Biology

Elongation factor Tu loads aminoacyl-tRNA into the A site

KEY TERMS:

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.

KEY CONCEPTS:

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 ? and ? 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 eEF1? 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 eEF1??, a counterpart to EF-Ts.

- Bypassing Involves Ribosome Movement
Certain sequences trigger a bypass event, when a ribosome stops translation, slides along mRNA with peptidyl-tRNA remaining in the P site, and then resumes translation (see Figure 7.30). This is a rather rare phenomenon, with only ~3 authenticated examples...

- Elongation Factors Bind Alternately To The Ribosome
KEY CONCEPTS:Translocation requires EF-G, whose structure resembles the aminoacyl-tRNA·EF-Tu·GTP complex. Binding of EF-Tu and EF-G to the ribosome is mutually exclusive. Translocation requires GTP hydrolysis, which triggers a change in EF-G, which...

- The Polypeptide Chain Is Transferred To Aminoacyl-trna
KEY TERMS:Peptidyl transferase is the activity of the ribosomal 50S subunit that synthesizes a peptide bond when an amino acid is added to a growing polypeptide chain. The actual catalytic activity is a property of the rRNA. Puromycin is an antibiotic...

- Use Of Fmet-trnaf Is Controlled By If-2 And The Ribosome
KEY TERMS:The context of a codon in mRNA refers to the fact that neighboring sequences may change the efficiency with which a codon is recognized by its aminoacyl-tRNA or is used to terminate protein synthesis. KEY CONCEPTS: IF-2 binds the initiator...

- Protein Synthesis Occurs By Initiation, Elongation, And Termination
KEY TERMS:The A site of the ribosome is the site that an aminoacyl-tRNA enters to base pair with the codon. The P site of the ribosome is the site that is occupied by peptidyl-tRNA, the tRNA carrying the nascent polypeptide chain, still paired with the...

Biology