RAkauskaite RNA 2011.pdf
Jonathan Dinman
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Abstract
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The paper investigates the role of specific nucleotides in the large ribosomal subunit (LSU) of yeast ribosomes during the accommodation of aminoacyl-tRNA (aa-tRNA) to the peptidyltransferase center (PTC). Through various mutagenesis studies and biochemical assays, the research identifies key conserved nucleotides within the ribosome that contribute to the accuracy and efficiency of protein synthesis. Findings suggest that local structural adjustments occur in response to nucleotide mutations, supporting the ribosome's adaptability and robustness in maintaining its function despite genetic variations.
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Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression
Molecular Cell, 2003
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On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Delivered on 20 October 2002 at the 28th FEBS Meeting in Istanbul
European Journal of Biochemistry, 2003
High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a twofold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3¢ end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.
Ribosomal crystallography: Peptide bond formation and its inhibition
Biopolymers, 2003
Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity.
Topology of the Ribosomal Binding Sites For tRNA as Revealed by Photoaffinity Labeling
Annals of the New York Academy of Sciences, 1980
Ribosomal catalysis: The evolution of mechanistic concepts for peptide bond formation and peptidyl-tRNA hydrolysis
RNA Biology, 2008
Over time the mechanistic concepts to describe the two principal chemical reactions that are catalyzed by the ribosome, peptide bond formation and peptidyl-tRNA hydrolysis, have undergone dramatic changes. While the initial models were based on a ribosomal protein-based mechanism, evidence for a direct functional contribution of ribosomal RNA for catalysis has accumulated over the past years. The presentation of high resolution crystallographic structures of the large ribosomal subunit at the beginning of the new millennium dramatically increased our molecular insight into the organization of the active center and finally placed the ribosome amongst the list of RNA enzymes. Combined with elaborate biochemical and biophysical approaches the translation field has made significant progress in understanding mechanistic details of ribosomal catalysis. While it seems that the mechanism of ribosome-catalyzed peptidyl-tRNA hydrolysis is just emerging, the knowledge on transpeptidation is already very advanced. It has been realized that intricate interactions between ribosomal RNA and the transfer RNA substrate are crucial for proton shuttling that is required for efficient amide bond formation.
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Biochimie, 1991
Chemical and photochemical affinity techniques have been used extensively to determine the positions of the tRNA binding sites on the Escherichia coil ribosome. Recent advances in our understanding of ribosome structure and function prompted us to critically review the data that have accumulated on tRNA-ribosome cross-links. As a result, we propose a new model of the tRNAribosome complex that accounts for nearly all of the pertinent evidence. affinity labeling / cross-linking techniques / tRNA-ribosome complex / peptidyi transferase center / decoding site
The Active Site of the Ribosome Is Composed of Two Layers of Conserved Nucleotides with Distinct Roles in Peptide Bond Formation and Peptide Release
Cell, 2004
