Genes VII

The ribosome remains in place while the polypeptide chain is elongated by transferring the polypeptide attached to the tRNA in the P site to the aminoacyl-tRNA present in the A site. The reaction is shown in Figure 6.21. The activity responsible for synthesis of the peptide bond is called peptidyl transferase.

The nature of the transfer reaction is revealed by the ability of the antibiotic puromycin to inhibit protein synthesis. Puromycin resembles an amino acid attached to the terminal adenosine of tRNA. Figure 6.22 shows that puromycin has an N instead of the O that joins an amino acid to tRNA. The antibiotic is treated by the ribosome as though it were an incoming aminoacyl-tRNA. Then the polypeptide attached to peptidyl-tRNA is transferred to the NH₂ group of the puromycin.

Because the puromycin moiety is not anchored to the A site of the ribosome, the polypeptidyl-puromycin adduct is released from the ribosome in the form of polypeptidyl-puromycin. This premature termination of protein synthesis is responsible for the lethal action of the antibiotic.

Peptidyl transferase is a function of the large (50S or 60S) ribosomal subunit. The transferase is part of a ribosomal site at which the ends of the peptidyl-tRNA and aminoacyl-tRNA are brought close together. Both rRNA and 50S subunit proteins are necessary for this activity. The catalytic activity is a property of the ribosomal RNA of the 50S subunit (see below).

The cycle of addition of amino acids to the growing polypeptide chain is completed by the translocation illustrated in Figure 6.23, in which the ribosome advances three nucleotides along the mRNA. The result of translocation is to expel the uncharged tRNA from the P site, so that the new peptidyl-tRNA can enter. The ribosome then has an empty A site ready for entry of the aminoacyl-tRNA corresponding to the next codon.

In bacteria the discharged tRNA leaves the ribosome via another site, the E site. In eukaryotes it is expelled directly into the cytosol. The process of relocating the discharged tRNA and peptidyl-tRNA takes place in two stages. First the aminoacyl ends of the tRNAs (located in the 50S subunit) move into the new sites (while the anticodon ends remain bound to their anticodons in the 30S subunit). At this stage, the tRNAs are effectively bound in hybrid sites, consisting of the 50S E/ 30S P and the 50S P/ 30S A sites. Then movement is extended to the 30S subunits, so that the anticodon-codon pairing region finds itself in the right site. The general model for two-part movement is called the hybrid states model.

The figure shows two possible ways to create the hybrid states. It could involve movement of tRNA relative to the ribosome, so that the aminoacyl end of tRNA moves within the 50S subunit; the anticodon end moves later when translocation occurs. Alternatively, the entire 50S subunit might move relative to the 30S subunit, so that translocation in effect involves two stages, the normal structure of the ribosome being restored by the second stage (444).

The ribosome faces an interesting dilemma at translocation. It needs to break many of its contacts with tRNA in order to allow movement. At least some of these contacts are needed to stabilize the inherently weak codon-anticodon interaction. But at the same time it must maintain pairing between tRNA and the anticodon (breaking the pairing of the deacylated tRNA only at the right moment). One possibility is that the ribosome switches between alternative, discrete conformations. The switch could consist of changes in rRNA base pairing. The accuracy of translation is influenced by certain mutations that influence alternative base pairing arrangements. The most likely interpretation is that the effect is mediated by the tightness of binding to tRNA of the alternative conformations (440).

Translocation requires GTP and another elongation factor, EF-G. This factor is a major constituent of the cell; it is present at a level of ~1 copy per ribosome (20,000 molecules per cell).

Ribosomes cannot bind EF-Tu and EF-G simultaneously, so protein synthesis follows the cycle illustrated in Figure 6.24 in which the factors are alternately bound to, and released from, the ribosome. So EF-Tu PGDP must be released before EF-G can bind; and then EF-G must be released before aminoacyl-tRNA PEF-Tu PGTP can bind.

Does the ability of each elongation factor to exclude the other rely on an allosteric effect on the overall conformation of the ribosome or on direct competition for overlapping binding sites? Figure 6.25 shows an extraordinary similarity between the structures of the ternary complex of aminoacyl-tRNA PEF-Tu PGDP and EF-G (928). The aminoacyl-tRNA is bound by EF-Tu around its amino acceptor stem. It is striking that the structure of the protein EF-G mimics the overall structure of the protein complexed with RNA in the ternary complex. This creates the immediate assumption that they compete for the same binding site (presumably in the vicinity of the A site). The need for each factor to be released before the other can bind ensures that the events of protein synthesis proceed in an orderly manner (445).

Both elongation factors are monomeric GTP-binding proteins that are active when bound to GTP but inactive when bound to GDP. The triphosphate form is required for binding to the ribosome, which ensures that each factor obtains access to the ribosome only in the company of the GTP that it needs to fulfill its function.

EF-G binds to the ribosome to sponsor translocation; and then is released following ribosome movement. It is an important part of the mechanism for translocation. The hydrolysis of GTP occurs before translocation and accelerates the ribosome movement. The most likely mechanism is that GTP hydrolysis causes a change in the structure of EF-G, which in turn forces a change in the ribosome structure. An extensive reorientation of EF-G occurs at translocation (947). Before translocation, it is bound across the two ribosomal subunits; after translocation, the domain (domain 4) that made most of the contacts with the 30S subunit is instead oriented toward the 50S subunit. Figure 6.26 shows the movement schematically.

EF-G can still bind to the ribosome when GMP-PCP is substituted for GTP; thus the presence of a guanine nucleotide is needed for binding, but its hydrolysis is not absolutely essential for translocation (although translocation is much slower in the absence of GTP hydrolysis). The hydrolysis of GTP is needed to release EF-G.

The need for EF-G release was discovered by the effects of the steroid antibiotic fusidic acid, which "jams" the ribosome in its post-translocation state (see Figure 6.24). In the presence of fusidic acid, one round of translocation occurs: EF-G binds to the ribosome, GTP is hydrolyzed, and the ribosome moves three nucleotides. But fusidic acid stabilizes the ribosome PEF-G PGDP complex, so that EF-G and GDP remain on the ribosome instead of being released. Because the ribosome then cannot bind aminoacyl-tRNA, no further amino acids can be added to the chain.

The eukaryotic counterpart to EF-G is the protein eEF-2, which functions in a similar manner, as a translocase dependent on GTP hydrolysis. Its action also is inhibited by fusidic acid. A stable complex of eEF-2 with GTP can be isolated; and the complex can bind to ribosomes with consequent hydrolysis of its GTP.

A unique reaction of eEF-2 is its susceptibility to diphtheria toxin. The toxin uses NAD (nicotinamide adenine dinucleotide) as a cofactor to transfer an ADPR moiety (adenosine diphosphate ribosyl) on to the eEF-2. The ADPR-eEF-2 conjugate is inactive in protein synthesis. The substrate for the attachment is an unusual amino acid, produced by modifying a histidine; it is common to the eEF-2 of many species.

The ADP-ribosylation is responsible for the lethal effects of diphtheria toxin. The reaction is extraordinarily effective: a single molecule of toxin can modify sufficient eEF-2 molecules to kill a cell.