Molecular biology

Eukaryotic Translation – Protein synthesis in Eukaryotic

THIS BLOG INCLUDES: hide 1 Eukaryotic Translation 2 Eukaryotic initiation factor 3 Elongation factor for Eukaryotic Translation 4 Steps of Eukaryotic Translation...

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Sourav Bio
This article writter by Sourav Bio on September 30, 2021

Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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Eukaryotic Translation
Eukaryotic Translation

Before going through Eukaryotic Translation steps, please take a look at our previous article Prokaryotic Translation Steps, Requirements, to check the components required for the translation procedure.

Eukaryotic Translation

  • Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes.
  • It consists of four phases: gene regulation, elongation, termination, and recycling.

Eukaryotic initiation factor

Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the initiation phase of eukaryotic translation. These proteins help stabilize the formation of ribosomal preinitiation complexes around the start codon and are an important input for post-transcription gene regulation. 

FactorFunction
eIF1 and eIF1AeIF1 and eIF1A both bind to the 40S ribosome subunit-mRNA complex. Together they induce an “open” conformation of the mRNA binding channel, which is crucial for scanning, tRNA delivery, and start codon recognition.
eIF2eIF2 is the main protein complex responsible for delivering the initiator tRNA to the P-site of the preinitiation complex, as a ternary complex containing Met-tRNAiMet and GTP (the eIF2-TC).
eIF3eIF3 independently binds the 40S ribosomal subunit, multiple initiation factors, and cellular and viral mRNA.
eIF4The eIF4F complex is composed of three subunits: eIF4A, eIF4E, and eIF4G. Each subunit has multiple human isoforms and there exist additional eIF4 proteins: eIF4B and eIF4H. eIF4G is a 175.5-kDa scaffolding protein that interacts with eIF3 and the Poly(A)-binding protein (PABP), as well as the other members of the eIF4F complex. eIF4E recognizes and binds to the 5′ cap structure of mRNA, while eIF4G binds PABP, which binds the poly(A) tail, potentially circularizing and activating the bound mRNA. eIF4A – a DEAD box RNA helicase – is important for resolving mRNA secondary structures.
eIF5eIF5 is a GTPase-activating protein, which helps the large ribosomal subunit associate with the small subunit. It is required for GTP-hydrolysis by eIF2 and contains the unusual amino acid hypusine.
eIF5AeIF5A is the eukaryotic homolog of EF-P. It helps with elongation and also plays a role in termination.
eIF5BeIF5B is a GTPase, and is involved in assembly of the full ribosome. It is the functional eukaryotic analog of bacterial IF2.
eIF6eIF6 performs the same inhibition of ribosome assembly as eIF3, but binds with the large subunit.

Elongation factor for Eukaryotic Translation

Elongation factors are a set of proteins that function at the ribosome, during protein synthesis, to facilitate translational elongation from the formation of the first to the last peptide bond of a growing polypeptide.

BacterialEukaryotic/ArchaealFunction
EF-TueEF-1 subunit αmediates the entry of the aminoacyl tRNA into a free site of the ribosome.
EF-TseEF-1 subunit βγserves as the guanine nucleotide exchange factor for EF-Tu, catalyzing the release of GDP from EF-Tu.
EF-GeEF-2catalyzes the translocation of the tRNA and mRNA down the ribosome at the end of each round of polypeptide elongation. Causes large conformation changes.
EF-PEIF5Apossibly stimulates formation of peptide bonds and resolves stalls.

Steps of Eukaryotic Translation

The eukaryotic translation is completed in the following steps;

  1. Initiation of Translation
  2. Elongation of peptide chain
  3. Termination of peptide chain

Initiation of Translation

The initiation of translation in eukaryotes is complex, involving at least ten eukaryotic initiation factors (eIFs). Some of the eIFs contain multiple (3-8) subunits. The process of translation initiation can be divided into four steps.

  1. Ribosomal dissociation.
  2. Formation of 43S preinitiation complex.
  3. Formation of 48S initiation complex.
  4. Formation of 80S initiation complex.

Ribosomal dissociation

  • The 80S ribosome dissociates to form 40S and 60S subunits.
  • Two initiating factors namely eIF3 and eIF-1A bind to the newly formed 40S subunit, and thereby block its reassociation with 60S subunit. For this reason, some workers name eIF-3 as anti-association factor.

Formation of 43S preinitiation complex

  • A ternary complex containing met-tRNAi and eIF-2 bound to GTP attaches to 40S ribosomal subunit to form 43S preinitiation complex.
  • The presence of eIF-3 and eIF-1A stabilizes this complex (Note : Met-tRNA is specifically involved in binding to the initiation condon AUGs; hence the superscripti is used in mettRNAi).

