Translation III

Elongation Factors

(See Table 27.4 on page 1044 and Table 28.7 on page 1101)

Molecular Mimicry

EF-Tu (a G protein) is the most abundant protein in E. coli and eEF-1A is also a very abundant protein in eukaryotic cells. EF-Tu and EF-G compete for overlapping binding sites on the ribosome; that is, either one or the other, but not both together, can be on the ribosome at any given time. When the crystal structures of EF-Tu and EF-G were solved, the structures suggested remarkable insights to the function of these two elongation factors (Figure AT); see also Figure 27.24 on page 1049). The distal domains 4 and 5 of EF-G are seen to have the same shape and size as the tRNA binding partner of EF-Tu. This remarkable molecular mimicry suggests why the two elongation factors work alternate shifts at the same binding site.

Elongation of Protein Synthesis

Figure 27.22 shows an overview of elongation (but see Figure AP below for a more accurate view). Elongation is controlled by three elongation factors (EFs). EF-Tu (eEF-1A in eukaryotes) forms a ternary complex with GTP and aminoacylated tRNAs and carries the aminoacylated tRNA to the ribosomal A-site for the decoding of mRNA by codon­anticodon interactions. When correct codon-anticodon recognition occurs, GTP hydrolysis on EF-Tu is stimulated by the ribosome and EF-Tu*GDP is released. EF-Tu has very low intrinsic GTPase activity. It works as a time-delayed molecular switch, and the GTPase activity is thus highly stimulated when the ternary complex interacts with ribosome. The nucleotide exchange factor EF-Ts (eEF-1B in eukaryotes) converts EF-Tu*GDP into active EF-Tu*GTP. After a proofreading step, the aminoacylated-tRNA is brought into contact with the peptidyl-tRNA in the ribosomal P-site, where peptide bond formation is catalysed adding one amino acid to the growing peptide. The last EF, EF-G (eEF-2 in eukaryotes), together with GTP controls the translocation of tRNAs and mRNA on the ribosome. It has been demonstrated that the EF-G*GTP complex mimics the ternary complex of EF-Tu*GTP with tRNA. Translocation takes place by a conformational change in EF-G so that its "tRNA mimicking part" will occupy the anticodon region of the A-site. Recent results suggest that this conformational change and translocation are subsequent to the GTP hydrolysis. Another interesting finding is that the main contacts between the ribosome and EF-Tu are between domain 1 and the 50 S subunit and between domain 2 and the 30 S subunit. This is interesting, not only because these two domains are common between EF-Tu and EF-G, but also because similar domains are present in all the translational G-proteins. The implication is that all of them will bind to the ribosome in the same way, and that the GTPase activity will be stimulated by the same mechanism.

Figure 27.23 EF-Ts (eEF-1B) is required to remove GDP from EF-Tu (eEF-1A) so that GTP can bind.

Figure AP2 shows the relative orientations of the A-, P-, and E-tRNAs and mRNA on the ribosome. The simultaneous reading of the two codons is accomodated by a kink in the mRNA backbone of about 45 degrees between the A and P codons.

Reconition of cognate tRNA in the ribosome by an induced fit

Figure AP3 shows molecular interactions important for selecting the correct tRNA at the A site. Figure AP4 shows these details at higher resolution. The binding of mRNA and cognate tRNA in the A site induces A1492 and A1493 to flip out of the internal loop of helix 44. This binding also causes the universally conserved base G530, which has been footprinted by A-site tRNA, to switch from the syn conformation present in the native structure to an anti conformation. In their new conformations, A1493 and A1492 interact, respectively, with the first and second base pairs of the codon-anticodon helix, whereas G530 interacts with both the second position of the anticodon and the third position of the codon. The result of these induced changes is that the first two base pairs of the codon-anticodon helix are closely monitored by the ribosome in a way that would be able to discriminate between Watson-Crick base pairing and mismatches, whereas the environment of the third, or "wobble," position appears to be suited for accommodating other base-pairing geometries.

tRNA binding sites on the ribosome are not static

Figure AP shows the hybrid sites occupied by tRNAs during elongation. EF-Tu "helps" to maintain the reading frame during elongation. Codon-anticodon base pairing is not sufficient to guarantee high accuracy of translation during elongation. Ribosomal interactions are important (protein S12 on the 30S subunit, for example) for accurate translation (see Figures AP3 and AP4 above). The loading of the correct tRNA (in the form of the ternary complex: aminoacyl-tRNA-EF-Tu-GTP) into the A site is also important. Aminoacyl-tRNAs with mismatched codon-anticodon interactions dissociate much more rapidly than correctly matched aminoacyl-tRNAs.

