Translation

Translation (mRNA → Protein)

Translation is the process of the synthesis of proteins using the genetic information in mRNA as a template. During this process, the sequence of nucleotides in the mRNA is decoded to form a specific sequence of amino acids, which are joined together to form a polypeptide (protein). This process occurs in the cytoplasm after the mRNA has left the nucleus through a nuclear pore.

All three types of RNA (tRNA, mRNA, and rRNA) are involved in transferring the information obtained from the sequence of nucleotides in DNA to the sequence of amino acids in the polypeptide chain of a protein.

Steps in translation

1. Activation of amino acids

2. Transfer of amino acids to tRNA

3. Initiation of protein synthesis

4. Elongation of the polypeptide chain

5. Chain termination


1. Activation of Amino Acids

In the cytoplasm, 20 different amino acids exist in an inactive form. Before they can participate in protein synthesis, they must be activated. This activation is catalyzed by a group of enzymes called aminoacyl-tRNA synthetases, each specific to a particular amino acid.

The amino acid (AA) reacts with ATP in the presence of the enzyme aminoacyl-tRNA synthetase. This reaction forms a high-energy intermediate called aminoacyl-adenylate (AA~AMP), which is enzyme-bound, and releases pyrophosphate (PPi).

Reaction:
                   AA+ ATP+ Aminoacyl-tRNA synthetase → AA∼AMP (enzyme-bound)+ PPi

where,        AA~AMP = Aminoacyl adenylate, PPi = Pyrophosphate

This step activates the amino acid, preparing it for attachment to its corresponding tRNA.


2. Transfer of Amino Acids to tRNA (Charging of tRNA)

This step is known as the charging of tRNA, where the activated amino acid is transferred to a specific tRNA molecule. Each tRNA has an amino acid attachment site at its 3’ end, specifically at the adenosine (A) of the CCA tail. The carboxyl (-COOH) group of the amino acid forms a high-energy ester bond with the 3’-OH group of the terminal adenosine of the tRNA. This results in the formation of aminoacyl-tRNA (AA~tRNA), which is now ready to participate in translation.

Example: tRNA^Ile carries isoleucine,  tRNA^Val carries valine

Reaction: AA∼AMP (enzyme-bound) + tRNA → AA∼tRNA + AMP + Enzyme    

where,  AA~tRNA = Aminoacyl-tRNA (charged tRNA)

This product carries the correct amino acid to the ribosome during protein synthesis.


3. Initiation of Protein Synthesis

Translation begins at the start codon AUG, which codes for formyl methionine (fMet) in prokaryotes. A special initiator tRNA called fMet-tRNA^fMet recognizes this codon. E.Coli cells have three initiation factors IF₁, IF₂, and IF₃.
IF₁ and IF₃ take part in ribosome dissociation.

Initiation Factors in E. coli

IF₁: Promotes dissociation of the ribosomal subunits.

IF₃: Binds to the free 30S subunit and prevents reassociation with the 50S subunit.

IF₂: Binds GTP and helps in the binding of fMet-tRNA to the 30S subunit along with mRNA.

Formation of 70S Initiation Complex:

30S subunit, along with IF₁, IF₂ (GTP-bound), and IF₃, binds to mRNA and fMet-tRNA. Once the complex is correctly assembled, the 50S subunit joins. This forms the complete 70S ribosome (functional ribosome). All initiation factors (IF₁, IF₂, IF₃) are then released, and GTP is hydrolyzed.

4. Elongation of Polypeptide Chain

After initiation, the ribosome begins adding amino acids one by one to elongate the polypeptide. Elongation consists of three repeated steps:

 
a. Binding of Aminoacyl-tRNA to A Site
Each new aminoacyl-tRNA binds to a complex with EF-Tu (elongation factor Tu) and GTP.
The Aminoacyl-tRNA·EF-Tu·GTP complex enters the A site of the ribosome.
GTP is hydrolyzed, and EF-Tu is released as EF-Tu·GDP.
EF-Ts (another elongation factor) regenerates EF-Tu·GTP from EF-Tu·GDP.

b. Peptide Bond Formation

The amino acid on the P site (already present) forms a peptide bond with the new amino acid at the A site. This reaction is catalyzed by the enzyme peptidyl transferase (present in the 50S subunit).The growing polypeptide chain is now transferred to the tRNA in the A site.

c. Translocation

The ribosome moves forward by one codon (three nucleotides) toward the 3' end of the mRNA.This shifts the tRNA from the A site to the P site, and the empty tRNA in the P site is released. The A site becomes available for the next aminoacyl-tRNA.

5. Chain Termination
Termination occurs when a stop codon on the mRNA is reached, like UAA, UAG, and UGA
Releasing Factors (RFs):
RF₁: Recognizes UAA and UAG
RF₂: Recognizes UAA and UGA
RF₃: Enhances the activity of RF₁ and RF₂
These proteins promote hydrolysis of the bond between the polypeptide and the tRNA in the P site. The completed polypeptide is released. The ribosome subunits dissociate, and translation ends.


Post-Translational Modifications

Proteins synthesized during translation are often non-functional in their initial form. After synthesis, they undergo various modifications to become biologically active and functional. These modifications may occur during or after translation and include structural trimming and various covalent changes. Together, these are referred to as post-translational modifications.

a. Proteolytic Degradation (Trimming)

 
Many proteins are synthesized as inactive precursors (proproteins or preproteins) that are larger than the final functional form. Specific segments of these precursor proteins are removed by proteolytic enzymes to produce the active form. This process is called proteolytic cleavage or trimming. Example: Insulin formation.
Insulin is synthesized initially as preproinsulin. The preproinsulin undergoes proteolytic cleavage to form proinsulin, and then is further trimmed to produce active insulin. Insulin consists of 51 amino acids, arranged in two chains (A and B) connected by two disulfide bridges.

b. Covalent Modifications

Proteins often undergo chemical changes by the addition or modification of certain groups. These covalent modifications can affect the protein’s activity, stability, location, and interactions.

Common Types of Covalent Modifications:

Phosphorylation: Addition of phosphate groups (commonly on serine, threonine, or tyrosine residues). Regulates enzyme activity and signaling pathways.

Glycosylation: Attachment of sugar moieties (mainly in secretory and membrane proteins). Important for protein folding, stability, and recognition.

Hydroxylation: Addition of hydroxyl groups (e.g., hydroxyproline in collagen). Involved in structural stability.

Acetylation: Addition of acetyl groups (often on histones). Affects gene regulation.

Methylation: Addition of methyl groups, influencing protein-protein interactions and gene expression.
Conclusion

Post-translational modifications are essential steps in protein maturation. They ensure that newly synthesized polypeptides are correctly folded, processed, and functionally active, enabling them to perform their roles in various cellular processes.