Elongation factor Tu loads aminoacyl-tRNA into the A site

  • Elongation factors (EF in prokaryotes, eEF in eukaryotes) are proteins that associate with ribosomes cyclically, during addition of each amino acid to the polypeptide chain.
  • EF-Tu is the elongation factor that binds aminoacyl-tRNA and places it into the A site of a bacterial ribosome.
  • GMP-PCP is an analog of GTP that cannot be hydrolyzed. It is used to test which stage in a reaction requires hydrolysis of GTP.
  • Kirromycin is an antibiotic that inhibits protein synthesis by acting on EF-Tu.
  • EF-Tu is a monomeric G protein whose active form (bound to GTP) binds aminoacyl-tRNA.
  • The EF-Tu·GTP·aminoacyl-tRNA complex binds to the ribosome A site. 

Once the complete ribosome is formed at the initiation codon, the stage is set for a cycle in which aminoacyl-tRNA enters the A site of a ribosome whose P site is occupied by peptidyl-tRNA. Any aminoacyl-tRNA except the initiator can enter the A site. Its entry is mediated by an elongation factor (EF-Tu in bacteria). The process is similar in eukaryotes. EF-Tu is a highly conserved protein throughout bacteria and mitochondria, and is homologous to its eukaryotic counterpart.
Just like its counterpart in initiation (IF-2), EF-Tu is associated with the ribosome only during the process of aminoacyl-tRNA entry. Once the aminoacyl-tRNA is in place, EF-Tu leaves the ribosome, to work again with another aminoacyl-tRNA. So it displays the cyclic association with, and dissociation from, the ribosome that is the hallmark of the accessory factors.

The pathway for aminoacyl-tRNA entry to the A site is illustrated in Figure 6.25. EF-Tu carries a guanine nucleotide. The factor is a monomeric G protein whose activity is controlled by the state of the guanine nucleotide (see introduction on 32.10 G proteins):
  • When GTP is present, the factor is in its active state.
  • When the GTP is hydrolyzed to GDP, the factor becomes inactive.
  • Activity is restored when the GDP is replaced by GTP.
The binary complex of EF-Tu·GTP binds aminoacyl-tRNA to form a ternary complex of aminoacyl-tRNA·EF-Tu·GTP. The ternary complex binds only to the A site of ribosomes whose P site is already occupied by peptidyl-tRNA. This is the critical reaction in ensuring that the aminoacyl-tRNA and peptidyl-tRNA are correctly positioned for peptide bond formation.
Aminoacyl-tRNA is loaded into the A site in two stages. First the anticodon end binds to the A site of the 30S subunit. Then codon-anticodon recognition triggers a change in the conformation of the ribosome. This stabilizes tRNA binding and causes EF-Tu to hydrolyze its GTP. The CCA end of the tRNA now moves into the A site on the 50S subunit. The binary complex EF-Tu·GDP is released. This form of EF-Tu is inactive and does not bind aminoacyl-tRNA effectively.
Another factor, EF-Ts, mediates the regeneration of the used form, EF-Tu·GDP, into the active form, EF-Tu·GTP. First, EF-Ts displaces the GDP from EF-Tu, forming the combined factor EF-Tu·EF-Ts. Then the EF-Ts is in turn displaced by GTP, reforming EF-Tu·GTP. The active binary complex binds aminoacyl-tRNA; and the released EF-Ts can recycle.
There are ~70,000 molecules of EF-Tu per bacterium (~5% of the total bacterial protein), which approaches the number of aminoacyl-tRNA molecules. This implies that most aminoacyl-tRNAs are likely to be present in ternary complexes. There are only ~10,000 molecules of EF-Ts per cell (about the same as the number of ribosomes). The kinetics of the interaction between EF-Tu and EF-Ts suggest that the EF-Tu·EF-Ts complex exists only transiently, so that the EF-Tu is very rapidly converted to the GTP-bound form, and then to a ternary complex.
The role of GTP in the ternary complex has been studied by substituting an analog that cannot be hydrolyzed. The compound GMP-PCP has a methylene bridge in place of the oxygen that links the β and γ phosphates in GTP. In the presence of GMP-PCP, a ternary complex can be formed that binds aminoacyl-tRNA to the ribosome. But the peptide bond cannot be formed. So the presence of GTP is needed for aminoacyl-tRNA to be bound at the A site; but the hydrolysis is not required until later.
Kirromycin is an antibiotic that inhibits the function of EF-Tu. When EF-Tu is bound by kirromycin, it remains able to bind aminoacyl-tRNA to the A site. But the EF-Tu·GDP complex cannot be released from the ribosome. Its continued presence prevents formation of the peptide bond between the peptidyl-tRNA and the aminoacyl-tRNA. As a result, the ribosome becomes "stalled" on mRNA, bringing protein synthesis to a halt.
This effect of kirromycin demonstrates that inhibiting one step in protein synthesis blocks the next step. The reason is that the continued presence of EF-Tu prevents the aminoacyl end of aminoacyl-tRNA from entering the A site on the 50S subunit (see Figure 6.31). So the release of EF-Tu·GDP is needed for the ribosome to undertake peptide bond formation. The same principle is seen at other stages of protein synthesis: one reaction must be completed properly before the next can occur.
The interaction with EF-Tu also plays a role in quality control. Aminoacyl-tRNAs are brought into the A site without knowing whether their anticodons will fit the codon. The hydrolysis of EF-Tu·GTP is relatively slow: because it takes longer than the time required for an incorrect aminoacyl-tRNA to dissociate from the A site, most incorrect species are removed at this stage. The release of EF-Tu·GDP after hydrolysis also is slow, so any surviving incorrect aminoacyl-tRNAs may dissociate at this stage. The basic principle is that the reactions involving EF-Tu occur slowly enough to allow incorrect aminoacyl-tRNAs to dissociate before they become trapped in protein synthesis.
In eukaryotes, the factor eEF1α is responsible for bringing aminoacyl-tRNA to the ribosome, again in a reaction that involves cleavage of a high-energy bond in GTP. Like its prokaryotic homologue (EF-Tu), it is an abundant protein. After hydrolysis of GTP, the active form is regenerated by the factor eEF1βγ, a counterpart to EF-Ts.