The tRNA "footprints" on 16S and 23S rRNA can be used as a direct assay for occupancy of the A, P, and E sites on the two ribosomal subunits. The state of occupancy of the tRNA binding sites has been examined during each step of a round of elongation in vitro. This study shows that the CCA end of peptidyl tRNA moves spontaneously from the P site to the E site of 23S rRNA upon peptide bond formation, while remaining in the P site with respect to 16S rRNA. This results in a hybrid state of binding, called the P/E state. Similarly, the CCA end of aminoacyl tRNA shifts from the A site to the P site on 23S rRNA, while remaining in the 16S rRNA A state. Thus, upon peptidyl transfer, the two tRNAs shift from A/A and P/P to A/P and P/E state, respectively. This movement occurs independently of factors or GTP. In the EF-G and GTP-dependent step, both tRNAs and mRNA move relative to 16S rRNA. Thus, translocation of tRNA occurs in two steps; in the first step, the tRNAs move relative to 23S rRNA, resulting in hybrid binding states, and the second step returns them to the nonhybrid state. Instead of two or three states of binding for tRNA, there are at least six: A/T, A/A, A/P, P/P, P/E, and E/E. Independent movement of the two ends of tRNA allows one end to be fixed while the other moves relative to the ribosome. Another important implication is that the nascent polypeptide chain remains in the same ribosomal location during the elongation cycle. The hybrid site model impies that there is relative motion between the 50S and 30S subunits during translocation, which would account for the fact that ribosomes are always made up of the two subunits.

Peptide bond formation is catalyzed by 23S rRNA

Peptidyl transferase is the sole catalytic activity that has unambiguously been shown to be a property of the ribosome itself. Peptidyl transferase activity has been demonstrated in severely protein-depleted 50S subunits, and one implication is that 23S rRNA itself has peptidyl transferase activity (RNAs with catalytic activity are called ribozymes). The generation of a model for the molecular structure of the large ribosomal subunit, from x-ray crystallographic studies at 2.4 Å resolution, is one of the most exciting biological advances in recent years. Of particular interest is the identification of the active site for peptidyl transfer based on the determination of the structure of a complex with a transition state analog (Yarus inhibitor). A key feature of this active site is a completely conserved adenosine (A 2451) that is proposed to act as a general base catalyst (Figure AP1). This adenosine has a markedly shifted pKa near 7.6.

Additional states in the elongation cycle

EF-G-dependent translocation is more complicated. Until recently, binding of EF-G-GTP to the ribosome was thought to induce translocation, followed by GTP hydrolysis and release of EF-G-GDP from the ribosome. This conclusion was based principally on the observation that a single round of translocation could occur with nonhydrolyzable GTP analogs, while GTP hydrolysis was required for release of EF-G after translocation. However, recent pre-steady state kinetic experiments show clearly that GTP hydrolysis occurs before translocation and accelerates translocation more than 50-fold relative to that observed with nonhydrolyzable GTP analogs. This suggests that a conformational transition in EF-G itself is in some way coupled to translocation.

The transition from GTP- to GDP-binding in the EF-Tu active site causes sizeable movement of the attached tRNA, consistent with models in which the tRNA of EF-Tu and the mimetic domain 4 of EF-G act as mechanical manipulators--something like robotic arms. GTP hydrolysis occurs five times faster than mRNA ratcheting. In other words, hydrolysis precedes translocation by quite a considerable time. The consequences of this finding are profound. It implies that the bond energy that is derived from the hydrolysis of GTP is stored in the protein, to be used late for mechanical work. The protein is like a compressed spring that is held against a trigger: ribosome-binding releases the trigger and the protein flies open, exerting force and allowing the phosphate that is generated as a result of GTP hydrolysis to exit from the active site. mRNA translocation is inhibited by mutagenic removal of domain 4 (the part of EF-G that mimics the tRNA of EF-Tu) whereas ribosome-activated GTP hydrolysis in unaffected. Domain 4 is also required for the dissociation and recycling of EF-G: deletion of domain 4 leaves that motor stranded in the ribosome site, able to perform a single round of GTP hydrolysis but unable to dissociate. So the mechanical motion of domain 4 seems to be necessary to drive the motor into its dissociating conformation.

EF-G-dependent translocation can be inhibited by several antibiotics

(1) Fusidic acid allows GTP hydrolysis and translocation but prevents release of EF-G-GDP. Domain 4 of EF-G is also required for the dissociation and recycling of EF-G: deletion of domain 4 leaves that motor stranded in the ribosome site, able to perform a single round of GTP hydrolysis but unable to dissociate. So the mechanical motion of domain 4 seems to be necessary to drive the motor into its dissociating conformation. Note: EF-G-GDP must be released before a new aminoacyl-tRNA-EF-Tu-GTP can bind to the ribosome.

(2) Viomycin allows GTP hydrolysis but prevents translocation. This is similar to the effect on mRNA translocation by mutagenic removal of domain 4 (the part of EF-G that mimics the tRNA of EF-Tu) where ribosome-activated GTP hydrolysis is unaffected but translocation is inhibited.