The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factordependent hydrolysis of peptidyl-tRNA. Unlike peptide bond formation, peptide release is strongly impaired by mutations of nucleotides within the active site, in particular by base exchanges at position A2602 (refs 1, 2). The 29-OH group of A76 of the peptidyl-tRNA substrate seems to have a key role in peptide release 3 . According to computational analysis 4 , the 29-OH may take part in a concerted 'proton shuttle' by which the leaving group is protonated, in analogy to similar current models of peptide bond formation 4-6 . Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D 2 O) of the two reactions catalysed by the active site of the Escherichia coli ribosome. The transition state of the release factor 2 (RF2)-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond. This finding argues against a concerted proton shuttle in the transition state of the hydrolysis reaction. In comparison, the proton inventory for peptide bond formation indicates the rate-limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eightmembered proton shuttle in the transition state 5 . Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.Peptide bond formation and peptide release involve the attack of different nucleophilic groups on the ester carbonyl carbon of peptidyltransfer (t)RNA in the P site of the ribosome; these nucleophilic groups are respectively the a-amino group of A-site-bound aminoacyl-tRNA and a water molecule. Unlike peptide bond formation, which does not require auxiliary factors, peptide release is assisted by termination (release) factors; in E. coli, these are RF1 and RF2. On recognition of a stop codon in the decoding site, the conserved GGQ motif of RF1/2 is inserted into the peptidyl transferase centre, augmenting the active site and inducing the hydrolysis of peptidyl-tRNA (see ref. 3 for a review). Release-factor binding induces a conformational change that involves conserved 23S ribosomal RNA residues, in particular U2506 and U2585 (refs 6-10), and opens the active site for the access of water 11 . The conserved Gln residue in RF1/2 has been attributed an essential function in positioning the hydrolytic water molecule and accounts for the high specificity of the active site for water as the nucleophile, discriminating against a larger amine 12 . Computational analysis suggested that the 29-OH of A76 of the P-site tRNA may take part in a concerted proton shuttle mechanism for the protonation of the leaving 39-O (ref. 4), in analogy to the mechanism suggested for peptide bond formation 5,13 . A fully concerted proton shuttle would ...

Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.