It is clear that a major contribution to catalysis of peptide bond formation by the ribosome derives from simply positioning two reactive substrates in close proximity to one another in an orientation favorable for catalysis (Jencks, 1969). Direct base-pairing interactions be-Medicine tween the CCA ends of the tRNA substrates and rRNA Baltimore, Maryland 21205 elements, the A and P loops, clearly play a central role in positioning the substrates for catalysis (Kim and Green, 1999; Nissen et al., 2000; Samaha et al., 1995). Indeed, Summary it is possible that the major function of this well-organized and densely packed active site is to position reac-Peptide bond formation and peptide release are catative substrates and possibly "buttress" motion along the lyzed in the active site of the large subunit of the reaction coordinate (Rajagopalan and Benkovic, 2002). ribosome where universally conserved nucleotides However, other mechanisms for promoting this reaction surround the CCA ends of the peptidyl-and aminoacylmay also be utilized by the ribosome, including general tRNA substrates. Here, we describe the use of an afacid base, metal ion-assisted, substrate-assisted, or finity-tagging system for the purification of mutant electrostatic catalysis, as has been observed in other ribosomes and analysis of four universally conserved protein and RNA enzymes. nucleotides in the innermost layer of the active site: From a chemical perspective, peptide release is a A2451, U2506, U2585, and A2602. While pre-steadymore challenging reaction than peptide bond formation state kinetic analysis of the peptidyl transferase activbecause of the lower nucleophilicity of water relative to ity of the mutant ribosomes reveals substantially the primary amine of an amino acid. Our current underreduced rates of peptide bond formation using the standing of peptide release relies heavily on the obminimal substrate puromycin, their rates of peptide served conservation of a GGQ motif in class I release bond formation are unaffected when the substrates factors from eukaryotes to bacteria (Frolova et al., 1999). are intact aminoacyl-tRNAs. These mutant ribosomes Based on crystal structures of eRF1 and RF2 (Song et do, however, display substantial defects in peptide al., 2000; Vestergaard et al., 2001), it has been proposed release. These results reveal a view of the catalytic that this highly conserved motif plays a critical role in center in which an inner shell of conserved nucleotides coordinating a water molecule in the active site of the is pivotal for peptide release, while an outer shell is ribosome for participation in hydrolysis. Recent cryoEM responsible for promoting peptide bond formation. data and tethered chemical probing experiments have provided compelling evidence that this conserved GGQ
Ribosomal proteins contacting with deacylated tRNA in the S-site of the translating ribosome
FEBS Letters, 1981
A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome
Proceedings of the National Academy of Sciences, 1985
A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome (tRNA cross-linking/aminacyl site/peptidyl site/transladonal fidelity/kfrromycin) ABSTRACT Previously, we reported that the antibiotic kirromycin induces two tRNA-binding sites on the elongation factor Tu. The classical binding site (site I) binds aminoacyl-tRNA and, with much less affinity, deacylated tRNA. The kirromycin-induced site II binds aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA with comparable affinities. Accordingly, 3'-oxidized tRNA can be cross-linked in the presence of the antibiotic to two specific sites of EF-Tu: Lvs-237 and Lys-208. Here, we report that 3'-oxidized tRNAh, bound to a ribosome-poly(U) complex, can also be crosslinked to either one of these two sites. When located in the ribosomal peptidyl site, it cross-links exclusively to Lys-208; when located in the ribosomal aminoacyl site, it cross-links exclusively to Lys-237, irrespective of the presence of kirromycin. Since no cross-linking could be detected in the absence of ribosomes and kirromycin, we conclude that the tRNA-binding site II is induced upon interaction of aminoacyl-tRNA-EF-Tu-GTP with the ribosome-mRNA complex. The results indicate that, on the ribosome, EF-Tu interacts with peptidyl-sitebound peptidyl-tRNA through tRNA-binding site II and with aminoacyl-site-bound aminoacyl-tRNA through tRNA-binding site I.
