The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination

The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively. We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis. While mutations at A2451, U2585, and C2063 (E. coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation. This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.     

Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome.

The fidelity of aminoacyl-tRNA (aa-tRNA) selection by the bacterial ribosome is determined by initial selection before and proofreading after GTP hydrolysis by elongation factor Tu. Here we report the rate constants of A-site binding of a near-cognate aa-tRNA. The comparison with the data for cognate aa-tRNA reveals an additional, important contribution to aa-tRNA discrimination of conformational coupling by induced fit. It is found that two rearrangement steps that limit the chemical reactions of A-site binding, i.e. GTPase activation (preceding GTP hydrolysis) and A-site accommodation (preceding peptide bond formation), are substantially faster for cognate than for near-cognate aa-tRNA. This suggests an induced-fit mechanism of aa-tRNA discrimination on the ribosome that operates in both initial selection and proofreading. It is proposed that the cognate codon-anticodon interaction, more efficiently than the near-cognate one, induces a particular conformation of the decoding center of 16S rRNA, which in turn promotes GTPase activation and A-site accommodation of aa-tRNA, thereby accelerating the chemical steps. As kinetically favored incorporation of the correct substrate has also been suggested for DNA and RNA polymerases, the present findings indicate that induced fit may contribute to the fidelity of template-programed systems in general.     

Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs

The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.     

Distinct functional classes of ram mutations in 16S rRNA

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed “accommodation”) or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.     

Structural dynamics of ribosomal RNA during decoding on the ribosome

Decoding is a multistep process by which the ribosome accurately selects aminoacyl-tRNA (aa-tRNA) that matches the mRNA codon in the A site. The correct geometry of the codon-anticodon complex is monitored by the ribosome, resulting in conformational changes in the decoding center of the small (30S) ribosomal subunit by an induced-fit mechanism. The recognition of aa-tRNA is modulated by changes of the ribosome conformation in regions other than the decoding center that may either affect the architecture of the latter or alter the communication of the 30S subunit with the large (50S) subunit where the GTPase and peptidyl transferase centers are located. Correct codon-anticodon complex formation greatly accelerates the rates of GTP hydrolysis and peptide bond formation, indicating the importance of crosstalk between the subunits and the role of the 50S subunit in aa-tRNA selection. In the present review, recent results of the ribosome crystallography, cryoelectron microscopy (cryo-EM), genetics, rapid kinetics and biochemical approaches are reviewed which show that the dynamics of the structure of ribosomal RNA (rRNA) play a crucial role in decoding.     

Trial for peptide bond formation using model molecules based on the interactions between the CCA sequence of tRNA and 23S rRNA.

The peptidyl transfer reaction catalyzed by the ribosome is a sophisticated product of evolution. The molecular mechanism of peptide bond formation has not been fully elucidated although the essential involvement of 23S rRNA has been established. The universal CCA sequence at the 3'-end of tRNA plays an important role in this process, by interacting with specific nucleotides in 23S rRNA. However, reconstitution of peptidyl transferase activity by a naked 23S rRNA (without the help of any of the ribosomal proteins) has not been reported. To investigate the possible evolutionary development of the peptidyl transfer reaction, we tried to obtain peptide bond formation using a piece of tRNA--an aminoacyl-minihelix--mixed with sequence-specific oligonucleotides that contained puromycin. This system reproduced conceptually the equivalent interactions between the CCA trinucleotide of tRNA and 23S rRNA. Peptide bond formation was detected by gel electrophoresis, TLC and mass spectrometry. These results have implications for the evolution of the peptidyl transfer reaction in biological system.     

The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release

Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.     

Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.     

SPARK--a novel method to monitor ribosomal peptidyl transferase activity

The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.     

Conformational Sampling of Aminoacyl-tRNA during Selection on the Bacterial Ribosome

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.     

Recognition and selection of tRNA in translation

Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.     

Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome.

In order to map the rRNA environment of the acceptor end of tRNA in th e ribosome, hydroxyl radicals were generated in situ from Fe(II) attached via an EDTA linker to the 5' end of tRNA. Nucleotides in rRNA cleaved by the radicals were identified by primer extension, and assigned to the ribosomal A, P and E sites by standard criteria. In the A site, cleavages were found in the 2555-2573 region of 23S rRNA, around bases previously shown to be protected by A site tRNA, and in the alpha-sarcin loop, the site of interaction of elongation factors EF-Tu and EF-G. P site cleavages occurred in the 2250 loop, where a base pair is made with C74 of tRNA; and around the 2493 region in domain V. Interestingly, two clusters of nucleotides in 23S rRNA are accessible to both A site and P site tRNA probes. The first cluster is in the 1940-1965 region of domain IV, around the site of affinity labeling by the 3' end of tRNA, and the second cluster is around the bulged adenosine A2602, whose accessibility to chemical probes is enhanced by P site tRNA and decreased by A site tRNA. From the E site, cleavages occur in the 2390-2440 region, surrounding C2394, a base protected from dimethyl sulfate by E site tRNA, and in the phylogenetically variable stem at positions 1860/1880 of domain IV. Unexpectedly, no cleavages were detected in the central loop of domain V of 23S rRNA.     

A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m⁵U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome.     

A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.     

The ribosomal peptidyl transferase center: structure, function, evolution, inhibition

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring ribozymes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

Toward Ribosomal RNA Catalytic Activity in the Absence of Protein

The ribosome is the ribonucleoprotein particle responsible for translation of genetic information into proteins. The RNA component of the ribosome has been implicated as the catalytic entity for peptide bond formation based on protease resistance and structural data indicating an all-RNA active site. Nevertheless, peptidyl transfer by ribosomal RNA (rRNA) alone has not been demonstrated. In an attempt to show such activity we generated a minimal construct that comprises much of the 23S rRNA peptidyl transferase center, including the central loop and the A- and P-loops. This minimal rRNA domain was inactive in peptide bond formation under all conditions tested. The RNA was subsequently subjected to six rounds of in vitro selection designed to enrich for this activity. The result was a mutated rRNA sequence that could catalyze the covalent linkage of an A-site and P-site substrate; however, the product did not contain a peptide bond. The current study is an example of an in vitro derived alternate function of rRNA mutants and illustrates the evolutionary possibility that the protoribosome may have used amino acids as substrates before it gained the ability to join them into peptides. Though peptidyl transferase activity in the absence of protein remains elusive, the ease with which alternate catalytic activity was selected from rRNA with a small number of mutations suggests that rRNA may have inherent activity. This study represents a step on the path toward isolating that native activity. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Niles Lehman]     

Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner.

Ribosomes complexed with synthetic mRNA and peptidyl-tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide-releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non-hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + P(i) was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.     

The Ribosomal Peptidyl Transferase Center: Structure,Function, Evolution,Inhibition

ABSTRACT

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.