On the Re-Creation of Protoribosome Analogues in the Lab.

The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world.

Three Biopolymers and Origin of Life Scenarios.

To track down the possible roots of life, various models for the initial living system composed of different combinations of the three extant biopolymers, RNA, DNA, and proteins, are presented. The suitability of each molecular set is assessed according to its ability to emerge autonomously, sustain, and evolve continuously towards life as we know it. The analysis incorporates current biological knowledge gained from high-resolution structural data and large sequence datasets, together with experimental results concerned with RNA replication and with the activity demonstrated by standalone constructs of the ribosomal Peptidyl Transferase Center region. The scrutiny excludes the DNA-protein combination and assigns negligible likelihood to the existence of an RNA-DNA world, as well as to an RNA world that contained a replicase made of RNA. It points to the precedence of an RNA-protein system, whose model of emergence suggests specific processes whereby a coded proto-ribosome ribozyme, specifically aminoacylated proto-tRNAs and a proto-polymerase enzyme, could have autonomously emerged, cross-catalyzing the formation of each other. This molecular set constitutes a feasible starting point for a continuous evolutionary path, proceeding via natural processes from the inanimate matter towards life as we know it.

Prebiotic Assembly of Cloverleaf tRNA, Its Aminoacylation and the Origin of Coding, Inferred from Acceptor Stem Coding-Triplets.

tRNA is a key component in life's most fundamental process, the translation of the instructions contained in mRNA into proteins. Its role had to be executed as soon as the earliest translation emerged, but the questions of the prebiotic tRNA materialization, aminoacylation, and the origin of the coding triplets it carries are still open. Here, these questions are addressed by utilizing a distinct pattern of coding triplets highly conserved in the acceptor stems from the modern bacterial tRNAs of five early-emerging amino acids. Self-assembly of several copies of a short RNA oligonucleotide that carries a related pattern of coding triplets, via a simple and statistically feasible process, is suggested to result in a proto-tRNA model highly compatible with the cloverleaf secondary structure of the modern tRNA. Furthermore, these stem coding triplets evoke the possibility that they were involved in self-aminoacylation of proto-tRNAs prior to the emergence of the earliest synthetases, a process proposed to underlie the formation of the genetic code. Being capable of autonomous materialization and of self-aminoacylation, this verifiable model of the proto-tRNA advent adds principal components to an initial set of molecules and processes that may have led, exclusively through natural means, to the emergence of life.

Origin of life: protoribosome forms peptide bonds and links RNA and protein dominated worlds.

Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC-MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome's construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life.

Coding triplets in the tRNA acceptor-TΨC arm and their role in present and past tRNA recognition.

The mechanism and evolution of the recognition scheme between key components of the translation system, that is, tRNAs, synthetases, and elongation factors, are fundamental issues in understanding the translation of genetic information into proteins. Statistical analysis of bacterial tRNA sequences reveals that for six amino acids, a string of 10 nucleotides preceding the tRNA 3' end carries cognate coding triplets to nearly full extent. The triplets conserved in positions 63-67 are implicated in the recognition by the elongation factor EF-Tu, and those conserved in positions 68-72, in the identification of cognate tRNAs, and their derived minihelices by class IIa synthetases. These coding triplets are suggested to have primordial origin, being engaged in aminoacylation of prebiotic tRNAs and in the establishment of the canonical codon set.

Hypothesis: Spontaneous Advent of the Prebiotic Translation System via the Accumulation of L-Shaped RNA Elements.

The feasibility of self-assembly of a translation system from prebiotic random RNA chains is a question that is central to the ability to conceive life emerging by natural processes. The spontaneous materialization of a translation system would have required the autonomous formation of proto-transfer RNA (tRNA) and proto-ribosome molecules that are indispensable for translating an RNA chain into a polypeptide. Currently, the vestiges of a non-coded proto-ribosome, which could have only catalyzed the formation of a peptide bond between random amino acids, is consensually localized in the region encircling the peptidyl transferase center of the ribosomal large subunit. The work presented here suggests, based on high resolution structures of ribosomes complexed with messenger RNA (mRNA) and tRNAs, that three types of L-shaped RNA building blocks derived from the modern ribosome, alongside with an L-shaped proto-tRNA, each composed of about 70-mer, could have randomly occurred in the prebiotic world and combined to form a simple translation system. The model of the initial coded proto-ribosome, which includes the active sites of both ribosomal subunits, together with a bridging element, incorporates less than 6% of the current prokaryotic rRNA, yet it integrates all of the ribosomal components that are vital for synthesizing the earliest coded polypeptides.

Sequence complementarity at the ribosomal Peptidyl Transferase Centre implies self-replicating origin.

A feasible scenario for the emergence of life requires the spontaneous materialization and sustainability of a proto-ribosome that could have catalysed the formation of the first peptides. Models of proto-ribosomes were derived from the ribosomal Peptidyl Transferase Centre (PTC) region, but the poor prebiotic copying abilities give rise to the question of their mode of replication. Here, complementarity is demonstrated in bacterial ribosomes, between nucleotides that constitute the two halves of the PTC cavity. The complementarity corroborates the dimeric nature of the proto-ribosome and is likely to underlie the symmetry of the PTC region. Furthermore, it indicates a simple and efficient replication mode; the strand of each monomer could have acted as a template for the synthesis of its counterpart, forming a self-replicating ribozyme.

