Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Abstract

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s ؊1 , regardless of the tRNA identity. The reactivity decreased in the order Lys ‫؍‬ Arg > Ala > Ser > Phe ‫؍‬ Val > Asp Ͼ Ͼ Pro, with Pro being by far the slowest. However, when Phe-tRNA Phe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ϳ7 s ؊1 , which corresponds to the rate of binding of Phe-tRNA Phe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyltRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.The enzymatic activity of the ribosome is to catalyze peptide bond formation. During the peptidyl transfer reaction, the ␣-amino group of aminoacyl-tRNA bound to the A site of the ribosome attacks the ester bond of peptidyl-tRNA in the P site, which results in peptidyl-tRNA extended by one amino acid in the A site and deacylated tRNA in the P site. The tRNA substrates are aligned in the active center of the ribosome by interactions of their CCA ends with 23 S rRNA bases (1-3). The ribosome lowers the activation entropy of the reaction (4, 5) by orienting the reacting groups precisely relative to each other (2, 3), providing an electrostatic environment that reduces the free energy of forming the transition state, shielding the reaction against bulk water (6, 7), or a combination of these effects (8).The peptidyl transfer reaction is modulated by conformational changes at the active site (3, 8 -10) as well as by the nature of the substrates. Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19). Early studies with 50 S ribosomal subunits indicated that efficient peptidyl transfer was observed with 3Ј-terminal RNase T1 fragments of N-acetylArg-tRNA Arg and fMet-tRNA fMet as model P-site substrates and an analog of aminoacyl-tRNA, puromycin (Pmn 4 ; O-methyltyrosine linked to N 6 -dimethyladenosine via an amide bond), as A-site substrate (20). In contrast,...

Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P

“…5B). This result is consistent with the findings that Pro-and Gly-tRNA in the A-site act as poor acceptors (3,4) and that Pro-tRNA in the P-site acts as a poor donor (1,2). Additionally, stalling is enhanced when peptidyl-Pro-Pro-tRNA is situated in the P-site, presumably because proline in the −1 position imparts additional conformational constraints on the P-site proline that are unfavorable for peptide bond formation (Fig.…”

“…However, the ribosome cannot form peptide bonds between all amino acids with the same efficiency; this is exemplified by the amino acid proline, which has an imino group instead of a primary amino group in other amino acids. Proline has been shown to be a particularly poor substrate for peptide-bond formation, both as a donor in the P-site and as an acceptor in the A-site (1)(2)(3)(4). In fact, ribosomes stall when attempting to incorporate three or more consecutive proline residues (PPP) into the polypeptide chain (5)(6)(7).…”

Activities of the peptidyl transferase center of ribosomes lacking protein L27

“…Because Pmn concentrations used in those experiments (1 µM) were far below the K M value for the ribosome-Pmn complex (∼1-30 mM, depending on the tRNA in the P site) (Youngman et al 2004;Beringer et al 2005;Wohlgemuth et al 2008), the observed decrease in the kinetics of the Pmn reaction might be due to a Pmn binding defect, rather than the reduced PT rate. To distinguish between the K M and k cat effects, we first reproduced the previous studies using fMet-tRNA fMet and Pmn as P-and A-site substrates.…”

“…Also in this case the rate of fMetPhe-Pmn tripeptide formation was independent of the presence of L27. To check whether L27 might be more important for less reactive substrates such as proline (Wohlgemuth et al 2008), which causes ribosomal stalling when more than two prolines have to be incorporated into the peptide (Doerfel et al 2013;Ude et al 2013), we checked the Pmn reaction with post-translocation complexes carrying a fMetPro-tRNA Pro in the P site. In this case, the rate of peptide-bond formation was slightly decreased in the absence of L27 (0.02 sec…”

The transition state for peptide bond formation reveals the ribosome as a water trap

“…The growing peptide chain is thereby transferred to the A-site tRNA and elongated by one amino acid. Recent years have witnessed considerable progress in our understanding of the peptidyl transfer process due to high-resolution crystallographic structures of the large ribosomal subunit with transition state (TS) analogs (1), as well as kinetic measurements (2)(3)(4)(5)(6)(7)(8)(9), mutagenesis data (4,(10)(11)(12)(13)(14), and computational studies (15)(16)(17). Hence, the current model of the peptidyl transfer reaction is that ribosomal nucleobases are not directly involved in bond making or breaking through acid-base catalysis (4,10,11,13,18,19), but that the A76 2′-OH group of the P-site substrate plays a key role in mediating proton transfer from the attacking nucleophile to the leaving 3′ ester oxygen (1,15,(20)(21)(22).…”