Peptidyl Transfer on the Ribosome

Abstract

Formation of the peptide bond is the central enzymatic activity of ribosomes. The active centre resides on the large ribosomal subunit, and is made of nucleotides from domain V of the 23S‐type rRNA.

Keywords: peptidyltransferase; rRNA, function in peptide‐bond formation; enzyme mechanism, template model; enzyme mechanism, chemical catalysis

Figure 1.

The ribosomal‐elongation cycle. The cycle is shown in the frame of the α–ε model, according to which the two tRNAs stably present on the ribosome are bound to a movable domain of the ribosome. The movable domain contains two binding regions α and ε (yellow and green, respectively). These two binding regions are exposed at the A and P sites of the ribosome in the PRE state and carries the tRNAs to the P and E sites, respectively, during translocation thus establishing the POST state. At the A site there is, in addition, the decoding centre δ (blue) that is not movable but fixed at this site. The decoding centre δ overlaps with the α region at the PRE state but stands alone at the POST state.

Figure 2.

The PTF reaction. The figure shows the four possible steps of peptide‐bond formation according to recent crystallographic and biochemical data (see Wilson and Nierhaus, , for further reading). The essential features are: (a) C74 and C75 of the P‐site tRNA (green) are Watson–Crick paired with G2252 and G2251, respectively, of the P loop (blue). Similarly, C75 from the A‐site substrate (red) forms a Watson–Crick base pair with G2553 (A‐loop). The inset is the Yarus inhibitor CCdAp‐puromycin (CCdApPmn), which was used to identify the PTF centre of the ribosome. The interactions of the Yarus inhibitor with the rRNA were deduced from 50S crystals of H. marismortui ribosomes after soaking the inhibitor into the crystals. Note that it was concluded that the protonated N3 of A2451 makes a H‐bridge to O2, which was thought to mark the position of the oxyanion of the tetrahedral intermediate (transition state) formed during peptide‐bond formation (Nissen et al., ; cf. with probably the correct representation in step (b)). The α‐NH2 function of the A‐site aminoacyl‐ tRNA is an ammonium ion at pH 7. (b) Deprotonation of the ammonium ion triggers the nucleophilic attack of the α‐amino function on the carbonyl group of the P‐site substrate, which results in the tetrahedral intermediate T±. The secondary α‐NH2 group forms a hydrogen bond with N3 of A2451 and a second with either the 2′‐OH of the A76 ribose at the P site (shown here) or alternatively with the 2′‐OH group of A2451. The oxyanion of the tetrahedral intermediate points away from the N3‐ A2451 (Hansen et al., ) and thus cannot, in contrast with the previous proposal, form a H‐bridge. (c) Further deprotonation of the secondary α‐ NH2 group leads to the tetrahedral intermediate T and the PTF reaction is completed by an elimination step. (d) The peptidyl residue is linked to the aminoacyl‐tRNA at the A site via a peptide bond.

Figure 3.

Secondary structure of the domain V of the E. coli 23S rRNA. Left, the A‐site (blue) and P‐site (green) regions that are related by 2‐fold symmetry, where the symmetry‐related residues within these regions are highlighted with the same colour. Right, the 2‐fold symmetry is illustrated from two different views using ribbon representations of the PTF centre from D. radiodurans 50S subunit, with the A‐ and P‐site CCA‐end ligands indicated in the corresponding colours. This figure was taken from Agmon et al. with permission.

Figure 4.

The A‐ and P‐site products in red and green, respectively, bound at the PTF centre of the 50S subunit. The proteins that reach within ∼20 Å of the PTF centre include proteins L2 (cyan), L3 (magenta), L4 (yellow) and L10e (blue).

Figure 5.

Peptide‐bond formation in model compounds with appropriate juxtaposed nucleophile. See text for explanations.

Figure 6.

Tight fixation of the CCA ends of the P‐ and A‐tRNAs observed in 50S subunit from H. marismortui in complex with (a) the Yarus inhibitor, and (b) the products following peptide‐bond formation. The CCA ends of the tRNAs in the A and P sites are coloured red and green, respectively. The N3 of A2451 (dark blue) is 3.4 Å from the O2 of the Yarus inhibitor (see also inset in Figure ), whereas the same O2 is only 2.8 Å from the 2′‐deoxy of A76 (arrowed). Selected rRNA residues of domain V of the 23S rRNA are coloured light blue, including the A‐ and P‐loop bases that participate in A‐ and P‐site CCA‐end fixation (E. coli numbering). In (b), the P‐site C74 and C75 have been omitted for clarity. Dashes indicate H‐bonding and rRNA nucleotides use the following colour scheme: oxygen, red; phosphorus, yellow; nitrogen, blue; carbon, dark blue.

Figure 7.

The outer (yellow) and the inner layer (blue) of universally conserved nucleotides at the peptidyl transferase centre. Those of the outer layer, the A and P loops (yellow) are involved in fixation of the CCA ends of the tRNAs at the P (green) and A sites (red), whereas those of the inner layer, A2451, U2506, U2585 and A2602, are involved in the release of the nascent protein mediated by the release factors.

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References

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Further Reading

Doudna JA and Lorsch JR (2005) Ribozyme catalysis: not different, just worse. Nature Structural and Molecular Biology 12: 395–402.

Erlacher MD, Lang K, Shankaran N et al. (2005) Chemical engineering of the peptidyl transferase center reveals an important role of the 2′‐hydroxyl group of A2451. Nucleic Acids Research 33: 1618–1627.

Green R and Lorsch JR (2002) The path to perdition is paved with protons. Cell 110: 665–668.

Wilson DN and Nierhaus KH (2003) The ribosome through the looking glass. Angewandte Chemie International Edition in English 42: 3464–3486.

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Nierhaus, Knud H, and Wilson, Daniel N(Jan 2006) Peptidyl Transfer on the Ribosome. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003951]