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The mechanism of protein synthesis has been studied thoroughly in E.coli. The process of protein synthesis on 70S ribosomes is described in detail below. The summary of the various steps in the mechanism of protein synthesis is shown in (Fig. 13).
Step 1 — Transcription:
One strand of DNA molecule functions as a template for the formation of mRNA. This mRNA contains the message for the protein to be synthesised. As soon as the mRNA is formed, it leaves the nucleus and reaches the cytoplasm where it attaches with the 30S subunit of ribosomes.
Step 2 — Attachment of mRNA to 30S Subunit of Ribosomes:
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In prokaryotic cells, ribosomes are found in a dissociated and an inactive state. The mRNA binds to the 30S subunit. Specific sequences of 16 S rRNA of the small subunit of the ribosome binds to complementary sequence of mRNA. The first amino acid, which is usually N formyl methionine-tRNA (F-met tRNA) attaches to the mRNA to form the initiation complex.
This process is aided by GTP and three protein factors (IF1, IF2 and IF3 factors). The ribosome is uniquely designed to bring mRNA and tRNA together. The mRNA is threaded through the small subunit tunnel, while the large and subunit fit together to form tRNA binding pockets. After the formation of the initiation complex, the 50S subunit joins with the smaller subunit to form the 70S ribosome.
Step 3 — Transfer of Amino Acids to the Site of Protein Synthesis:
During this step, the amino acids are transported from the amino acid pool to the ribosomes. But the amino acids occur in the cytoplasm in an inactive state. Each amino acid is activated by a specific activating enzyme known as amino acyl synthetase and ATP.
The cell has at least 20 aminoacyl synthetase enzymes for the 20 amino acids. Each enzyme is specific and activates the correct amino acid. The enzyme is recognised by the enzyme site of the tRNA and the amino acid residue is transferred to the amino acid attachment site of the tRNA. The amino acid is linked to the 3-OH at the -CCA end of tRNA. As a result, the AMP and enzyme are released and the final product amino acyl-tRNA is formed. This moves towards the site of protein synthesis.
Step 4 — Initiation of Protein Synthesis:
The initiation involves the formation of the 70S complex and is aided by initiation factors.
The mRNA usually has AUG as its first codon. AUG codes for methionine. The methionine is formulated and plays an important role in initiating the process of protein synthesis. After the protein synthesis is completed, the formyl methionine is detached by the activity of the hydrolytic enzyme.
The function of the formyl methionine is to ensure that protein synthesis progresses in a specific direction. This is because, in formyl methionine, the NH2 is blocked by the formyl group leaving the -COOH end to react with the -NH2 group of the second amino acid.
The process of initiation differs significantly in prokaryotes and eukaryotes. Three initiation factors are involved in prokaryotes – IF1, IF2 and IF3. Initiation in eukaryotes is more complex and requires 8 initiation factors. (Table 8). The first amino acid is methionine in eukaryotes.
Step 5 — Elongation of Polypeptide Chain:
During elongation, amino acids are linked to one another. The process of elongation requires elongation factors, EF-Tu, Ef-G and EF-Ts. A charged tRNA with the anticodon complementary to the codon of mRNA is brought to the A site of the ribosome. Peptide formation occurs between the amino acids in the P site and the A site in the presence of the enzyme, peptidyl transferase.
The peptidyl transferase reaction transfers the amino acid, from the P site onto the amino acyl-tRNA in the A site. The second tRNA now carries a dipeptide and the first tRNA is without an amino acid.
The next step of elongation is the movement of ribosomes by one codon along the mRNA. This is called translocation. The enzyme involved in this reaction is translocase. Translocation requires energy, which is provided by the hydrolysis of GTP. As translocation occurs, the tRNA with the dipeptide translocates to the P site and a new codon is available in the A site. The first tRNA now moves to the ‘E’ site. This tRNA is ‘kicked out’ by the action of transferase, which is also responsible for flipping the formyl-methionine to the tRNA present now at the P site.
In short, the whole process involves arrival of tRNA-amino acid complex, peptide bond formation and translocation. As the ribosomes moves over the mRNA in the 5′→3′ direction, all the codons are exposed at the A site one after the other and there is subsequent growth of the polypeptide chain.
Thus, the language of DNA is transcribed into the language of mRNA which is later translated into the language of polypeptides. The newly synthesised polypeptide is known as nascent polypeptide. The sequence of events in elongation occurs very rapidly and under optimal conditions a polypeptide chain of 40 amino acids can be produced in 20 seconds.
Step — 6 Termination and Release of Polypeptide Chain:
At the end of the mRNA chain there is a stop or terminator codon. There are three terminator codons – UAA, UGA and UAG.
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When the information processing region encounters a terminator codon, it signals the stop of protein synthesis. A release factor joins the stop codon and this aids, the hydrolysis of the completed polypeptide chain from the P site. The ribosome separate from the mRNA chain and the ribosome dissociates into two subunits.
Step — 7 Modification of the Released Polypeptide:
The released polypeptide is in its primary form. It undergoes coiling and acquires a secondary and tertiary structure. It may combine with other polypeptides to assume a quaternary structure. Proteins known as chaperones help in the folding of the proteins to become functional.
Proteins synthesised on free ribosomes are released into the cytoplasm and function as structural and enzymatic proteins. Proteins formed on attached ribosomes, pass through the tunnel into the channels of ER and are exported as cell secretions by exocytosis after packaging in the Golgi apparatus.
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The mRNA and ribosome are reusable. Sometimes many ribosomes read one mRNA molecule at one time. They form polysomes or polyribosomes producing many identical polypeptides. The polypeptides when released form proteins which may assume secondary, tertiary or quaternary structure. The sequence of DNA which codes for a polypeptide chain is called a cistron or gene.
