Important Contribution to Catalysis of Peptide Bond Formation by a Single Ionizing Group within the Ribosome

Katunin, Vladimir I.; Muth, Gregory W.; Strobel, Scott A.; Wintermeyer, Wolfgang; Rodnina, Marina V.

doi:10.1016/s1097-2765(02)00566-x

Cited by 161 publications

(260 citation statements)

References 23 publications

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“…2). The rate of the faster reaction component agrees with previous literature values (19,47,48). The origin of the slow reacting component has yet to be delineated.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondsupporting

confidence: 89%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

454

575

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Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by Ϸ6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components. P rotein synthesis, catalyzed by the ribosome, is rapid, processive, and highly regulated. In bacteria, two RNA-protein subunits consisting of a small 30S subunit and a large 50S subunit assemble around a mRNA template into a roughly spherical 70S particle that translates the nucleotide sequence into a polypeptide chain through repetitive, codon-dependent binding of aminoacylated tRNA.The landmark structures of the 30S (1, 2), 50S (3, 4), and 70S (5) ribosomal particles provide a molecular basis for understanding ribosome function. tRNA molecules bind to the ribosome in a solvent-accessible channel at the subunit interface. Three binding sites for tRNA, called the aminoacyl site (A site), peptidyl site (P site), and exit site (E site), have been identified on both the large and small subunit (Fig. 1). Each tRNA is separated from its neighbor at the elbow region (the site of Ϸ90°b ending) by 25-45 Å (5, 6). The anticodon stem loops of A-and P-site tRNA form Watson-Crick base pairs with adjacent mRNA codons on the 30S subunit (5, 7), whereas the 3Ј CCA terminal residues of A-and P-site tRNAs base-pair with conserved ribosomal RNA (rRNA) loops (8-10) within the 50S subunit peptidyltransferase center, the site of peptide bond formation (11). Additional interactions with rRNA and ribosomal proteins position tRNA molecules on the ribosome (5).The essential components and principal steps of translation have been delineated through genetic, biochemical, and kinetic methods (12). The process of polypeptide elongation has been most extensively characterized (13). Binding of aminoacyl-tRNA (aa-tRNA) to the A site occurs as a ''ternary complex'' with the GTPase elongation ...

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“…2). The rate of the faster reaction component agrees with previous literature values (19,47,48). The origin of the slow reacting component has yet to be delineated.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondsupporting

confidence: 89%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

454

575

View full text Add to dashboard Cite

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“…36 The ribosome contributes positional catalysis to peptide bond formation as well as to polypeptide elongation: by providing the architectural means for accurate substrate positioning and alignment; by the design of an exact path for the A-to P-site passage; and by the guidance of the rotatory motion along this path. No ribosomal component is required for the mere chemical events of peptide bond formation, but specific ribosomal moieties could have a major influence on the rate of protein biosynthesis, which plays a key role in cell vitality, 37,38 as well as on the process following the formation of the peptide bond, namely the elongation of the nascent chains.…”

Section: Spectacular Ribosomal Architecture Global Motionsmentioning

confidence: 99%

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

Zarivach

Bashan

Berisio

et al. 2004

J of Physical Organic Chem

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High-resolution structures of both ribosomal subunits revealed that most stages of protein biosynthesis, including decoding of genetic information, are navigated and controlled by the elaborate ribosomal architecturaldesign. Remote interactions govern accurate substrate alignment within a flexible active-site pocket [peptidyl transferase center (PTC)], and spatial considerations, due mainly to a universal mobile nucleotide, U2585, ensure proper chirality by interfering with D-amino-acids incorporation. tRNA translocation involves two correlated motions: overall mRNA/tRNA (messenger and transfer RNA) shift, and a rotation of the tRNA single-stranded aminoacylated-3 0 end around the bond connecting it with the tRNA helical-regions. This bond coincides with an axis passing through a sizable symmetry-related region, identified around the PTC in all large-subunit crystal structures. Propelled by a bulged universal nucleotide, A2602, positioned at the two-fold symmetry axis, and guided by a ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry optimal for peptide-bond formation and in geometry ensuring nascent proteins entrance into their exit tunnel. Hence, confirming that ribosomes contribute positional rather than chemical catalysis, and that peptide bond formation is concurrent with A-to P-site tRNA passage. Connecting between the PTC, the decoding center, the tRNA entrance and exit points, the symmetry-related region can transfer intra-ribosomal signals between remote functional locations, guaranteeing smooth processivity of amino acids polymerization. Ribosomal proteins are involved in accurate substrate placement (L16), discrimination and signal transmission (L22) and protein biosynthesis regulation (CTC). Residing on the exit tunnel walls near its entrance, and stretching to its opening, protein-L22 can mediate ribosome response to cellular regulatory signals, since it can swing across the tunnel, causing gating and elongation arrest. Each of the protein CTC domains has a defined task. The N-terminal domain stabilizes the intersubunit-bridge confining the A-site-tRNA entrance. The middle domain protects the bridge conformation at elevated temperatures. The C-terminal domain can undergo substantial conformational rearrangements upon substrate binding, indicating CTC participation in biosynthesiscontrol under stressful conditions.

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“…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).…”

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confidence: 99%

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

Wallin

Åqvist

2010

Proc. Natl. Acad. Sci. U.S.A.

103

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Recent progress in elucidating the peptide bond formation process on the ribosome has led to notion of a proton shuttle mechanism where the 2'-hydroxyl group of the P-site tRNA plays a key role in mediating proton transfer between the nucleophile and leaving group, whereas ribosomal groups do not actively participate in the reaction. Despite these advances, the detailed nature of the transition state for peptidyl transfer and the role of several trapped water molecules in the peptidyl transferase center remain major open questions. Here, we employ high-level quantum chemical ab initio calculations to locate and characterize global transition states for the reaction, described by a molecular model encompassing all the key elements of the reaction center. The calculated activation enthalpy as well as structures are in excellent agreement with experimental data and point to feasibility of an eight-membered "double proton shuttle" mechanism in which an auxiliary water molecule, observed both in computer simulations and crystal structures, actively participates. A second conserved water molecule is found to be of key importance for stabilizing developing negative charge on the substrate oxyanion and its presence is catalytically favorable both in terms of activation enthalpy and entropy. Transition states calculated both for six-and eight-membered mechanisms are invariably late and do not involve significant charge development on the attacking amino group. Predicted kinetic isotope effects consistent with this picture are similar to those observed for uncatalyzed ester aminolysis reactions in solution.density functional theory | protein synthesis T he peptide bond formation step in protein synthesis is catalyzed by the peptidyl transferase center (PTC) on the large ribosomal subunit. This reaction involves the attack of the α-amino group of an aminoacyl-tRNA molecule bound to ribosomal A-site on the ester carbon of the peptidyl-tRNA in the adjacent P-site of the ribosome. 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 (2-9), mutagenesis data (4, 10-14), 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). Furthermore, it was shown that the lower free energy barrier for the ribosome reaction, compared to an uncatalyzed reference reaction in water, is entirely due to a less negative activation entropy (3, 6, 7). Besides contributions from substrate proximity and...

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Important Contribution to Catalysis of Peptide Bond Formation by a Single Ionizing Group within the Ribosome

Cited by 161 publications

References 23 publications

tRNA dynamics on the ribosome during translation

tRNA dynamics on the ribosome during translation

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

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

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