A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

Abstract

The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.

Figure 1

A schematic diagram of the elongation phase of the ribosome-catalyzed translation. A peptidyl-tRNA is bound to the P site and the deacylated tRNA is in the ribosomal E site. The elongation factor EF-Tu complexed with GTP (orange) delivers an aminoacyl-tRNA to the A site. The deacylated tRNA dissociates from the E site on binding of the aminoacyl-tRNA to the A site. Upon codon recognition the GTP-ase activity of EF-Tu is stimulated and this causes a conformational change in EF-Tu upon which the factor dissociates from the ribosome. If the appropriate codon-anticodon interaction is established the CCA-end of the A-site aminoacyl-tRNA undergoes conformational change in a process known as accommodation, whereas the non-cognate tRNA is rejected at this point. After accommodation a free α-amino group of the aminoacyl-tRNA is oriented properly for the nucleophilic attack onto the acyl-ester link of the peptidyl-tRNA in the P site. The peptidyl transfer reaction occurs rapidly yielding a lengthened peptidyl-tRNA bound to the A site and the deacylated tRNA in the P site. The translocation of the reaction products and mRNA is promoted by the elongation factor EF-G in a GTP-dependent manner. The peptidyl-tRNA moves from the A site into the P site, whereas the deacylated tRNA moves from the P site into the E site. Also, the ribosome has now shifted in the 3’ direction of the mRNA and a new codon occupies the A site on the 30S subunit. After dissociation of the EF-G:GDP complex from the ribosome a new round of peptide synthesis ensues. Once the ribosome encounters the translational stop codon the termination phase of protein synthesis is initiated (not shown).

Figure 2

The structure of the ribosomal PTC and the mode of tRNA recognition. (A) A cartoon diagram of the 50S subunit from H. marismortui showing the exact location of the PTC [116]. The two-fold symmetry of the active site is evident. The P loop is blue, the A loop is red, whereas the floor and the walls of the PTC are orange. The arrow points to the tunnel entrance. rRNA is light blue, while the proteins are colored in light green. (B) The A loop (green) binds CCA of the A-site substrate analog CC-Pmn (blue) [26]. C75 makes Watson-Crick interactions with G2588 (G2553) and C75 base stacks with U2590 (U2555). (C) The P-loop (green) bases G2285 (G2252) and G2284 (G2251) form Watson-Crick base pairs with C74 and C75 of the peptidyl-tRNA mimic CCA-Pcb (blue), respectively [26]. In A and B hydrogen bonds are shown with dashed lines.

Figure 3

Identification of the pre-translocation intermediate in catalytically active crystals of the 50S subunit from H. marismortui. (A) Crystals of Hma50 were soaked with a mixture of substrates shown on the left hand side with the expectation that the active 50S ribosome would yield products shown on the right hand side. (B) Examination of the resulting electron density map revealed that the lengthened peptidyl-tRNA analog, CCA-Pmn-Pcb (red), was produced and bound to the A site (dark green), whereas CCA (blue) was found in the P site (light green). The figure is adapted from the reference .

Figure 4

A schematic diagram of the revised hybrid state model of translation. (I) The peptidyl-tRNA is bound in the P site and the A site is empty. (II) The elongation factor EF-Tu (light red) delivers an aminoacyl-tRNA to the ribosomal A-site in a GTP-dependent manner. Upon GTP hydrolysis EF-Tu (dark red) dissociates from the ribosome. The anticodon and the CCA-end of the aminoacyl-tRNA are bound to the A sites in the 50S and the 30S subunit, respectively. Likewise, the ends of the peptidyl-tRNA are attached to the P sites producing the so-called P/P and A/A states. (III) Upon peptidyl transfer the peptidyl-tRNA is now attached to the A site, whereas the deacylated tRNA is in the P site. Again, both the anticodon and the CCA-ends of the product tRNAs are bound to the same ribosomal sites suggesting that the tRNAs are in their P/P and A/A states after peptidyl-transfer. This state is observed in the crystals of Hma50 (see Fig. 3) and is termed as the pre-translocation state. (IV) CCA of the deacylated tRNA moves into the E site, while the CCA-end of the peptidyl tRNA translocates into the P site. The corresponding anticodon loops, however, remain attached to the A and P sites giving a rise to the A/P and P/E hybrid state. (V) Finally, the elongation factor EF-G (green) promotes the translocation of the anticodon loops in a GTP-dependent fashion. The figure is adapted from reference .

