Elongation Factor Tu Loads Aminoacyl-tRNA into the A Site

KEY CONCEPTS
- EF-Tu is a monomeric G protein whose active form (bound to GTP) binds to aminoacyl-tRNA.
- The EF-Tu–GTP–aminoacyl-tRNA complex binds to the ribosome’s A site.
Once the complete ribosome is formed at the initiation codon, the stage is set for an elongation cycle in which an aminoacyl-tRNA enters the A site of a ribosome whose P site is occupied by a peptidyl-tRNA. Any aminoacyl-tRNA except the initiator can enter the A site; the one that does enter is determined by the mRNA codon in 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 among bacteria and mitochondria and is homologous to its eukaryotic counterpart.
Just like its counterpart in the initiation stage (IF-2), EF-Tu is associated with the ribosome only during the process of aminoacyltRNA entry. Once the aminoacyl-tRNA is in place EF-Tu leaves the ribosome to work again with another aminoacyl-tRNA. Thus, it displays the cyclic association with, and dissociation from, the ribosome that is the hallmark of the accessory factors.
Figure 1 depicts the role of EF-Tu in bringing aminoacyl-tRNA to the A site. EF-Tu is a monomeric GTP-binding protein that is active when bound to GTP and inactive when bound to guanine diphosphate (GDP). The binary complex of EF-Tu–GTP binds to aminoacyl-tRNA to form a ternary complex of aminoacyl-tRNA–EFTu–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 peptidyltRNA are correctly positioned for peptide bond formation.

FIGURE 1. EF-Tu–GTP places aminoacyl-tRNA on the A site of ribosome and then is released as EF-Tu–GDP. EF-Ts is required to mediate the replacement of GDP by GTP. The reaction consumes GTP and releases GDP. The only aminoacyl-tRNA that cannot be recognized by EF-Tu–GTP is fMet-tRNA_f , whose failure to bind prevents it from responding to internal AUG or GUG codons.
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 base pairing 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.
The guanine nucleotide exchange factor, EF-Ts, mediates the regeneration of the inactive 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 to an aminoacyl-tRNA, and the released EF-Ts can recycle.
Each cell has about 70,000 molecules of EF-Tu (which is about 5% of the total amount of bacterial protein), which approaches the number of aminoacyl-tRNA molecules. This implies that most aminoacyl-tRNAs are likely to be in ternary complexes. Each cell has only about 10,000 molecules of EF-T, about the same as the number of ribosomes. The kinetics of the interaction between EFTu 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 GTPbound 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 that binds aminoacyl-tRNA to the ribosome can be formed. However, the peptide bond cannot be formed, so the presence of GTP is needed for aminoacyl-tRNA to be bound at the A site. 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 aminoacyltRNA to the A site. However, 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 the mRNA, bringing translation to a halt.
This effect of kirromycin demonstrates that inhibiting one step in translation 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. Thus, 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 translation: One reaction must be properly completed 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 regard for whether their anticodons will fit the codon. The hydrolysis of EFTu– GTP is relatively slow; it takes longer than the time required for an incorrect aminoacyl-tRNA to dissociate from the A site, so most incorrect aminoacyl-tRNAs are removed at this stage. The release of EF-Tu–GDP after hydrolysis is also slow, so any remaining 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 translation.
In eukaryotes, the factor eEF1a is responsible for bringing aminoacyl-tRNA to the ribosome, also in a reaction that involves cleavage of a high-energy bond in GTP. Like its prokaryotic homolog (EF-Tu), it is abundant in the cell. After hydrolysis of GTP, the active form is regenerated by the factor eEF1βγ, a counterpart to EF-Ts.