Allosteric Three-site Model Research Articles

Further in Vitro Exploration Fails to Support the Allosteric Three-site Model

Ongoing debate in the ribosome field has focused on the role of bound E-site tRNA and the Shine-Dalgarno-anti-Shine-Dalgarno (SD-aSD) interaction on A-site tRNA interactions and the fidelity of tRNA selection. Here we use an in vitro reconstituted Escherichia coli translation system to explore the reported effects of E-site-bound tRNA and SD-aSD interactions on tRNA selection events and find no evidence for allosteric coupling. A large set of experiments exploring the role of the E-site tRNA in miscoding failed to recapitulate the observations of earlier studies (Di Giacco, V., Márquez, V., Qin, Y., Pech, M., Triana-Alonso, F. J., Wilson, D. N., and Nierhaus, K. H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 10715-10720 and Geigenmüller, U., and Nierhaus, K. H. (1990) EMBO J. 9, 4527-4533); the frequency of miscoding was unaffected by the presence of E-site-bound cognate tRNA. Moreover, our data provide clear evidence that the reported effects of the SD-aSD interaction on fidelity can be attributed to the binding of ribosomes to an unanticipated site on the mRNA (in the absence of the SD sequence) that provides a cognate pairing codon leading naturally to incorporation of the purported "noncognate" amino acid.

Ribosomal tRNA Binding Sites: Three-Site Models of Translation

The first models of translation described protein synthesis in terms of two operationally defined tRNA binding sites, the P-site for the donor substrate, the peptidyl-tRNA, and the A-site for the acceptor substrates, the aminoacyl-tRNAs. The discovery and analysis of the third tRNA binding site, the E-site specific for deacylated tRNAs, resulted in the allosteric three-site model, the two major features of which are (1) the reciprocal relationship of A-site and E-site occupation, and (2) simultaneous codon-anticodon interactions of both tRNAs present at the elongating ribosome. However, structural studies do not support the three operationally defined sites in a simple fashion as three topographically fixed entities, thus leading to new concepts of tRNA binding and movement: (1) the hybrid-site model describes the tRNAs' movement through the ribosome in terms of changing binding sites on the 30S and 50S subunits in an alternating fashion. The tRNAs thereby pass through hybrid binding states. (2) The α-ε model introduces the concept of a movable tRNA-binding domain comprising two binding sites, termed α and ε. The translocation movement is seen as a result of a conformational change of the ribosome rather than as a diffusion process between fixed binding sites. The α-ε model reconciles most of the experimental data currently available.

Models of the elongation cycle: an evaluation.

The central process for the transfer of the genetic information from the nucleic acid world into the structure of proteins is the ribosomal elongation cycle, where the sequence of codons is translated into the sequence of amino acids. The nascent polypeptide chain is elongated by one amino acid during the reactions of one cycle. Essentially, three models for the elongation cycle have been proposed. The allosteric three-site model and the hybrid-site model describe different aspects of tRNA binding and do not necessarily contradict each other. However, the alpha-epsilon model is not compatible with both models. The three models are evaluated in the light of recent results on the tRNA localization within the ribosome: the tRNAs of the elongating ribosome could be localized by two different techniques, viz. an advanced method of small-angle neutron scattering and cryo-electron microscopy. The best fit with the biochemical and structural data is obtained with the alpha-epsilon model.

Are the current three-site models valid descriptions of the ribosomal elongation cycle?

In the early eighties a third tRNA binding site, the E site, was found in addition to the classical A and P sites; the E site seems to be a universal feature of ribosomes (for review see ref. 1). Two models exist that describe different aspects of the three sites. The hybrid-site model is based on protection experiments (2) introducing hybrid states of tRNA binding: The tRNAs move alternately on one or the other subunits through the tRNA binding sites in the course of elongation. An analysis of the functional interplay of the three ribosomal sites led to the second model, the allosteric three-site model. The key features are that the deacylated tRNA is stably bound to the E site in a codon-dependent manner and that the A site and the E site are reciprocally linked (3). Both models highlight different features of the elongation cycle and do not necessarily exclude each other.

