The Process of Protein Synthesis or Translation

Translation the process by which a ribosome synthesizes a protein using the information encoded mRNA that occurs in the cytoplasm.

The mRNA acts as a template, and tRNA molecules bring specific amino acids to the ribosome based on the mRNA sequence.

The ribosome reads the mRNA codons, matches them with tRNA anticodons, and links the amino acids together.

This process plays a crucial role in gene expression, is the second stage in the process of gene expression and protein synthesis

It allowing the conversion of genetic information into functional proteins.

in the 5′ to 3′ direction of the mRNA molecule, ensuring accurate protein synthesis. The speed of translation can vary depending on several factors

In prokaryotes, translation can occur rapidly, with speeds of up to 20 amino acids per second (900 residues per minute). Eukaryotic translation tends to be slower, with rates ranging from 2 to 10 amino acids per second (900 residues per minute).

Translation involves three main steps: initiation, elongation, and termination.

Initiation of Translation

the small ribosomal subunit binds to the mRNA molecule at the start codon. This is facilitated by initiation factors and the binding of initiator tRNA carrying methionine. The large ribosomal subunit then joins, forming a functional ribosome ready for protein synthesis.

1. Amino acid activation

It is also known as aminoacylation or tRNA charging.

An aminoacyl-tRNA synthetase enzyme recognizes a specific amino acid and attaches it to the corresponding tRNA molecule, forming an aminoacyl-tRNA complex. This process requires ATP, which provides the energy for the reaction.

An enzyme aminoacyl-tRNA synthetase binds ATP (adenosine triphosphate) to a corresponding amino acid, forming a reactive aminoacyl adenylate intermediate (AMP-amino acid) and releasing inorganic pyrophosphate (iP)

ATP + amino acid → aminoacyl adenylate (AMP-amino acid) + iP

The aminoacyl adenylate intermediate remains bound to the enzyme, while the PPi is expelled from the active site

aminoacyl adenylate + tRNA → aminoacyl-tRNA + AMP

this process requires Mg2+ as enzyme activator.

2. Ribosome Binding

In prokaryotes, initiation starts with the binding of the small ribosomal subunit (30S) to a specific sequence called the Shine-Dalgarno sequence on the mRNA. In eukaryotes, the small ribosomal subunit (40S) binds to the 5′ cap of the mRNA.

2. Formation of Initiation Complex

In both prokaryotes and eukaryotes, initiation factors assist in recruiting the initiator tRNA (carrying methionine in most cases) to the start codon on the mRNA. The initiator tRNA (In prokaryotes) recognizes the start codon (usually AUG) through base-pairing with the mRNA. In eukaryotes, the initiator tRNA (tRNAiMet) is guided by the ribosome scanning along the mRNA until it reaches the start codon.

3. Ribosome Assembly

The large ribosomal subunit (50S in prokaryotes, 60S in eukaryotes) associates with the small subunit, forming a complete ribosome. This process requires additional initiation factors.

Once the initiation complex is formed, the ribosome is ready for the elongation phase of translation, where amino acids are added to the growing polypeptide chain. The initiation step is crucial for ensuring the proper start of protein synthesis and allows for accurate reading of the genetic code.

initiation factors are required for protein synthesis. Here are some of the key initiation factors involved in protein synthesis:

Prokaryotic Initiation Factors (in bacteria):

  • Initiation Factor 1 (IF-1): It binds to the small ribosomal subunit, promoting its dissociation from the large subunit and preventing premature reassembly.
  • Initiation Factor 2 (IF-2): It binds to the initiator tRNA and helps in its proper placement at the start codon on the mRNA.
  • Initiation Factor 3 (IF-3): It binds to the small ribosomal subunit, preventing premature association with the large subunit.

