Is the three-site model for the ribosomal elongation cycle sound?

The release of deacylated tRNA from the ribosome as a result of translocation has been studied. Translating ribosomes prepared with poly(U)-S-S-Sepharose columns have been used. It has been shown that deacylated tRNA released from the ribosomal P site as a result of translocation rebinds with the vacated A site. Consistent with the known properties of the A site of the ribosome, this interaction is reversible, Mg2+-dependent, codon-specific and is inhibited by the antibiotic tetracycline. It has been concluded that the proposed three-site model of the ribosomal elongation cycle (Rheinberger and Nierhaus (1983) Proc. Natl. Acad. Sci. USA 80, 4213-4217) is not sound: the experimentally observed 'retention' of the deacylated tRNA on the ribosome after translocation can be explained by a codon-dependent rebinding to the A site, rather than by its transition to the 'E site', i.e., in terms of the classical two-site model.     

Testing the classical two-tRNA-site model for the ribosomal elongation cycle

An experimental system where the elongation of a polypeptide (polyphenylalanine) is performed stepwise and synchronously by purified Escherichia coli ribosome in a matrix-coupled poly (U) column is proposed for testing the number of non-overlapping tRNA binding sites on the elongating ribosome. If phenylalanyl[3H]tRNA is introduced into the column and bound with the ribosomes at the beginning of a given elongation cycle, deacylated [3H]tRNA is shown to be released from the ribosomes and comes out from the column at the translocation step of the next elongation cycle. The result obtained is fully predicted by the classical two-tRNA-site model and contradicts any model involving more than two non-overlapping high-affinity tRNA binding sites in the ribosomal elongation cycle.     

Spontaneous intersubunit rotation in single ribosomes

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.     

The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes

The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in [32P]orthophosphoric acid for about four generations. The cells were harvested in the logarithmic phase, resuspended in a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees C in the presence of 3H-labelled amino acids. This procedure preferentially labels growing peptidyl chains. Polysomes were isolated, the fraction in the post-translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis. The data revealed that at least 75% of post-translocational ribosomes in isolated native polysomes carry a tRNA in their E site. The results are consistent with the allosteric three-site model for the elongation cycle but disagree with the two-site model.     

The function of the translating ribosome: allosteric three-site model of elongation.

During the last decade, a new model for the ribosomal elongation cycle has emerged. It is based on the finding that eubacterial ribosomes possess 3 tRNA binding sites. More recently, this has been confirmed for archaebacterial and eukaryotic ribosomes as well, and thus appears to be a universal feature of the protein synthetic machinery. Ribosomes from organisms of all 3 kingdoms harbor, in addition to the classical P and A sites, an E site (E for exit), into which deacylated tRNA is displaced during translocation, and from which it is expelled by the binding of an aminoacyl-tRNA to the A site at the beginning of the subsequent elongation round. The main features of the allosteric 3-site model of ribosomal elongation are the following: first, the third tRNA binding site is located 'upstream' adjacent to the P site with respect to the messenger, ie on the 5'-side of the P site. Second, during translocation, deacylated tRNA does not leave the ribosome from the P site, but co-translocates from the P site to the E site--when peptidyl-tRNA translocates from the A site to the P site. Third, deacylated tRNA is tightly bound to the E site in the post-translocational state, where it undergoes codon--anticodon interaction. Fourth, the elongating ribosome oscillates between 2 main conformations: (i), the pre-translocational conformer, where aminoacyl-tRNA (or peptidyl-tRNA) and peptidyl-tRNA (or deacylated tRNA) are firmly bound to the A and P sites, respectively; and (ii), the post-translocational conformer, where peptidyl-tRNA and deacylated tRNA are firmly bound to the P and E sites, respectively. The transition between the 2 states is regulated in an allosteric manner via negative cooperatively. It is modulated in a symmetrical fashion by the 2 elongation factors Tu and G. An elongating ribosome always maintains 2 high-affinity tRNA binding sites with 2 adjacent codon--anticodon interactions. The allosteric transition from the post- to the pre-translocational state is involved in the accuracy of aminoacyl-tRNA selection, and the maintenance of 2 codon--anticodon interactions helps to keep the messenger in frame during translation.     