Figure 5

The binding of the A-site substrate induces a conformational change in the PTC, which in turn promotes peptidyl transfer. (A) When the peptidyl-tRNA is in the P site and the A site is empty, the acyl-ester link is sequestered from water by the 23S rRNA. The peptidyl-tRNA mimic CCA-Pcb is colored green, the PTC is shown as a beige surface, the 23S rRNA bases C2104 (C2063), A2486 (A2541) and U2620 (U2585) are shown as beige sticks as well. Two water molecules (red spheres) are modeled at the putative attack locations and it is clear that due to steric clashes water cannot access the acyl-ester link when the A site is empty. (B) The binding of either the transition-state analog (TSA) (red) or CC-hPmn (pink) to the A site induces the conformational change in the PTC, whereas C-hPmn (beige) does not promote the conformational change. C-hPmn binds somewhat higher in the A site when compared to both CC-hPmn and TSA, and its α-amino group is positioned far from the carbonyl carbon of the aminoacyl-tRNA (blue sphere). (C) The induced-fit conformational change in the residues G2618-U2620 (G2553-U2585) and U2541 (U2506) allows the rotation of the carbonyl oxygen. The rotation is the largest in the case of the transition analogue soak (blue) allowing the nucleophilic attack by the free α-amino group onto the carbonyl carbon. The rotation occurs in the presence of CC-hPmn (light blue) as well but to a somewhat lesser degree. In the presence of C-hPmn (beige), however, the carbonyl oxygen prevents the nucleophilic attack. The direction of the nucleophilic attack is shown with a red arrow. In B and C, the PTC residues are colored as following: orange for the TSA soak, light orange for the CC-hPmn soak and grey for the C-hPmn soak. The figure is adapted from reference .

Figure 6

The stereochemistry and mechanism of the peptidyl transfer reaction. (A) The complex with the transition state analog revealed that the oxyanion oxygen points away from A2486 (A2451). The structure further suggested that the reaction proceeds through an intermediate with S-chirality. The base of U2637 (U2602) and the ribose of U2619 (U2584) coordinate a water molecule that forms an oxyanion hole. (B) A schematic diagram of the transition-state analogue shown in A. (C) The mechanism of the peptidyl transfer based on the structural and kinetic data. The attack of the α-amino group of the aminoacyl-tRNA (red) onto the carbonyl carbon of the peptidyl-tRNA (blue) occurs in concert with the proton shuttle. The O3’ oxygen and the O2’ hydroxyls of the P-site A76 as well as the α-amino group of the aminoacyl-tRNA are involved in the proton shuttle. The reaction proceeds through a six-membered intermediate state (in brackets) in which the formation of the new C-N bond is commensurate to the dissolution of the N-H bond. Finally, the intermediate collapses into the reaction products shown on far right. Panels A and B are adapted from reference , whereas panel C is adapted from reference .

Figure 7

Peptide release is promoted by the induced-fit conformational change in the PTC. (A) The A-site CCA (light blue) promotes peptide release by coordinating the hydrolytic water molecule (red sphere) through its O3’ hydroxyl group. Also, the carbonyl oxygen of the peptidyl-tRNA analog, CCA-Pcb (blue), forms a hydrogen bond with the base of A2486 (A2451), which allows the nucleophilic attack by the water. (B) On the other hand, CA (light blue) binds higher in the A site and its O3’ hydroxyl is positioned too remotely from the P-site acyl-ester bond to be able to catalyze peptide release. Also, the carbonyl oxygen forms a hydrogen bond with a water molecule and its not oriented properly for the nucleophilic attack. In A and B, the 23S rRNA residues are colored in green. (C) The binding of both CA and CCA to the A site induces the conformational change in the PTC. The P site is oriented toward the reader, whereas the A site is behind the U2619-U2620 (U2584-U2585). CCA-Pcb is in blue and the CCA oligonucleotide in the A site is cyan. The induced-fit conformation of the PTC is colored orange, whereas the apo-PTC is grey. (D) A conformational change is observed in the PTC of the T. thermophilus 70S ribosome when in complex with RF2. The induced-fit change is similar to that observed in the 50S subunit complexed with the appropriate A-site substrate. The 70S-tRNA complex is grey, whereas the ternary 70S-tRNA-RF2 complex is in red. The GGQ motif of RF2 is orange with the side-chain of Q240 shown as sticks. Panels A and B were adapted from reference 25, while panel D was adapted from reference .