An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA

Abstract

The large ribosomal subunit catalyses the reaction between the alpha-amino group of the aminoacyl-tRNA bound to the A site and the ester carbon of the peptidyl-tRNA bound to the P site, while preventing the nucleophilic attack of water on the ester, which would lead to unprogrammed deacylation of the peptidyl-tRNA. Here we describe three new structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with peptidyl transferase substrate analogues that reveal an induced-fit mechanism in which substrates and active-site residues reposition to allow the peptidyl transferase reaction. Proper binding of an aminoacyl-tRNA analogue to the A site induces specific movements of 23S rRNA nucleotides 2618-2620 (Escherichia coli numbering 2583-2585) and 2541(2506), thereby reorienting the ester group of the peptidyl-tRNA and making it accessible for attack. In the absence of the appropriate A-site substrate, the peptidyl transferase centre positions the ester link of the peptidyl-tRNA in a conformation that precludes the catalysed nucleophilic attack by water. Protein release factors may also function, in part, by inducing an active-site rearrangement similar to that produced by the A-site aminoacyl-tRNA, allowing the carbonyl group and water to be positioned for hydrolysis.

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

“…Like A2602, also U2585 has been shown to be a structurally flexible residue in the catalytic cavity of the PTC in various 50S subunit crystal structures (6,7,9,11,26,45). Furthermore, mutations at this base also showed reductions in peptide release activities, albeit to a weaker extent compared to A2602 (24,25).…”

“…In fact all of them showed slightly enhanced (∼ 2-fold) peptide release rates (Table 1). Based on crystallographic studies it has been suggested that the base of U2585 protects the ester bond of P-site-bound peptidyl-tRNA from premature hydrolysis during the elongation phase of protein synthesis (9). We therefore tested if the accelerated peptide release rates of the abasic 2585 subunit are the result of an RF-independent peptidyl-tRNA hydrolysis.…”

mtRF1a Is a Human Mitochondrial Translation Release Factor Decoding the Major Termination Codons UAA and UAG

“…In the absence of translation termination, nascent proteins will be inserted into the membrane but will also still be attached to the ribosome via the P site bound peptidyl-tRNA. It is understood that this ester bond can be spontaneously hydrolyzed over time, but release factor activity at the ribosome dramatically accelerates this natural process (Schmeing et al., 2005). However, it is possible that subtle temporal effects on the assembly process following siRNA-mediated knockdown may lead to assembled complexes with minor defects in electron transfer, leading to increased loss of electrons in the form of ROS.…”

Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide

“…The conformations of 23S rRNA nucleotides U2584 and U2585 were taken from the equivalent nucleotides of the Haloarcula marismortui 50S subunit in complex with the unaccommodated A-site tRNA mimic (PDB1VQ6) 21,22 after the PDB was fitted as a rigid body into the ErmBL-SRC density map. The conformation of U2506 was similar to the equivalent nucleotide of the Haloarcula marismortui 50S subunit in complex with the accommodated A-site tRNA mimic (PDB1VQN) 21,22 , however required manual rotation of the base to prevent clashing with the unaccommodated position of U2585. In contrast, the conformation of A2062 present in the ErmBL-SRC is unique, being distinct from the tunnel “in” (PDB3CC2) 38 and tunnel “out” (PDB1VQ6) 21,22 conformations observed in previously reported ribosome structures.…”