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP^R) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP^R-eEF2 to examine the effects of DT in vivo. ADP^R of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP^R-eEF2.     

Ribosome + protein elongation factors of the ribosomal cycle as a single enzyme. Why are protein factors needed?]

Model of ribosomal cycle with participation of protein elongation factors is discussed. According to this model both elongation factors should help to ribosome to discriminate substrates versus products of reactions. For aminoacyl site it is a discrimination of aa-tRNA versus tRNA, and for peptidyl site-discrimination of peptidyl-tRNA versus tRNA.     

Allosteric interactions between the ribosomal transfer RNA-binding sites A and E

We have previously proposed a three-site model for the elongation cycle. The model is characterized by the presence of two tRNAs on the ribosome before and after translocation. We have already shown a first consequence of the model, namely that the translocation reaction is not coupled with a release of deacylated tRNA. Here we demonstrate the following conclusions. Occupation of the A site triggers the tRNA release from the E site, i.e. the A site occupation induces a drastic decrease in the affinity of the E site for deacylated tRNA. In the concentration range of deacylated tRNA in which a ribosome binds a second tRNA in addition to that one already present at the P site the deacylated tRNA does not compete for one and the same binding site with an A site ligand (AcPhe-tRNA) at 37 degrees C. It follows that the second deacylated tRNA binds to a site, the E site, which is physically distinct from the A site. When the ribosome binds a deacylated tRNA at the E site (in addition to a tRNA at the P site), the A site cannot be occupied by AcPhe-tRNA at 0 degree C and only poorly by the ternary complex elongation factor Tu . Phe-tRNA . guanyl-5'-yl imidodiphosphate. At 37 degrees C a significant A site binding is observed, with a corresponding tRNA release from the E site. In contrast, if the E site is free and only the P site occupied, the A site can bind significant amounts of charged tRNA already at 0 degree C. It follows that an occupied E site induces a low-affinity state of the A site. Thus, the ribosome always contains two high-affinity binding sites, which are A and P sites before and P and E sites after translocation. A and E sites are allosterically linked in a bidirectional manner.     

Interaction of tRNA with ribosomes

Definition of the site of tRNA-binding to ribosomes is suggested on the basis of a free energy of tRNA-ribosome interaction. From this point of view disagreements that have arisen in recent years concerning the numbers of tRNA binding sites on the ribosome, their distribution between subunits, the properties of the third site E in ribosomes and the compatibility of new experimental data with different models of elongation cycle are discussed. The observation of the third site in the ribosome (messenger independent and with a presumably exit function) is not a refutation but an extension of Watson's model of translating ribosome.     

Cotranslational Folding of Proteins

We suppose that folding of proteins occurs cotranslationally by the following scheme. The polypeptide chains enter the folding sites from protein translocation complexes (ribosome, translocation machinery incorporated in membranes) directionally with the N-terminus and gradually. The chain starts to fold as soon as its N-terminal residue enters the folding site from the translocation complex. The folding process accompanies the translocation of the chain to its folding site and is completed after the C-terminal residue leaves the translocation complex. Proteins fold in sequential stages, by translocation of their polypeptide into folding compartments. At each stage a particular conformation of the N-terminal part of the chain that has emerged from the translocation complex is formed. The formation of both the particular conformations of the N-terminal chain segment at each folding stage and the final native protein conformation at the last stage occurs in a time that does not exceed the duration of the fastest elongation cycle on the ribosome.     

Kinetic and thermodynamic parameters for tRNA binding to the ribosome and for the translocation reaction.

