Gly Leu

Gly Leu landing site

Figure 12.10. The ribosome hopping over 50 nucleotides during translation of bacteriophage T4 DNA topoisomerase mRNA (gene 60 transcript) (W.M. Huang, S.Z. Ao, S. Casjens, R. Orlandi, R. Zeikus, R. Weiss, D. Winge & M. Fang, Science 239, 1005-1012 , 1988).

(Adapted from R. Weiss, D. Dunn, J. Atkins & R. Gesteland, in "The Ribosome: Structure, Function, and Evolution ", W.E. Hill, A. Dahlberg, R.A. Garrett, P.B. Moore, D. Schlessinger & J.R. Warner, eds., p.p. 534-540, ASM Press, Washington, DC).

phenomenon can be designated "trans translation" (Atkins & Gesteland, 1996). E. coli cells and cell-free extracts contain an RNA species called 10Sa RNA (363-nucleotide long) that encodes for a decapeptide (Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala) found as a C-terminal extension of different incomplete (truncated) proteins. This extension serves as a "tag" recognized by a proteolytic degradation system of the bacterium. At the same time the 10Sa RNA has been shown to be aminoacylated at its 3'-end by alanine, like tRNA. When the ribosome reads a truncated mRNA or a synthetic message without stop codon, it halts at the 3'-end; the A site becomes empty. The 10Sa RNA enters the empty, codon-free A site; as a result of transpeptidation, its alanyl residue is found to be added to the nascent polypeptide encoded by the truncated mRNA, and the elongated peptidyl-10Sa RNA occupies the position of a peptidyl-tRNA in the pre-translocation state ribosome (Fig. 12.11). Seemingly during translocation of the 10Sa RNA from the A site to the P site the ribosome hops on the first codon of the coding sequence of the 10Sa RNA. Then it normally translates ten codons of this coding sequence and terminates at the stop codon of the 10Sa RNA. Thus 11 amino acids become added to the C-terminus of a truncated protein, one (non-encoded, "junction" alanine) being brought by the 10Sa RNA as its 3'-bonded residue and other ten being annexed as the 10Sa RNA-encoded sequence (see the sequence above).

The facts of exclusively downstream movement of mRNA during ribosome hopping may suggest the existence of some mechanism or force for active one-directional displacement of a message through the ribosome, independent of the translocation of tRNA residues. This cannot be excluded, especially taking in mind that mRNA is a polar polymer and therefore the movements forward and backward can make difference (a ratchet). However, a simple explanation of the "downstream driving force" for mRNA is possible: in the polyribosome, if a ribosome pauses or stops, the following ribosome will approach the preceding one and push it downstream along mRNA (or, what is the same, pull out mRNA from it) during translocation.

Figure 12.11. Model for the involvement of 10Sa RNA in tagging truncated proteins by trans-translation. (Reproduced from B. Felden, H. Himeno, A. Muto, J.P. McCutcheon, J.F. Atkins & R.F. Gesteland, RNA 3, 89-103, 1997, with permission). When the ribosome translates a truncated mRNA or a synthetic polynucleotide without a stop codon, it stalls at the 3'-end of the message without termination. In this case the polypeptide synthesized remains bound to a tRNA in the P site. 10Sa RNA acylated by alanine binds to the vacant A site of the ribosome. Transpeptidation between the peptidyl-tRNA of the P site and the alanyl-10Sa RNA results in the formation of the alanine-elongated peptidyl-10Sa RNA. During translocation the ribosome switches messages and start to translate the open reading frame of the 10Sa RNA producing the elongation of the polypeptide by an additional amino acid sequence AlaAsnAspGluAsnTyrAlaLeuAlaAla. This sequence serves as a tag for rapid degradation of the released polypeptide by cellular proteases. (G.-F. Tu, G.E. Reid, J.-G. Zhang, R.L. Moritz & R.J. Simpson, J. Biol. Chem. 270, 9322-9326, 1995; K.C. Keiler, P.R.H. Waller & R.T. Sauer, Science 271, 990-993, 1996; A. Muto, M. Sato, T. Tadaki, M. Fukushima, C. Ushida & H. Himeno, Biochimie 78: 985-991, 1996).