Formation of 48S initiation complex

  • The binding of mRNA to 43S preinitiation complex results in the formation of 48S initiation complex through the intermediate 43S initiation complex. This, however, involves certain interactions between some of the eIFs and activation of mRNA.
  • eIF-4F complex is formed by the association of eIF-4G, eIF-4A with eIF-4E.
  • The so formed eIF-4F (referred to as cap binding protein) binds to the cap of mRNA.
  • Then elF-4A and elF-4B bind to mRNA and reduce its complex structure.
  • This mRNA is then transferred to 43S complex.
  • For the appropriate association of 43S preinitiation complex with mRNA, energy has to be supplied by ATP.

Recognition of initiation codon: 

  • The ribosomal initiation complex scans the mRNA for the identification of appropriate initiation codon. 5c-AUG is the initiation codon and its recognition is facilitated by a specific sequence of nucleotides surrounding it. 
  • This marker sequence for the identification of AUG is called as Kozak consensus sequence. 
  • In case of prokaryotes the recognition sequence of initiation codon is referred to as Shine- Dalgarno sequence.

Formation of 80S initiation complex

  • 48S initiation complex binds to 60S ribosomal subunit to form 80S initiation complex.
  • The binding involves the hydrolysis of GTP (bound to eIF-2). This step is facilitated by the involvement of eIF-5.
  • As the 80S complex is formed, the initiation factors bound to 48S initiation complex are released and recycled. 
  • The activation of eIF-2 requires eIF-2B (also called as guanine nucleotide exchange factor) and GTP. 
  • The activated eIF-2 (i.e. bound to GTP) requires eIF2C to form the ternary complex.

Regulation of initiation

  • The eIF-4F, a complex formed by the assembly of three initiation factors controls initiation, and thus the translation process.
  • eIF4E, a component of eIF-4F is primarily responsible for the recognition of mRNA cap. And this step is the rate-limiting in translation.
  • eIF-2 which is involved in the formation of 43S preinitiation complex also controls protein biosynthesis to some extent.

Elongation of Translation

Ribosomes elongate the polypeptide chain by a sequential addition of amino acids. The amino acid sequence is determined by the order of the codons in the specific mRNA. Elongation, a cyclic process involving certain elongation factors (EFs), may be divided into three steps.

  1. Binding of aminoacyl t-RNA to A-site.
  2. Peptide bond formation.
  3. Translocation.

Binding of aminoacyl—tRNA to A-site

  • The 80S initiation complex contains met-tRNAi in the P-site, and the A-site is free.
  • Another aminoacyl-tRNA is placed in the A-site.  This requires proper codon recognition on the mRNA and the involvement of elongation factor 1a (EF-Ia) and supply of energy by GTP. 
  • As the aminoacyl-tRNA is placed in the A-site, EF-1D and GDP are recycled to bring another aminoacyl-tRNA.

Peptide bond formation

  • The enzyme peptidyltransferase catalyses the formation of peptide bond. 
  • The activity of this enzyme lies on 28S RNA of 60S ribosomal subunit. It is therefore the rRNA (and not protein) referred to as ribozyme that catalyses the peptide bond formation. 
  • As the amino acid in the aminoacyl-tRNA is already activated, no additional energy is required for peptide bond formation. The net result of peptide bond formation is the attachment of the growing peptide chain to the tRNA in the A-site.

Translocation

  • As the peptide bond formation occurs, the ribosome moves to the next codon of the mRNA (towards 3c-end). This process called translocation, basically involves the movement of growing peptide chain from A-site to P-site.
  • Translocation requires EF-2 and GTP.
  • GTP gets hydrolysed and supplies energy to move mRNA. EF-2 and GTP complex recycles for translocation.
  • In recent years, another site namely exit site (E-site) has been identified in eukaryotes. The deacylated tRNA moves into the E-site, from where it leaves the ribosome.
  • In case of prokaryotes, the elongation factors are different, and they are EF-Tu, EF-Ts (in place of of EF-1a) and EF-G (instead of EF-2).

Incorporation of amino acids

  • It is estimated that about six amino acids per second are incorporated during the course of elongation of translation in eukaryotes.
  • In case of prokaryotes, as many as 20 amino acids can be incorporated per second.
  • Thus the process of protein/polypeptide synthesis in translation occurs with great speed and accuracy.

Termination of Translation

Termination is a simple process when compared to initiation and elongation. After several cycles of elongation, incorporating amino acids and the formation of the specific protein/ polypeptide molecule, one of the stop or termination signals (UAA, UAG and UCA) terminates the growing polypeptide.

The termination codons which act as stop signals do not have specific tRNAs to bind. As theb termination codon occupies the ribosomal A-site, the release factor namely eRF recognizes the stop signal. eRF-GTP complex, in association with the enzyme peptidyltransferase, cleaves the peptide bond between the polypeptide and the tRNA occupying P-site. In this reaction, a water molecule, instead of an amino acid is added. This hydrolysis releases the protein and tRNA from the P-site. The 80S ribosome dissociates to form 40S and 60S subunits which are recycled. The mRNA is also released.

Steps of Eukaryotic Translation
Steps of Eukaryotic Translation

References

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Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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