Kinetic analyses of tRNA binding to the ribosome and of the translocation reaction showed the following results. 1) The activation energy for the P site binding of AcPhe-tRNA to poly(U)-programmed ribosomes is relatively high (Ea = 72 kJ mol-1; 15 mM Mg2+). If only the P site is occupied with deacylated tRNA(Phe), then the E site can be filled more easily with tRNA(Phe) (no activation energy measurable) than the A site with AcPhe-tRNA (Ea = 47 kJ mol-1; 15 mM Mg2+). 2) A ribosome with blocked P and E sites represents a standard state of the elongation cycle, in contrast to a ribosome with only a filled P site. The two states differ in that AcPhe-tRNA binding to the A site of a ribosome with prefilled P and E sites requires much higher activation energy (87 versus 47 kJ mol-1). The latter reaction simulates the allosteric transition from the post- to the pretranslocational state, whereby the tRNA(Phe) is released from the E site upon occupation of the A site (Rheinberger, H.-J., and Nierhaus, K. H. (1986) J. Biol. Chem. 261, 9133-9139). The reversed transition from the pre- to the posttranslocational state (translocation reaction) requires about the same activation energy (90 kJ mol-1). 3) Both elongation factors EF-Tu and EF-G drastically reduce the respective activation energies. 4) The rate of the A site occupation is slower than the rate of translocation in the presence of the respective elongation factors. The data suggest that the A site occupation rather than, as generally assumed, the translocation reaction is the rate-limiting step of the elongation cycle.     

The enzymatic binding of aminoacyl-tRNA to reticulocyte ribosomes: The stimulatory effect of donor site bound peptidyl-tRNA

The affinity of ribosomes for the elongation factors EF₁ and EF₂ changes while the ribosome is going through the different steps of the elongation cycle. In this communication we provide evidence that the affinity of the EF₁-aa-tRNA-GTP complex for the ribosomal acceptor site differs for ribosomes having their donor site either vacant or occupied by peptidyl-tRNA or by uncharged tRNA. Ribosomes having peptidyl-tRNA at their donor site bind the EF₁ complex with the highest affinity.Results are discussed in light of recent findings that the two elongation factors are not bound to the ribosome simultaneously.     

The two main states of the elongating ribosome and the role of the alpha-sarcin stem-loop structure of 23S RNA.

According to the allosteric three-site model of the elongation cycle the ribosome oscillates between two main-functional states, viz the pre-translocational state with occupied A and P sites (E site with low affinity) and the post-translocational state with occupied P and E sites (A site with low affinity). This proposition could be confirmed by a determination of the thermodynamic parameters. High activation-energy barriers were found between both states, namely about 90 kJ mol-1 at 15 mM Mg2+ for either transition (post----pre transition = A-site binding and pre----post transition = translocation). The various A-site states (binding of ternary complex, EF-Tu dependent GTP cleavage, peptide-bond formation) are not separated by significant activation-energy barriers. The rate-limiting step of the elongation cycle is A-site binding, and not translocation as assumed previously. The principal role of both elongation factors is the reduction of the respective activation-energy barrier, thus accelerating the rate of the elongation cycle by several orders of magnitude. Cleavage of a single phosphodiester bond after G2661 of 23S rRNA by the RNase alpha-sarcin abolishes the functions of both elongation factors on the ribosome. This observation implies that the alpha-sarcin stem-loop structure plays an important role in the ribosomal conformational changes involved in the allosteric transitions. Indeed we could demonstrate that suitable oligodeoxynucleotide probes complementary to the alpha-sarcin region induce a conformational change in the 50S subunits; this conformational change causes an irreversible dissociation of tightly coupled ribosomes upon sucrose-gradient centrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elongation factors on the ribosome

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.     

Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA

Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNA^Val and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.     

Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome

GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation. The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome. Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis. Another conserved arginine, R59, which is homologous to the catalytic arginine of G(alpha) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation. These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different.     

Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu.GDP and aurodox

Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.     

Mechanism of ribosomal translocation. tRNA binds transiently to an exit site before leaving the ribosome during translocation

Escherichia coli ribosomes have a site (E) to which deacylated tRNA binds transiently before leaving the ribosome during translocation. The affinity of the site is Mg2+ dependent and low at physiological Mg2+ concentrations. Correct codon-anticodon interaction is unnecessary in this site. With these features, the E site cannot reduce frameshift errors through additional mRNA anchorage. Occupancy of the A site does not influence the tRNA binding in the E site, although a conformational change of elongation factor G, brought about by GTP hydrolysis, is necessary for efficient tRNA release. The tRNA can dissociate unhindered from the E site when the elongation factor is bound to the ribosome by fusidic acid. During elongation, the thermodynamically stable state is not attained, since E site occupation inhibits translocation. However, the E site can aid elongation by providing an intermediate state for tRNA dissociation, dispersing the process into more than one step.