12.5. Mechanics and Energetics of Translocation 12.5.1.Stereochemistry and Mechanics

The evidence available on the structure of tRNA and the ribosome as well as on the properties of pre-translocation and post-translocation complexes can be used to formulate a plausible stereochemical model of the interactions between the ribosome, two tRNAs, and mRNA, and of the changes in these relationships during translocation. As has been noted in stereochemical consideration of the codon-anticodon interaction (Section 10.1.5), the anticodons of two ribosome-bound tRNAs form double-helical structures of the A-form type with two adjacent mRNA codons (e.g., as in Fig. 10.3). The 3'-ends of the two tRNA molecules are brought into dose proximity, while their corners are somewhat apart, so that the planes of the two tRNA molecules are at an angle to each other (see Figs. 9.12 and 9.14). This situation continues after transpeptidation, except the 3'-ends of the tRNA residues are now shifted inside a limited region of peptidyl transferase center and its neighborhood (Section 11.4). Therefore the pre-translocation state ribosome contains a complex between deacylated tRNA (in the P site, with the 3'-end in the e-site) and peptidyl-tRNA (in the A site, with the ester group and the 3'-end in the p-site) joined by a complementary hexaplet of mRNA. In the case of the R-type orientation (Section 9.5), the corner of the peptidyl-tRNA may be positioned close to the heads of the 30S and 50S ribosomal subunits, while the comer of the deacylated tRNA molecule would be located in the region of the base of the L7/L12 stalk of the 50S subunit (Fig. 9.15 upper). If the S-type orientation is accepted, the positions of the corners of the two tRNAs should be exchanged (Fig. 9.15 lower).

Translocation can be conceived as an operation of the helical displacement of the two tRNAs (Rich, 1974; Spirin, 1986). In the case of the R-type orientation, the displacement will include a clockwise turn (if one looks from their anticodons) and translation along the axis connecting the anticodons with the acceptor ends. In the case of the S-type orientation, the turn would be counterclockwise, with a similar axial translation. As a result, the deacylated tRNA is displaced from the P site and dissociates from the complex with its codon; the peptidyl-tRNA is then in the P site, and the A site is now vacant. This is the post-translocation state (Fig. 12.1 right).

Which force is responsible for the movement of the peptidyl-tRNA from the A site to the P site during translocation? If the pathway of the displacement of the complex between two tRNAs and mRNA is determined by the construction of the ribosome, then the movement itself may be a consequence of just thermal motion. Since the displacement is followed by dissociation of the deacylated tRNA, it should result in an entropy gain. At any rate, in the case of factor-free (non-enzymatic) translocation, there appears to be no other motive forces except thermal motion. It is likely that thermal motion similarly induces the displacements in the course of EF2:GTP-catalyzed translocation, but the attachment of EF2:GTP to the ribosome creates a specific structural environment wherein steric and energy barriers in the transition pathway are decreased.


The energy aspect of translocation was misunderstood for a long time due to various historical factors and due to traditional ways of thinking among biochemists. The participation of GTP in translocation was determined earlier than all other facts concerning this stage of the elongation cycle. Consideration of translocation as the process of mechanical displacement of large molecular masses and the observation of coupled cleavage of GTP into GDP and orthophosphate suggested an analogy to muscle contraction, which proceeds at the expense of energy of the ATP hydrolysis into ADP and orthophosphate. This analogy created a powerful psychological stimulus for inventing special problems of energy supply for translocation, which had to be solved at the expense of GTP cleavage. Most of the models of translocation proposed thus far assume that it is the energy of EF-G-mediated GTP hydrolysis that is used in one way or another for mechanical work involving the active movement of ligands (tRNA, mRNA) along the ribosome or at least the active removal of ribosomal ligands (tRNA) from their binding sites; correspondingly, the function of contractile proteins is often ascribed to EF-G or to protein L7/L12. According to some models, the energy of GTP through EF-G is applied to peptidyl-tRNA occupying the A site, and the developing force moves this tRNA together with its codon toward the P-site, displacing deacylated tRNA from the P site. In other models, GTP energy is realized through the EF-G initially for the removal (pushing out) of the deacylated tRNA from the P site; then the peptidyl-tRNA undergoes spontaneous transition from the A site to the vacant P site, to which it has a greater affinity. There is also a class of dilettante models where EF-G is considered as "an authentic molecular motor", "exerting force", working "like a spring" and directly "ratcheting the mRNA tape".

