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.     

Single-molecule study of viomycin's inhibition mechanism on ribosome translocation

Viomycin belongs to the tuberactinomycin family of antibiotics against tuberculosis. However, its inhibition mechanism remains elusive. Although it is clear that viomycin inhibits the ribosome intersubunit ratcheting, there are contradictory reports about whether the antibiotic viomycin stabilizes the tRNA hybrid or classical state. By using a single-molecule FRET method to directly observe the tRNA dynamics relative to ribosomal protein L27, we have found that viomycin trapped the hybrid state within certain ribosome subgroups but did not significantly suppress the tRNA dynamics. The persistent fluctuation of tRNA implied that tRNA motions were decoupled from the ribosome intersubunit ratcheting. Viomycin also promoted peptidyl-tRNA fluctuation in the posttranslocation complex, implying that, in addition to acylated P-site tRNA, the decoding center also played an important role of ribosome locking after translocation. Therefore, viomycin inhibits translocation by trapping the hybrid state in the pretranslocation complex and disturbing the stability of posttranslocation complex. Our results imply that ribosome translocation is possibly a synergistic process of multiple decoupled local dynamics.     

Periodic conformational changes in rRNA: monitoring the dynamics of translating ribosomes

In protein synthesis, a tRNA transits the ribosome via consecutive binding to the A (acceptor), P (peptidyl), and E (exit) site; these tRNA movements are catalyzed by elongation factor G (EF-G) and GTP. Site-specific Pb2+ cleavage was applied to trace tertiary alterations in tRNA and all rRNAs on pre- and posttranslocational ribosomes. The cleavage pattern of deacylated tRNA and AcPhe-tRNA changed individually upon binding to the ribosome; however, these different conformations were unaffected by translocation. On the other hand, translocation affects 23S rRNA structure. Significantly, the Pb2+ cleavage pattern near the peptidyl transferase center was different before and after translocation. This structural rearrangement emerged periodically during elongation, thus providing evidence for a dynamic and mobile role of 23S rRNA in translocation.     

Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.     

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.     

Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues

The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA [Wagner, T., Cramer, F., & Sprinzl, M. (1982) Biochemistry 21, 1521-1529]. This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site. In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited. The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA. If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

Nonenzymic translocation and spontaneous release of noncognate peptidyl transfer ribonucleic acid from Escherichia coli ribosomes

Poly(uridylic acid)-programmed ribosomes have been used to synthesize the noncognate peptidyl-tRNA Ac-Phe-Tyr-tRNATyr and its cognate counterpart Ac(Phe)2-tRNAPhe. After synthesis, Ac(Phe)2-tRNAPhe remains, as expected, in the ribosomal acceptor (A) site, but the noncognate AcPhe-Tyr-tRNATyr does not; part of it spontaneously falls off the ribosome and the rest translocates, without elongation factor (EF) G, to the ribosomal donor site. The inhibitor of translocation viomycin prevents both the spontaneous release and the nonenzymatic translocation by confining the noncognate peptidyl-tRNA to the A site. Under these conditions, the interaction of AcPhe-Tyr-tRNATyr with the A site appears to be similar to that of Ac(Phe)2-tRNAPhe without the antibiotic, and EF-G promotes the translocation and subsequent elongation of both peptidyl-tRNAs to comparable extents. The results indicate that, without viomycin, the noncognate peptidyl-tRNA is weakly held in the ribosomal A site and support the proposal that the release of peptidyl-tRNA occurring during protein synthesis in vivo is related to a ribosomal editing mechanism which discards mistranslated nascent proteins [Menninger, J. R. (1977) Mech. Ageing Dev. 6, 131].     

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.     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.     

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.     

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.     

The distance between the anticodon loops of two tRNAs bound to the 70 S Escherichia coli ribosome.

Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNA_Y^Phe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNA_Pf^Phe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNA_Y^Phe or tRNA_Pf^Phe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.     

tRNA binding sites on the subunits of Escherichia coli ribosomes

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.     

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.     

Escherichia coli ribosomes having peptidyl-tRNA and deacylated tRNA at the A- and P-sites, respectively, may not be competent in translocation]

Ribosomes can have two states at 0 degree C: competent and noncompetent in translocation. In both states poly(U)-programmed ribosomes bind phenylalanyl-tRNA to A and P sites and form peptide bond. Elongation factor G promotes fast translocation in competent ribosomes and makes them noncompetent ones. Initial correlation between competent and noncompetent ribosomes is 2:1. Addition of deacylated tRNA does not influence phenomenon described as well as thermal reactivation of the ribosomes before beginning of the experiments. The possibility of deacylated tRNA translocation is shown. The translocation does not occurred provided that at least one of the ribosome sites is filled with shortened tRNA analog (tRNA with truncated CCA-end or tRNA anticodon arm).     

Function of the ribosomal E-site: a mutagenesis study

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.     

Binding of the 3'' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.