Could a Proto-Ribosome Emerge Spontaneously in the Prebiotic World?

An indispensable prerequisite for establishing a scenario of life emerging by natural processes is the requirement that the first simple proto-molecules could have had a realistic probability of self-assembly from random molecular polymers in the prebiotic world. The vestige of the proto-ribosome, which is believed to be still embedded in the contemporary ribosome, is used to assess the feasibility of such spontaneous emergence. Three concentric structural elements of different magnitudes, having a dimeric nature derived from the symmetrical region of the ribosomal large subunit, were suggested to constitute the vestige of the proto-ribosome. It is assumed to have materialized spontaneously in the prebiotic world, catalyzing non-coded peptide bond formation and simple elongation. Probabilistic and energetic considerations are applied in order to evaluate the suitability of the three contenders for being the initial proto-ribosome. The analysis points to the simplest proto-ribosome, comprised of a dimer of tRNA-like molecules presently embedded in the core of the symmetrical region, as the only one having a realistic statistical likelihood of spontaneous emergence from random RNA chains. Hence it offers a feasible starting point for a continuous evolutionary path from the prebiotic matter, through natural processes, into the intricate modern translation system.

A model for the role of isomerization in nascent peptide movement through the ribosomal tunnel.

The ribosome is a molecular machine whose manner of controlling the progression of the nascent chain through the ribosomal exit tunnel is currently unknown. A novel model for the mechanism driving the nascent chain motion is hereby presented, in which the ∼180° rotatory motion performed by each C-terminal amino acid of the nascent chain during its translocation from the A site to the P site, is suggested to twist the newly formed peptide bond into cis conformation. By catalyzing the cis to trans isomerization, the ribosome is proposed to release the potential energy stored within the cis conformer and to utilize it to push the chain down the tunnel, thus operating as a molecular motor. This hypothetical isomerization mechanism is supported by its ability to provide an explanation for the peculiar conduct observed in translational events of nascent chains with C-terminal prolines: the slow peptide bond formation with puromycin, translation arrest, and tmRNA tagging.

The dimeric proto-ribosome: Structural details and possible implications on the origin of life.

A symmetric pocket-like entity, composed of two L-shaped RNA units, encircles the peptide synthesis site within the contemporary ribosome. This entity was suggested to be the vestige of a dimeric proto-ribosome, which could have formed spontaneously in the prebiotic world, catalyzing non-coded peptide bond formation and elongation. This structural element, beyond offering the initial step in the evolution of translation, is hypothesized here to be linked to the origin of life. By catalyzing the production of random peptide chains, the proto-ribosome could have enabled the formation of primary enzymes, launching a process of co-evolution of the translation apparatus and the proteins, thus presenting an alternative to the RNA world hypothesis.

Ribosome's mode of function: myths, facts and recent results.

Ribosomes translate the genetic code into proteins in all living cells with extremely high efficiency, owing to their inherent flexibility and to their spectacular architecture. During the last 6 decades, extensive effort has been made to elucidate the molecular mechanisms associated with their function, and a quantum jump has been made in recent years, once the three dimensional structures of ribosomes and their functional complexes have been determined. These illuminated key issues in ribosome function, confirmed various biochemical, genetic, and medical findings, and revealed mechanistic details beyond previous expectation, thus leading to conceptual revolutions, and turning old myths into actual facts.

Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center.

During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.

The transition state for formation of the peptide bond in the ribosome.

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 approximately 18 kcal/mol. 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.

Symmetry at the active site of the ribosome: structural and functional implications.

The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3'-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3'-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2'-hydroxyl.

Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism.

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.

Ribosomal crystallography: peptide bond formation and its inhibition.

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. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

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 two-fold 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.

Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.

Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.

Antibiotics targeting ribosomes: crystallographic studies.

Resistance to antibiotics is a major problem in modern therapeutics. Ribosomes, the cellular organelle catalyzing the translation of the genetic code into proteins, are targets for several clinically relevant antibiotics. The ribosomes from eubacteria are excellent pathogen models. High resolution structures of the large and small ribosomal subunits were used as references that allowed unambiguous localization of almost a dozen antibiotic drugs, most of which are clinically relevant. Analyses of these structures showed a great diversity in the antibiotics' modes of action, such as interference with substrate binding, hindrance of the mobility required for the biosynthetic process and the blockage of tunnel which provides the path of exit for nascent proteins. All antibiotics studied by us were found to bind primarily to ribosomal RNA and, except for one allosteric effect, their binding did not cause major conformational changes. Antibiotics of the small ribosomal subunit may hinder tRNA binding, decoding, translocation, and the initiation of the entire biosynthetic process. The large subunit agents may target the GTPase center, interfere with peptide bond formation, or block the entrance of the nascent protein exit tunnel. The overall structure of the peptidyl transferase center and the modes of action of the antibiotic agents indicate that the ribosome serves as a template for proper positioning of tRNAs, rather than participating actively in the catalytic events associated with the creation of peptide bonds.