As already mentioned, there is no apparent coupling between GTP hydrolysis and translocation. In addition, it has been shown that the hydrolysis involves the direct transfer of the phosphate residue from GTP to water without the formation of a phosphorylated intermediate that could be responsible for such coupling (Webb & Eccleston, 1981). Thus, another mechanism should be assumed: translocation is coupled with the adsorption of EF2 on the ribosome whereas GTP hydrolysis is required for the desorption of EF2. If work had been required to effect the transition of the ribosome from the pre-translocation to the post-translocation state, it might be thought that the work is performed at the expense of the energy of the complex formation between the ribosome and EF2:GTP, and the adsorption energy is then compensated by the energy of GTP hydrolysis. Such a mechanism would imply that the energy of GTP hydrolysis eventually is responsible for the work done, but not through direct coupling but by "lending" with the subsequent return.

In reality, it has been demonstrated that translocation can proceed spontaneously, without EF-G and GTP (non-enzymatic translocation). This implies that the process is thermodynamically permissible (downhill process) or, in other words, that the thermodynamic potential (free energy) of the pre-translocation state is higher than that of the post-translocation state. There is no need to explain that in this situation energy expenditure for performing work (increasing potential) is not required. Thus, any thermodynamic contribution of EF2 with GTP in translocation should be rejected.

Nevertheless, in EF2-catalyzed translocation, EF2 with GTP binds to the ribosome, the GTP is subsequently hydrolyzed, and so additional free energy is expended. But what is the purpose of this energy expenditure? It is apparent that energy generally can be expended either on some useful work against thermodynamic potential (uphill process), or on overcoming barriers in a spontaneous (downhill) process without accumulation of productive work. If the first of the two alternatives is excluded, it has to be recognized that the contribution of GTP is a purely kinetic one: at first, the interaction of GTP with EF2 provides for the attachment of EF2:GTP to the ribosome and thereby decreases barriers in the course of translocation; thereafter, the GTP hydrolysis removes the barrier created by the EF-G itself for the completion of the translocation (the decay of the translocation intermediate). Thus, GTP energy is expended solely to overcome barriers, and eventually it dissipates completely into heat (Chetverin & Spirin, 1982). This process is called the catalysis of translocation. A peculiar feature of catalysis in this case is its energy dependence, which is similar to the catalysis of aminoacyl-tRNA binding with the participation of EF-Tu.

12.6. Inhibitors of Translocation

As expected from the involvement of both ribosomal subunits in translocation, this step of elongation cycle can be inhibited by agents specifically acting either on the small or the large subunit. Three types of inhibitory mechanisms can be anticipated: fastening the tRNA residues at their A and/or P sites on the small subunit; preventing the EF2:GTP interaction with the factor-binding site on the large subunit; directly intervening in the events of intraribosomal molecular movements, including possible inter-subunit movements. A number of specific inhibitors of translocation are described among antibiotics (for reviews, see Pestka, 1969; Cundliffe, 1980, 1990; Gale et al, 1981).

12.6.1. Aminoglycosides and Aminocyclitols

Neomycin, kanamycin and gentamycin (Fig. 10.10) were reported as inhibitors of translocation, together with their action on the aminoacyl-tRNA binding step and their miscoding effect (see Section 10.3.2). Prokaryotic ribosomes seem to be markedly more sensitive to the drugs as compared with eukaryotic ribosomes. The inhibitory effect on translocation is connected with their specific binding to the small ribosomal subunit. Concerning the mechanism of this effect, it may be that these polycationic antibiotics bound on the ribosome increase the affinity of the ribosomal A site to tRNA and thus hamper the exit of the tRNA residue of peptidyl-tRNA from the A site during translocation.

Spectinomycin (Fig. 12.12) is a representative of a related group of antibiotics, aminocyclitols. Contrary to aminoglycosides, it inhibits only bacterial ribosomes. Its binding site is also localized on the

30S ribosomal subunit, and again the rRNA is mainly responsible for the binding. Spectinomycin protects positions C1063-G1064 of helix 34 near the A-site part of the decoding center (Fig. 9.3) (Noller et al., 1990). Mutations at position C1192 in the same helix confer the resistance against the drug (Cundliffe, 1990). Mutations of ribosomal protein S5 also confer the resistance against the antibiotic. The drug neither inhibits aminoacyl-tRNA binding to the ribosome, nor induces misbinding. It is believed to affect the translocation step of the elongation cycle. The mechanism of action is unknown, but, by analogy with aminoglycosides, it can be thought to impede the exit of the tRNA residue from the A site during translocation.

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