Different substrate-dependent transition states in the active site of the ribosome

Abstract

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 ...

Activities of the peptidyl transferase center of ribosomes lacking protein L27

“…) was in agreement with that previously determined using wt (MRE600) ribosomes (Kuhlenkoetter et al 2011;Indrisiunaite et al 2015).…”

“…Both models are consistent with the experimental data available so far, including the pH/rate profiles for different substrates, mutational analysis, and KIE and KSIE measurements (Katunin et al 2002;Youngman et al 2004;Beringer and Rodnina 2007;Lang et al 2008;Wohlgemuth et al 2008;Hiller et al 2011;Kuhlenkoetter et al 2011;Zaher et al 2011). Both models suggest that the proton from the attacking amine is transferred to the leaving group through the 2 ′ -OH of the P-site tRNA A76; however, the impact of L27 and the exact path of proton transfer is different (we note that at a time when the proton shuttle model was suggested, the positions of the water molecules in the catalytic site were not known with precision).…”

“…Ribosomes, initiation factors, EF-Tu, EF-G, tRNAs, and RF2 were prepared as previously described (Rodnina et al 1995;Gromadski and Rodnina 2004;Kuhlenkoetter et al 2011 Phe or fMetPro-tRNA Pro in the P site were formed by reacting ICs with a twofold excess of the suitable TC and catalytic amounts of EF-G for 5 min at 37°C. Ribosome complexes were purified by centrifugation through sucrose cushions (1.1 M sucrose in buffer A) at 259,000g for 2 h. Pellets were dissolved in buffer A and stored at −80°C in small aliquots.…”

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

“…It is generally assumed that the ribosome emerged from the RNA world, when the proteins did not exist yet, and RNA acted as catalyst in chemical reactions (for recent reviews see refs 34,35). In fact, the peptidyl transferase centre of the ribosome, which appears to be a relic from that ancient time 36,37 , is built of RNA 38 , and its activity is not appreciably affected by the r-proteins (for review, see ref. 39).…”

Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

“…4F), suggesting that proton transfer from the hydrolytic water is not rate-limiting for GTP hydrolysis on the ribosome, consistent with theoretical predictions (13). For comparison, a KSIE of about 4 with one proton in flight in the TS was observed for the hydrolysis of peptidyl-tRNA in the catalytic center on the 50S subunit (55).…”