Eukaryotic Initiation Factors (in eukaryotes):

  • Eukaryotic Initiation Factor 1 (eIF-1): It encourages the big ribosomal subunit’s separation from the small subunit and prevents an early association.
  • Eukaryotic Initiation Factor 2 (eIF-2): It is similar to prokaryotic IF-2, it binds to the initiator tRNA and plays a role in its correct placement on the start codon.
  • Eukaryotic Initiation Factor 3 (eIF-3): It binds to the small ribosomal subunit and prevents its premature association with the large subunit.
  • Eukaryotic Initiation Factor 4 (eIF-4): It is a complex of proteins that promotes the binding of the small ribosomal subunit to the mRNA and facilitates ribosome scanning.

GTP: GTP is necessary for translation initiation, as well as elongation and termination to occur

ATP: ATP is necessary for adding a specific amino acid to tRNA molecules, for charging aminoacyl-tRNAs, and for creating more GTP for translation factors in peptide bond formation

Elongation of Translation

the ribosome moves along the mRNA molecule, reading the codons and matching them with complementary tRNA molecules carrying the corresponding amino acids. Peptide bonds form between adjacent amino acids, extending the growing polypeptide chain . Here is a brief explanation of the elongation process:

1. Codon Recognition

The ribosome reads the mRNA codons in a 5′ to 3′ direction. Each codon corresponds to a specific amino acid. Aminoacyl-tRNA molecules, carrying the corresponding amino acids, enter the ribosome and base-pair with the codons on the mRNA. GTP is used as energy to improve codon recognition accuracy.

2. Peptide Bond Formation

The ribosome catalyzes the formation of a peptide bond between the amino acid carried by the incoming aminoacyl-tRNA and the growing polypeptide chain.

The Process of Protein Synthesis or Translation

at this process, the amino acid is moved from the tRNA at the aminoacyl site (A-site) to the polypeptide chain in the peptidyl site (P-site).

Using energy from GTP hydrolysis, the ribosome catalyses the creation of the peptide bond.

3. Translocation

The ribosome translocates, or moves, along the mRNA in a 5′ to 3′ direction.

The tRNA molecules are moved from the A-site to the P-site and from the P-site to the E-site (exit site) via this movement. The ribosome then releases the empty tRNA in the E-site.

4. Repeat

The process of codon recognition, peptide bond formation, and translocation repeats, allowing the ribosome to continue adding amino acids to the growing polypeptide chain. This process continues until a stop codon is reached on the mRNA.

The Process of Protein Synthesis or Translation
Source- Wikimedia commons.

Elongation factors are essential proteins that assist in the elongation phase of protein synthesis, ensuring the accurate and efficient addition of amino acids to the growing polypeptide chain.

1. EF-Tu (Elongation Factor Tu): In prokaryotes, EF-Tu is responsible for delivering the aminoacyl-tRNA (charged tRNA) to the ribosome.

It attaches to the aminoacyl-tRNA in the cytoplasm and aids during its precise insertion into the ribosome’s A-site (aminoacyl site). EF-Tu, along with GTP (guanosine triphosphate), undergoes a conformational change upon binding to aminoacyl-tRNA, allowing it to enter the ribosome.

2. EF-Ts (Elongation Factor Ts): EF-Ts plays a role in the recycling of EF-Tu. It catalyzes the exchange of GDP (guanosine diphosphate) for GTP on EF-Tu, replenishing the GTP-bound form of EF-Tu required for its activity.

3. EF-G (Elongation Factor G): EF-G is involved in the translocation step of elongation. It encourages the ribosome to proceed along the mRNA, moving the deacylated tRNA from the P-site to the E-site and the peptidyl-tRNA from the A-site. This movement exposes the next codon for aminoacyl-tRNA binding.

4. eEF1A (Eukaryotic Elongation Factor 1A): eEF1A is the eukaryotic counterpart of EF-Tu.It brings the aminoacyl-tRNA to the ribosome’s A-site by binding to it. It also interacts with GTP for its activity.

5. eEF1B (Eukaryotic Elongation Factor 1B): eEF1B assists in the exchange of GDP for GTP on eEF1A, ensuring its activation for further rounds of elongation.

