm)-23S T1 oligonucleotide] (Fig. 2A). The rate of reaction of the covalently bound substrate is similar to that observed in a standard reaction with 50S subunits in which free puromycin is supplied at a saturating concentration. CrosslinkedB. stearothermophilus 50S–s4TCPm complexes were also reactive in a peptidyl transferase reaction (8). Thus, the A-site substrate s4TCPm is crosslinked to 23S rRNA in its biologically active configuration.

Figure 2

Peptidyl transferase activity of the crosslinked s4TCPm-50S complex. (A) Peptidyl transferase reactivity of s4TCPm covalently bound to ribosomes with P-site substrate CACCA-(N-Ac-Phe). Reaction products were analyzed by RNase T1 digestion of 23S rRNA and resolved on a 24% polyacrylamide gel containing 6 M urea (11, 18). The reaction time and extent of reaction (Frxn.) are indicated. (B) Antibiotic inhibition of crosslinking by s4TCPm to 23S rRNA (19). Values were normalized to 1.0 for the reaction mixture with no antibiotics. (C) Antibiotic inhibition of peptidyl transferase activity of crosslinked complex (s4TCPm-50S) (19). Values were normalized as in (B). Chlor, chloramphenicol; Carbo, carbomycin; Clinda, clindamycin; Erythro, erythromycin; Neo, neomycin; Sparso, sparsomycin; Puro, puromycin.

A number of peptidyl transferase–specific antibiotics, including chloramphenicol, carbomycin, clindamycin, sparsomycin, and puromycin, specifically inhibited the crosslinking of s4TCPm to the 2555 loop of 23S rRNA; erythromycin and neomycin, two antibiotics that do not target peptidyl transferase, had no effect (Fig. 2B) (19). However, only chloramphenicol, carbomycin, and clindamycin strongly inhibited the peptidyl transferase reactivity of the crosslinked complex (Fig. 2C) (19). Sparsomycin and puromycin, both known to directly compete with aminoacyl-tRNA binding to the A site, predictably had no effect on the peptidyl transferase activity of the crosslinked complex (20, 21). These effects provide further evidence that the synthetic substrate, s4TCPm, is crosslinked to its physiologically correct binding site.

Crosslinking of s4TCPm is strongly dependent on occupancy of the P site with deacylated tRNAPhe (Fig.3A). To determine which particular features of P-site tRNA are required for binding to the A site, we constructed several mutant versions of tRNAPhe containing alterations of the CCA terminus (22). None of the altered tRNAs, when bound to the P site, was able to support efficient crosslinking of s4TCPm in the A site (Fig. 3A). Requirement for a specific Watson-Crick interaction in the P site between C74 of tRNA and G2252 of 23S rRNA (3) was demonstrated by the inability of C74A mutant tRNA to stimulate efficient A-site s4TCPm crosslinking except in the context of G2252U mutant ribosomes (Fig. 3B). A properly engaged, intact P-site–bound tRNA is required for formation of this highly efficient A-site crosslink. Thus, the A site on the 50S subunit may be incompletely formed (or is inaccessible) in the absence of P-site–bound tRNA or the P-site tRNA itself provides one or more of the A-site binding determinants. Cooperative interactions between P-site– and A-site–bound tRNAs have been reported (21,23).

Figure 3

Crosslinking of s4TCPm to the A site of the ribosome depends on occupancy of the P site by an intact tRNA. (A) Analysis of tRNAPhe species with mutant CCA acceptor ends. Ribosomal complexes were formed (7) with the indicated alterations of the CCA acceptor end of deacylated tRNAPhe in the P site and limiting [32P]s4TCPm in the A site and exposed to UV light. Radioactivity incorporated into 23SrRNA was normalized to the amount incorporated in the absence of any tRNAPhe. (B) Suppression of C74A tRNAPhe crosslinking deficiency with mutant ribosomes [G2252 to U (G2252U)]. Ribosomal complexes were formed with saturating s4TCPm and various tRNAPhe species [lane 1, none; lane 2, wild type (CCA); lane 3, C74A (ACA); lane 4, C75A (CAA)] and exposed to UV light. Allele-specific primer extension (PSII) was used to compare the extent of 23S rRNA crosslinking in wild-type and G2252U mutant ribosomal populations (3).

Our findings have strong implications for the displacement and hybrid state (24) models for the movements of tRNA substrates in the translational elongation cycle. Both models invoke coupling of the peptidyl transferase reaction with movement of the acceptor end of A-site–bound tRNA into the P site of the 50S subunit. The efficient catalysis performed by the crosslinked complex suggests that either the crosslinked product retains sufficient mobility to undergo movement (which may be modest), that movement is coupled to intramolecular rearrangement of 23S rRNA, or that peptidyl transfer and movement are sequential rather than concerted.

The active site of the ribosome is thus composed of at least three distal elements of 23S rRNA dispersed across the secondary structure of domain V (Fig. 4): the 2451–2452 region of the central loop of domain V must be located near the aminoacyl moiety of P-site–bound tRNA (2), the 2250 loop interacts directly with C74 of P-site–bound tRNA (3), and, here, the 2555 loop has been localized to the A-site–bound tRNA. Such data argue for a primary role for 23S rRNA in peptidyl transferase and force consideration of the steric limitations on the potential involvement of other 23S rRNA or ribosomal protein elements in this catalytic site.

Figure 4

Secondary structure of domains IV (part) and V of 23S rRNA. The footprints of A and P site tRNAs (closed circles for RNA-protected moieties and open circles for acyl-protected moieties) are indicated (15). Several sites crosslinked by the acceptor end of P- and A-site–bound tRNAs are indicated with small arrows (29). Two specific nucleotides, G2252 and G2553, known to form close contact with the CCA end of P- and A-site–bound tRNAs, respectively, are indicated; the loops in which these nucleotides are found are designated the P loop (30) and the A loop.

  • * Present address: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

  • To whom correspondence should be addressed. E-mail: harry{at}


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