The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli.

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.

Solution of the ribosome riddle: how the ribosome selects the correct aminoacyl‐tRNA out of 41 similar contestants

Three tRNA binding sites, the A, P and E sites, have been demonstrated on ribosomes of bacterial, archaebacterial and eukaryotic origin. In all these cases the first and the third site, the A and the E site, are allosterically coupled in the sense of a negative co-operativity. Therefore, the allosteric three-site model seems to be a generally valid description of the ribosomal elongation phase, where in a cycle of reactions the nascent peptide chain is prolonged by one amino acid. The molecular concept of the allosteric three-site model explains the astonishing ability of the ribosome to select the correct substrate out of a large number of very similar substrates, and it provides a framework within which the mechanisms of the elongation factors could be understood.

The two main states of the elongating ribosome and the role of the α-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 Mg 2+ 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 α-sarcin abolishes the functions of both elongation factors on the ribosome. This observation implies that the α-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 complemetary to the α-sarcin region induce a conformational change in the 50S subunits; this conformational change causes an irreversible dissociation of tightly coupled ribosomes upon sucrose-gradient cetrifugation. The 50S remained in its inactive state even after removal of the hybridized DNA probe. The results suggest that the α-sarcin region plays a pivotal role in lowering the activation barriers of the conformational transitions between the post- and pre-translocational states. However, the presence of an elongation factor is essential for establishing a defined pre- or post-translocational state.

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 cooperativity. 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.

Similarities and differences in the inhibition patterns of thiostrepton and viomycin: Evidence for two functionally different populations of P sites when occupied with AcPhe-tRNA

According to the allosteric three-site model for the ribosomal elongation cycle, the reactions from the pre- to the post-translocational state and vice versa represent allosteric transitions which are catalyzed by elongation factor (EF)-G and EF-Tu, respectively. It has been shown recently that the non-related antibiotics thisotrepton and viomycin inhibit protein biosynthesis via a surprisingly similar mechanism. Both drugs primarily block the allosteric transitions in either direction (Hausner et al. (1988) J. Biol. Chem. 263, 13103–13111). Here we show that the secondary effects of these antibiotics differ strikingly. When the P site of poly(U) programmed ribosomes is quantitatively filled with AcPhe-tRNA, thiostrepton stimulates the rate of the formation of AcPhe-puromycin 2-fold, whereas viomycin inhibits the puromycin reaction (up to 75% inhibition). The thiostrepton-dependent stimulation is only observed when the drug is given before the P site is occupied; when thiostrepton is added after pre-filling the P site, the peptidyltransferase activity is not affected, in contrast to the translocation reaction, which is blocked irrespective of whether the drug is administered before or after tRNA is bound. The effects of both drugs became distinctly more pronounced when the P sites were saturated with AcPhe-tRNA as compared to half-saturated ribosomes. We conclude that roughly one half of the ribosomes, which first bind AcPhe-tRNA to the P site, carry this ligand in a different orientation to that of the second half of the ribosome population. These two populations probably reflect the P site in the pre- and post-translocational state, respectively.

The allosteric three-site model for the ribosomal elongation cycle: features and future

The ribosome contains three binding sites for tRNA, viz., the A site for aminoacyl-tRNA (decoding site), the P site for peptidyl-tRNA, and the E site for deacylated tRNA (E for exit). The surprising finding of an allosteric linkage between the E and A sites in the sense of a negative cooperativity has three consequences: (a) it improves the proper selection of aminoacyl-tRNAs while preventing interference from noncognate aminoacyl-tRNAs in the decoding process, (b) it provides an explanation for the ribosomal accuracy without having to resort to the proofreading hypothesis, and (c) it has deepened our understanding of the mode of action of some antibiotics.