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Contacts between the growing peptide chain and the 23S RNA in the 50S ribosomal subunit
Nucleic Acids Research, 1994
Peptides of defined length carrying a diazirine photoaffinity label attached either to the a-NH 2 group of the N-terminal methionine residue, or to the e-NH 2 group of an immediately adjacent lysine residue, were prepared in situ on Escherichia coli ribosomes in the presence of a synthetic mRNA analogue. Peptide growth was stopped simply by withholding the aminoacyl-tRNA cognate to an appropriate downstream codon. After photo-activation at 350 nm the sites of cross-linking to ribosomal RNA were determined by our standard procedures; the C-terminal amino acid of each peptide was labelled with tritium, in order to confirm whether the individual cross-linked complexes contained the expected 'full-length' peptide, as opposed to shorter products. The shortest peptides became cross-linked to sites within the 'peptidyl transferase ring' of the 23S RNA, namely to positions 2062, 2506, 2585 and 2609. However, already when the peptide was three or four residues long, a new crosslink was observed several hundred nucleotides away in another secondary structural domain; this site, at position 1781, lies within one of several RNA regions which have been implicated in other studies as being located close to the peptidyl transferase ring. Further application of this approach, combined with modelbuilding studies, should enable the path of the nascent peptide through the large ribosomal subunit to be definitively mapped. MATERIALS AND METHODS Preparation of mRNA, and tRNA derivatives An mRNA analogue was prepared by T7 transcription from a synthetic DNA template (9,11). This mRNA had the sequence GGG AGA AAG AAA AUG AAA UUC GAA CUG GAC ACC, carrying codons for methionine, lysine, phenylalanine and glutamic acid (M, K, F, E, underlined). Individual tRNA species
Quality control by the ribosome following peptide bond formation
Nature, 2009
The overall fidelity of protein synthesis has been thought to rely on the combined accuracy of two basic processes: the aminoacylation of transfer RNAs with their cognate amino acid by the aminoacyl-tRNA synthetases, and the selection of cognate aminoacyl-tRNAs by the ribosome in cooperation with the GTPase elongation factor EF-Tu. These two processes, which together ensure the specific acceptance of a correctly charged cognate tRNA into the aminoacyl (A) site, operate before peptide bond formation. Here we report the identification of an additional mechanism that contributes to high fidelity protein synthesis after peptidyl transfer, using a well-defined in vitro bacterial translation system. In this retrospective quality control step, the incorporation of an amino acid from a non-cognate tRNA into the growing polypeptide chain leads to a general loss of specificity in the A site of the ribosome, and thus to a propagation of errors that results in abortive termination of protein synthesis.
Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer
The EMBO Journal, 2000
Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the ®xation of the 3¢-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.
Structure of the ribosome with elongation factor G trapped in the pretranslocation state
Proceedings of the National Academy of Sciences, 2013
Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection
Proceedings of the National Academy of Sciences, 2020
The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond for...
Two Distinct Conformations of the Conserved RNA-rich Decoding Center of the Small Ribosomal Subunit Are Recognized by tRNAs and Release Factors
Cold Spring Harbor Symposia on Quantitative Biology, 2006
[Mechanism of tRNA translocation on the ribosome]
Molekuliarnaia biologiia
During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970-80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes of the ribosome and of EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.
The 2'-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC
The EMBO journal, 2011
The ribosome accelerates the rate of peptidyl transfer by >10(6)-fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2'-OH of the peptidyl-tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2'-OH of the peptidyl-tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100-fold slower with the dA76-substituted peptidyl-tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl-tRNA lacks the 2'-OH group. Additionally, the 2'-OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and ...
The transition state for formation of the peptide bond in the ribosome
Proceedings of the National Academy of Sciences, 2006
Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3 end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, E a, is 35.5 kcal (1 kcal ؍ 4.18 kJ)͞mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be Ϸ18 kcal͞mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2 OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments. quantum mechanics ͉ ribosomal symmetry ͉ activation energy ͉ quantum crystallography ͉ catalytic OH R ibosomal crystallography has revealed the detailed structure of the ribosome, the universal cell organelle translating the genetic code into proteins. Thus, the ribosome is a ribozyme exerting substrate positioning and promoting substratemediated catalysis (1-5).
Interactions of the ribosome with mRNA and tRNA
Current Opinion in Structural Biology, 2010
Recent collection of high-resolution crystal structures of the 70S ribosome with mRNA and tRNA substrates enhances our knowledge of protein synthesis principles. A novel network of interactions between the ribosome in the elongation state and mRNA downstream from the A codon suggests that mRNA is stabilized and aligned at the entrance to the decoding center. The X-ray studies clarify how natural modifications of tRNA are involved in the stabilization of the codon-anticodon interactions, prevention of frame-shifting and also expansion of the decoding capacity of tRNAs. In addition, the crystal structures provide the view that tRNA in the A and P sites communicate through a protein rich environment and suggest how these tRNAs are controlled through the intersubunit bridge formed by protein L31.
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