Protein synthesis in Eukaryotes (Eukaryotic translation)
Definition: The process by which individual amino acids are connected to each other in a specific order dictated by the nucleotide sequence in DNA.
Site of translation process: Translation occurs in the cytoplasm.
Template for Protein synthesis: mRNA sequence is used as a template to guide the synthesis of a chain of amino acids that form a protein.
Polarity of mRNA: 5’ to 3’ (ribosome moves along the mRNA in this direction).
Eukaryotic mRNA has only one translation start site. Eukaryotic mRNA is always monocystronic.
Monocistronic mRNA: mRNA can acts as the template for the synthesis of single protein.
Direction of polypeptide synthesis: Polypeptides grow from the N terminus toward the C terminus.
In eukaryotic cells transcription and translation will not take place simultaneously.
Transcription and translation are separate process, transcription occurs in the nucleus whereas translation occurs in the cytoplasm.
Pre-mRNA must be processed and transported to the cytoplasm in eukaryotes before translation is initiated. Thus, there is ample opportunity for the formation of complex secondary structures that must be removed to expose signals in the mature mRNA.
Life span of mRNA: Life span of mRNA long, few hours to a day or sometimes more.
Protein synthesis in eukaryotic cells may continue long after transcription has ended.
Ribosome type in Eukaryotes: 80S ribosome
Endoplasmic reticulum is present. So protein synthesizing ribosome usually attached to the ER.
Ribosomal components: 60S larger subunit and 40S smaller subunit
Sub components of ribosomal subunit: rRNA (5S rRNA, 28S rRNA, 18S rRNA and 5.8SrRNA) + Proteins
Enzymes involved in protein synthesis: Aminoacyl tRNA synthetase, Peptidyl transferase and tRNA deacylase enzyme
Codons involved in protein synthesis: 3 stop codons (UAA, UAG and UGA) and 61 aminoacid coding codons.
Speed of translation: ~1 amino acid/second
Protein synthesis can be divided into four phases: Binding of aminoacid to appropriate tRNAs, initiation, elongation and termination.
Activation of the amino acid: Charging of tRNA with its cognate aminoacid
- Attachment of the correct amino acid to the tRNA by specific enzyme called aminoacyl tRNA synthetases which is an Mg2+ dependent enzyme.
- Process occurs in the cytoplasm utilizing ATP as energy source.
- The amino acid is attached by its carboxyl end to the 3’ end of the tRNA.
- Upon attachment of the amino acid, the tRNA becomes activated or charged.
Lecture notes on activation of aminoacid: http://easylifescienceworld.com/activation-of-amino-acids-by-aminoacyl-trna-synthetases/
Type of translation initiation mechanism: Cap dependent initiation and cap independent initiation
Lecture notes on Cap independent translation initiation: http://easylifescienceworld.com/cap-independent-translation-initiation-in-eukaryotes/
Start site of translation: Always have only one start site which is located towards the 5’ region of mRNA.
Kozak sequence: Kozak sequence is present in the mRNA which is located few nucleotide upstream of start site. Kozak sequence assists initiation process of translation.
Smaller subunit of ribosome (40S) recognize the 5’ cap of mRNA during initiation
Initiation codon: Initiation codon is always AUG.
The AUG nearest the 5′ end of mRNA is usually selected as the start site.
First aminoacid: Methionine (No formylation of methionine)
First tRNA: Met – tRNAmet
Initiation factors: 11 factors
eIF3, eIF1A, eIF6, eIF2, eIF2B, eIF4F complex (eIF4E, eIF4G and eIF4A), eIF4B, eIF4H, eIF5B
Steps involved in initiation: Formation of 80S initiation complex
- Formation of 43S pre initiation complex (40S + initiator tRNA with aminoacid + Initiation factors)
- The 40S subunit that already containing the initiator tRNA attaches to the 5’ – end of the mRNA and tracks along the mRNA in the 5′ to 3′ direction until the AUG start codon is recognized.
- The recognition of the AUG occurs mainly through base pairing with the CUA anticodon on the bound Met – tRNAi Met. Then the 60S subunit binds to the complex of Met-tRNAi – mRNA – 40S subunit.
Small subunit binds stably to mRNA only after initiator met – tRNA has bound to 40S subunit.
(The initiator tRNA must bind to the small subunit before it binds the mRNA)
Lecture notes on translation initiation: http://easylifescienceworld.com/initiation-of-protein-synthesis-in-eukaryotes-translation-initiation-in-eukaryotes/
Elongation Factors: Elongation factors are eEF1 and eEF2
eEF-1 has two subunits: α subunit and βγ subunit.
- α acts as counterpart to prokaryotic EF-Tu.
Function: Mediating the entry of the aminoacyl tRNA into a free site of the ribosome.
- βγ acts as counterpart to prokaryotic EF-Ts.
Function: Serving as the guanine nucleotide exchange factor for α (GEF). It catalyzes the release of GDP from α.
eEF – 2: The counterpart to prokaryotic EF-G.
Function: Catalyzing the translocation of the tRNA and mRNA down the ribosome at the end of each round of polypeptide elongation.
Steps involved in elongation: The lengthening of the growing polypeptide chain by one amino acid is called elongation.
Each amino acid is added to the nascent polypeptide chain in a three-step cycle
1) Positioning the correct aminoacyl-tRNA in the A site of the ribosome: The anticodon of the second incoming aminoacyl tRNA base pair with complementary codon on mRNA which is located in the A site.
2) Peptide bond formation: An rRNA molecule of the large subunit (28SrRNA) catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site.
3) Translocation: The tRNA in the A site bearing the two-peptide protein shifts into the P site, allowing another tRNA with its amino acid to bind to a newly exposed codon at the A site. The empty tRNA in the P site is moved to the E site, where it is released.
The mRNA is moved through the ribosome in one direction only (5′ to 3′). The ribosome moves along the mRNA one codon at a time.
Figure 2: Elongation of polypeptide chain in eukaryotes
Termination Factors: Termination is facilitated by only one release factor eRF1
eRF1 recognizes all three stop codons.
eRF3 is a ribosome-dependent GTPase that helps eRF1 release the completed polypeptide.
Steps involved in termination: The overall process of termination is similar in prokaryotes.
Protein synthesis ends when the ribosome reaches a stop codon at the A site, which causes the completed protein to detach from the final tRNA.
Then the tRNA an mRNA are released from the ribosome and the ribosome is disassembled into its subunits.
Lecture notes on translation termination: http://easylifescienceworld.com/translation-termination-in-eukaryotes-termination-of-protein-synthesis/
Post translational modification: Post translational modification usually takes place in the endoplasmic reticulum or Golgi bodies or in the cytoplasm
(Details will be updated in next lecture)
Important point: Usually the un-formylated first methionine as such is removed from the polypeptide after protein synthesis