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 allosteric three-site model for the ribosomal elongation cycle: Analysis with a heteropolymeric mRNA

Abstract Ribosomal tRNA binding studies and functional tests were performed at 6 mM Mg2+ using the mRNA analogue C17AUGA4C17 which contains three unique codons in its central region. The following results were obtained. 1) The relative binding affinities of 20 different deacylated tRNAs to nonprogrammed 70 S ribosomes were assessed and were found to vary substantially. 2) When added as the first tRNA, fMet-tRNA and deacylated tRNAs (but not N-acetylated aminoacyl-tRNAs) can bind to internal codons of the mRNA and are therefore suitable for setting the reading frame via codon-anticodon interaction in the peptidyl-tRNA site (P site). 3) After prefilling the P site with deacylated tRNA, the exit site for deacylated tRNA (E site) can be quantitatively occupied only if the cognate codon is present at that site. 4) The translocation of peptidyl-tRNA from the aminoacyl-tRNA site (A site) to the P site is not accompanied by a release of deacylated tRNA. The codon sequence excludes a release and rebinding of deacylated tRNA to the newly exposed A site. Rather, the deacylated tRNA is cotranslocated from the P to the E site where it remains stably bound. 5) After one round of elongation, addition of an A site ligand triggers the dissociation of deacylated tRNA from the E site. Conversely, E site occupation reduces the affinity of the A site for N-acetylated aminoacyl-tRNA. Thus, A and E sites are allosterically linked via negative cooperativity. The results support the allosteric three-site model as an appropriate description of the ribosomal elongation cycle.

The allosteric three-site model for the ribosomal elongation cycle. New insights into the inhibition mechanisms of aminoglycosides, thiostrepton, and viomycin.

According to the allosteric three-site model for the ribosomal elongation cycle (Rheinberger, H.J. and Nierhaus, K.H. (1986) J. Biol. Chem. 261, 9133-9139), two types of A site (aminoacyl-tRNA site) occupation exist. First is the A site occupation after initiation (i-type), with only one site, the P site (peptidyl-tRNA site), being prefilled with a tRNA (initiator tRNA). Second is the A site occupation after an elongation cycle (e-type), with two prefilled sites, namely the P and E sites containing peptidyl-tRNA and deacylated tRNA, respectively. The individual reactions of the elongation cycle were tested, including both types of A site occupation in the presence of various antibiotics. A test system was used allowing the functional studies to be made with quantitative tRNA binding at 6 mM Mg2+. The following results were obtained: 1) thiostrepton (5 x 10(-6) M) induced a complete block of both EF-(elongation factor) G dependent and EF-G independent translocation, in agreement with older observations. The A-site occupation of the e-type was severely inhibited in contrast to that of the i-type. Thus, thiostrepton blocks the allosteric transitions in both directions, i.e. the transition from pre- to post-translocational state (translocation) and that from the post- to the pre-translocational state (A site occupation of the e-type). In addition the ribosomal binding of EF-G.[3H] GMPPNP was inhibited by about 60%. 2) Similarly, viomycin (5 x 10(-5) M) appears to be an inhibitor of both allosteric transitions, since it strongly inhibited the e-type (but not the i-type) A site occupation in addition to translocation. 3) The aminoglycosides streptomycin, hygromycin B, neomycin, kanamycin, and gentamicin prevented A site occupation of the e-type (residual activity below 15%). Neomycin and hygromycin, in addition, blocked the translocation reaction. Only marginal effects were observed with A site occupation of the i-type. It appears that the inhibition of the A site binding of the e-type (allosteric transition from the post- to the pretranslocational state) is the predominant effect of the misreading-inducing aminoglycosides.

The Ribosomal E Site at Low Mg2+: Coordinate Inactivation of Ribosomal Functions at Mg2+Concentrations Below 10 mM and its Prevention by Polyamines

Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM. The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J. Biol. Chem. 261, 9133 (1986] are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.