Translation - Protein Synthesis

Translation is the process in which genetic information encoded in the sequence of nucleotides is tra in messenger RNA (mRNA) is translated into the sequence of amino acids in a polypeptide chain. Let's understand the biosynthesis of protein. 

1.0Central Dogma 

The Central Dogma of molecular biology is a framework that describes the flow of genetic information within a biological system. It was proposed by Francis Crick. The Central Dogma outlines how DNA is transcribed into RNA, which is then translated into protein, summarizing the directional flow of genetic information as:

DNA to RNA (Transcription): Genetic information stored in DNA is transcribed into messenger RNA (mRNA) in the nucleus. During transcription, RNA polymerase reads the DNA strand and synthesizes a complementary strand of mRNA.

RNA to Protein (Translation): The mRNA is then transported out of the nucleus into the cytoplasm, where it is translated into a protein.  

2.0Translation : Step by Step

Activation of Amino Acids

• Protein synthesis involves the participation of 20 distinct amino acids.
• In the activation process, an amino acid combines with ATP (Adenosine Triphosphate) to form an "Aminoacyl-AMP enzyme complex," commonly referred to as the "Activated Amino Acid." This critical reaction is facilitated by a specific enzyme known as "Aminoacyl-tRNA Synthetase."
• Each type of amino acid is matched with its unique Aminoacyl-tRNA Synthetase enzyme, ensuring precise activation and subsequent incorporation into proteins.

Amino acid + ATP → Aminoacyl AMP + PP

Charging of tRNA (Loading of tRNA)

• The specific activated amino acid is identified by its corresponding tRNA molecule.
• The amino acid is then linked to the 'Amino Acid Attachment Site' on its designated tRNA, during which the AMP and enzyme dissociate from the complex. This results in the formation of an Aminoacyl-tRNA complex, also referred to as 'charged tRNA.'
• Once charged, the Aminoacyl-tRNA is transported to the ribosome, where it plays a pivotal role in protein synthesis.   

Initiation of Polypeptide Chain

The initiation phase requires the assembly of the 30S and 50S ribosomal subunits to form a complete ribosome, GTP (Guanosine Triphosphate), Mg²⁺ ions, charged tRNA (specifically carrying the amino acid methionine in prokaryotes), mRNA, and various initiation factors. In prokaryotic organisms, three key initiation factors are involved: IF1, IF2, and IF3.

This "30S mRNA complex" interacts with the "Formyl-methionyl tRNA complex," resulting in the formation of a "30S mRNA-Formyl-methionyl tRNA complex." 

Following the formation of the "30S mRNA-Formyl-methionyl tRNA complex," the larger 50S ribosomal subunit associates with this complex. Upon this association, the initiation factors are released, resulting in the formation of a complete 70S ribosome.

Within the 50S subunit of the ribosome, there are three designated sites for tRNA binding:

• 'P' site (Peptidyl site): This site holds the tRNA carrying the growing polypeptide chain.
• 'A' site (Aminoacyl site): It is the entry point for a new tRNA carrying an amino acid to be added to the polypeptide chain.
• 'E' site (Exit site): This is where tRNAs, after donating their amino acid, move to before exiting the ribosomes.

At the start of translation, the mRNA starting codon aligns close to the 'P' site of the ribosome, allowing the tRNA with the formyl-methionine amino acid to first attach to the 'P' site. Consequently, the next codon of the mRNA positions near the 'A' site, prepared for the arrival of a new tRNA carrying the subsequent amino acid.

Special Points

In prokaryotes, the "SD sequence" (Shine-Dalgarno sequence) plays a vital role in guiding the mRNA to recognize the smaller ribosomal subunit. Located upstream of the start codon on the mRNA, typically 4-12 nucleotides away, the SD sequence consists of a specific arrangement of approximately 8 nucleotides. This sequence is purine-rich, enhancing its functionality in the initiation process.

The smaller subunit of the ribosome contains a complementary sequence to the SD sequence, known as the "Anti-Shine-Dalgarno sequence" (ASD sequence), found within the 16S rRNA component. The interaction between the SD sequence on the mRNA and the ASD sequence on the 16S rRNA facilitates the precise alignment of the mRNA with the ribosomal subunit.

This complementary pairing between the SD and ASD sequences ensures that the mRNA is correctly positioned relative to the ribosome, allowing for the accurate initiation of protein synthesis.

In eukaryotes, the Kozak sequence is a specific nucleotide sequence surrounding the start codon (AUG) of mRNA that plays a crucial role in the initiation of translation, akin to the Shine-Dalgarno sequence in prokaryotes. 

5’- ACC(A/G)CCAUGG -3’ In this sequence, the AUG codon is the start codon for translation. The Kozak sequence works by guiding the ribosomal 40S subunit to the correct start codon, ensuring that translation begins at the right place to produce the correct protein. This sequence is a key element in the complex mechanism of translation initiation in eukaryotes, involving various initiation factors and the assembly of the ribosomal subunits at the start codon.

Elongation Chain

A new tRNA carrying a new amino acid attaches at the 'A' site of the ribosome. During this process:

• The bond between the amino acid at the 'P' site and its tRNA is cleaved, freeing the carboxyl (COOH) group of the amino acid at the 'P' site.
• A peptide bond forms between the COOH group of the amino acid at the 'P' site and the amino (NH₂) group of the amino acid at the 'A' site.
• The 23s rRNA within the ribosome catalyzes the peptide bond formation, acting enzymatically. Therefore, it is referred to as a "Ribozyme."
• After the peptide bond is established, the tRNA previously at the 'P' site exits the ribosome through the 'E' site, leaving the dipeptide attached to the tRNA at the 'A' site.
• The tRNA at the 'A' site, now carrying the nascent polypeptide chain, moves to the 'P' site, vacating the 'A' site for a new tRNA.
• The ribosome advances along the mRNA strand from the 5' end towards the 3' end. This movement ensures that new codons are continuously presented at the 'A' site, allowing successive amino acids to be added to the growing polypeptide chain.
• The enzyme Translocase aids in the ribosome's movement (translocation) along the mRNA, with GTP providing the necessary energy for this sliding motion.
• The elongation phase also involves specific protein factors known as "Elongation Factors." In prokaryotes, these include EF-Tu, EF-Ts, and EF-G, each playing a crucial role in facilitating the elongation process.

Termination of Polypeptide Chain

The elongation of the polypeptide chain concludes when one of the three stop codons (UAA, UAG, or UGA) occupies the 'A' site of the ribosome. These stop codons do not correspond to any amino acids but signal the end of protein synthesis. They are identified by specific proteins known as release factors (RFs).

In the bacterium E. coli, two distinct release factors, RF-1 and RF-2, play this role. RF-1 is responsible for recognizing the stop codons UAA and UAG, whereas RF-2 identifies UAA and UGA. This mechanism ensures that protein synthesis is halted at the appropriate moment.

In contrast, eukaryotic cells utilize a single release factor (eRF) that is capable of recognizing all three stop codons. This universal release factor facilitates the termination of protein synthesis by prompting the release of the newly synthesized polypeptide from the ribosome.

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