These elongation factors, along with other components of the translation machinery, facilitate the accurate and efficient elongation of the polypeptide chain during protein synthesis.

Termination of Translation

Termination is the final stage of protein synthesis, which occurs when a stop codon (UAA, UAG, or UGA) enters the A site of the ribosome. Stop codons do not specify any amino acid, and instead, one of two release factors binds to the stalled ribosome and causes the release of peptidyl-tRNA

the ribosome encounters a stop codon on the mRNA. Release factors bind to the ribosome, leading to the detachment of the completed protein and the disassembly of the ribosome-mRNA complex.

1. Stop Codon Recognition

When the ribosome encounters a stop codon (UAA, UAG, or UGA) on the mRNA, it does not have a corresponding tRNA to bind to the codon. Instead, release factors recognize the stop codon and bind to the ribosome.

2. Release Factor Binding

The release factor proteins bind to the A-site of the ribosome in place of the tRNA, such as RF1 (Release Factor 1), RF2 (Release Factor 2), or eRF1 (Eukaryotic Release Factor 1) in eukaryotes.

3. Peptide Release

The binding of release factors triggers the hydrolysis of the bond between the completed polypeptide chain and the tRNA in the P-site. This process releases the polypeptide from the ribosome.

4. Ribosome Dissociation

After peptide release, the ribosome subunits dissociate from the mRNA template, preparing for another round of translation or disassembling into their individual subunits.

Prokaryotic Release Factors:

  • RF1 (Release Factor 1): Recognizes the UAA and UAG stop codons in prokaryotes and binds to the A-site of the ribosome, triggering peptide release.
  • RF2 (Release Factor 2): Recognizes the UAA and UGA stop codons in prokaryotes and also binds to the A-site of the ribosome to promote peptide release.
  • RF3 (Release Factor 3): Assists in the recycling of RF1 and RF2, ensuring their availability for subsequent rounds of translation.

Eukaryotic Release Factors:

  • eRF1 (Eukaryotic Release Factor 1): Recognizes all three stop codons (UAA, UAG, and UGA) in eukaryotes.It facilitates the hydrolysis of the connection between the polypeptide chain and the tRNA, releasing the peptide, by attaching to the ribosome’s A-site.
  • eRF3 (Eukaryotic Release Factor 3): Similar to RF3 in prokaryotes, eRF3 helps in the recycling of eRF1 and ensures efficient termination.

The Role of Mg2+ and GTP in Protein Synthesis

Guanosine triphosphate (GTP) and magnesium ions (Mg2+) are essential for protein synthesis.

Magnesium ions (Mg2+):

Ribosome Function: Ribosomes, the biological machinery in the process of protein synthesis, require Mg2+ ions to function properly. They support ribosomal RNA (rRNA) structural stabilisation and ribosome assembly and function.

tRNA Binding: Mg2+ ions facilitate the binding of transfer RNA (tRNA) to the ribosome, allowing accurate and efficient delivery of amino acids to the growing polypeptide chain.

Enzymatic Reactions: Mg2+ ions act as cofactors for various enzymatic reactions during translation, such as peptidyl transferase activity, which forms peptide bonds between amino acids.

Guanosine Triphosphate (GTP):

Initiation: GTP is involved in the initiation of translation. During the formation of the initiation complex, GTP is hydrolyzed to GDP (guanosine diphosphate) when initiation factors bind to the small ribosomal subunit and other components. GTP provides the energy required for these initiation steps.

Elongation: GTP is also required during the elongation phase of translation. Elongation factors, such as EF-Tu and EF-G, use GTP as an energy source for their activity. GTP hydrolysis powers the movement of tRNA molecules and the ribosome along the mRNA during peptide bond formation and translocation.

  • Mg2+ is necessary for guanine nucleotide binding and GTP hydrolysis in GTP-binding proteins
  • Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5′-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly
  • High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation
  • GTP is necessary for various stages of protein synthesis, including initiation, elongation, and termination

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