Let us make an in-depth study of the protein synthesis in eukaryotes.
The basic mechanism of protein synthesis in eukaryotes is similar to prokaryotes, but there are a number of differences occurring in individual steps.
The mechanism offers greater complexity due to the involvement of some components that do not exist in prokaryotes.
The eukaryotic ribosome has a sedimentation coefficient of 80S, comprising of a small 40S and a large 60S subunit. The smaller subunit contains a single 18S rRNA of about 1900 nucleotides and 30-35 proteins. The larger subunit contains 3 RNA chains, 5S, 5.8S and 28S, consisting of 120, 160 and about 2800 nucleotides respectively and about 45-50 proteins. However, the ribosomes of mitochondria and chloroplasts resemble the prokaryotic ones.
Eukaryotic protein synthesis involves more protein components and some steps are more complex. The initiating codon is eukaryotes is also AUG. The eukaryotic mRNAs do not have a purine-rich sequence (ribosome binding sequence) on the 5′ end. The AUG nearest to the 5′ end of the mRNA is usually selected as the start site. The 40S ribosomal subunit attaches to the cap at the 5′ end of the eukaryotic mRNA and searches for an AUG codon by moving stepwise in the 3′ direction.
The scanning process is driven by the hydrolysis of ATP. The anticodon of met-tRNA; bound to the 40S subunit pairs with the AUG codon, signifying that the target has been found. An eukaryotic message has only one start site and hence, is template for a single protein. It is because all eukaryotic mRNAs are monocistronic. In contrast, most prokaryotic mRNAs are polycistronic, hence they have multiple start sites and synthesise several proteins.
Like prokaryotes, eukaryotes too have two kinds of tRNA that bind methionine. The initiator tRNA is called tRNAimet which recognizes AUG and inserts methionine, and the other one is called tRNAmmet which inserts methionine in internal positions. Each of the tRNA species is aminoacylated with methionine by the corresponding methionyl-tRNA synthetizes.
Eukaryotes require many more initiation factors compared to prokaryotes, and their interplay is much more intricate. At least nine initiation factors are known, many of which consist of multiple subunits. The prefix eIF denotes a eukaryotic initiation factor. The 40S subunit with initiation components binds to the 5′ cap of the mRNA, and ATP hydrolysis drives the 40S subunit to the start signal AUG. The factor eIF2, a GTP binding protein, brings the initiator tRNA (met- tRNAimet), mRNA and the 40S subunit together with the help of eIF4, eIF3 and cap binding proteins.
The cap binding proteins bind to the cap of mRNA. ATP is hydrolyzed by eIF4 which drives the 40S subunit initiation complex along the start AUG codon. The complex is joined by eIF3 which helps in finding the AUG closest to the 5′ end. Subsequently, eIF5 induces the release of eIF2 and eIF3 following pairing of met-tRNA; with the start codon. eIF5 triggers the hydrolysis of GTP bound to eIF2, and the reaction results in the release of eIF2-GDP along with other initiation factors. Finally, the 60S subunit joins the initiation complex involving tRNA, mRNA, and 40S subunit to form the active SOS ribosome. (Fig. 7.18)
Eukaryotic Elongation Factors:
Chain elongation factors in eukaryotes roughly correspond to the elongation factors (EF) of E. coli. At least four factors are required, namely EF1α, EP1β, EF1y , and EF2. EF1α, EF1β are analogous to EF-Tu and EF-Ts in prokaryotes, while EF2 is a counterpart of EF-G. EF1α is GTP bound and delivers aminoacyl tRNA to the A site of ribosome, while EFlβ catalyzes GTP to release bound GDP. Eukaryotic EF2 mediated GTP driven translocation is accomplished in the same way as does prokaryotic EF-G.
Protein termination in eukaryotes is carried out by a single release factor eRF, a GTP driven protein, compared with two in prokaryotes. Finally, eIF3, like IF3 in prokaryotes, prevents re-association of ribosome when synthesis is completed. It should be noted that phosphorylation of eIF2 proteins play an important role in the initiation process. Dephosphorylation of eIF2 activates and its dephosphorylation deactivates initiation process.
Inhibitors of Protein Synthesis in Eukaryotes:
We have discussed inhibitors of protein synthesis in prokaryotes, and it is found that some antibiotics are active against bacterial as well as eukaryotic cells. Chloramphenicol is one of them which inhibits bacterial and mitochondrial ribosomes for their peptidyl transferees activity. However, the cytoplasmic ribosomes are insensitive. The drug cycloheximide is a potent inhibitor of peptide bond formation since it inhibits peptidyl transferase activity of 60S subunit. Similarly, the toxin from the bacterium causing diphtheria inhibits elongation factor required for translocation.