Translation:- It takes place at ribosomes. Includes 4 steps-
1. Charging of tRNA (aminoacylation of tRNA):-
· Formation of peptide bond requires energy obtained from ATP.
· For this, amino acids are activated (amino acid + ATP) and linked to their cognate tRNA in the presence of aminoacyl tRNA synthetase. So the tRNA becomes charged.
2. Initiation:-
· It begins at the 5’-end of mRNA in the presence of an initiation factor.
· The mRNA binds to the small subunit of ribosome. Now the large subunit binds to the small subunit to complete the initiation complex.
· Large subunit has 2 binding sites for tRNA- aminoacyl tRNA binding site (A site) and peptidyl site (P site).
· Initiation codon for methionine is AUG. So methionyl tRNA complex would have UAC at the Anticodon site.
3. Elongation:-
· At the P site the first codon of mRNA binds with anticodon of methionyl tRNA complex.
· Another aminoacyl tRNA complex with an appropriate amino acid enters the ribosome and attaches to A site. Its anticodon binds to the second codon on the mRNA and a peptide bond is formed between first and second amino acids in presence of an enzyme, peptidyl transferase.
· First amino acid and its tRNA are broken. This tRNA is removed from P site & send to E site and second tRNA at the A site is pulled to P site along with mRNA. This is called translocation.
· Then 3rd codon comes on A site and a suitable tRNA with 3rd amino acid binds at the A site. This process is repeated.
· A group of ribosomes associated with a single mRNA for translation is called a polyribosome (polysomes).
4. Termination:-
· When aminoacyl tRNA reaches the termination codon like UAA, UAG & UGA, the termination of translation occurs. The polypeptide and tRNA are released from the ribosomes.
· The ribosome dissociates into large and small subunits at the end of protein synthesis.
· An mRNA has additional sequences that are not translated (untranslated regions or UTR). UTRs are present at both 5’-end (before start codon) and 3’-end (after stop codon). They are required for efficient translation process.
Prokaryotic versus Eukaryotic Translation:-
1. Process:- In prokaryotes transcription and translation are continuous processes and occurs simultaneously in the cytoplasm. While in eukaryotes transcription and translation are separate process, transcription occurs in the nucleus whereas translation occurs in the cytoplasm.
2. Starting:- In prokaryotes 5' end of mRNA is immediately available for translation. While in eukaryotes the primary transcript is processed after transcription and then it is transported to the cytoplasm, then only the cytoplasmic ribosomes can initiate translation.
3. Ribosome:- 70S type ribosomes in prokaryotes. While 80S type ribosomes in eukaryotes.
4. Ribosome sub-units:- In prokaryotes 50S larger sub-unit and 305 smaller sub-unit. While in eukaryotes ribosome composed of 60S larger subunit and 40S smaller subunit.
5. rRNA of larger ribosome sub-unit:- In prokaryotes two rRNA molecules 5S and 23S rRNA. While in eukaryotes three rRNA molecules, 5S, 5.8S and 28S rRNA.
6. rRNA of smaller ribosome sub-unit:- In prokaryotes one rRNA molecule, 16S rRNA. While in prokaryotes one rRNA molecule18S rRNA.
7. Smaller ribosome sub-unit proteins:- In prokaryotes 21 proteins. While in eukaryotes 33 proteins.
8. Larger ribosome sub-unit proteins:- In prokaryotes 36 proteins. While in eukaryotes 49 proteins.
9. Ribosome mass:- In prokaryotes 2700 kd. While in eukaryotes 4200 kd.
10. Endoplasmic reticulum:- In prokaryotes it is absent and hence protein synthesizing ribosome freely distributed in the cytoplasm. While in eukaryotes Endoplasmic reticulum is present, protein synthesizing ribosome usually attached to the ER.
11. mRNA:- It is polycystronic in prokaryotes means mRNA can acts as the template for the synthesis of many polypeptides. While in eukaryotes it is always monocystronic means mRNA can act as the template for a single polypeptide.
12. Translation initiation:- In prokaryotes single type which is cap independent initiation. While in eukaryotes two types of translation initiation mechanisms- (i) Cap depended and (ii) Cap independent.
13. Start site:- Prokaryotes may have many start sites. While eukaryotes always have only one start site.
14. SD Sequence (Shine-Dalgarno sequence):- In prokaryotes SD sequence present 8 nucleotide upstream of start codon. SD sequence act as the ribosome binding site. Smaller subunit of ribosome (30S) recognize the SD sequence in the mRNA during translation initiation. While in eukaryotes SD sequence is absent in mRNA.
15. Kozak sequence:- In prokaryotes kozak sequence absent in mRNA. While in eukaryotes 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
16. Initiation codon:- In prokaryotes it is usually AUG, occasionally GUG or UUG. While in eukaryotes initiation codon is AUG, occasionally GUG or CUG.
17. First tRNA:- In prokaryotes first tRNA is special type namely Met-tRNAf. While in eukaryotes first tRNA is Met-tRNA.
18. First amino acid:- In prokaryotes first amino acid in the protein synthesis (methionine) will be formylated. While in eukaryotes no fomylation of methionine, the first amino acid, will occurs.
19. Initiation factors:- In prokaryotes only three initiation factors are required, they are IF1, IF2,IF3. While in eukaryotes seven types of initiation factors are required for translation, they are eIF1, eIF2, eIF3, eIF4, eIF5A, eIF5B, eIF6.
20. Elongation factors:- In prokaryotes two types of elongation factors, EF - Tu and EF -Ts. While in eukaryotes elongation factors are eEF1and eEF2.
21. Speed of translation:- In prokaryotes 20 amino acids / second. While in eukaryotes 1amino acid / second.
22. Termination:- In prokaryotes it is facilitated by three release factors, RF1, RF2, RF3. While in eukaryotes termination is facilitated by only one release factor eRF1.
23. Removal of first amino acid:- In prokaryotes only the formyl group from the first amino acid (methionine) is removed from the polypeptide after protein synthesis. While in ekaryotes usually the un-formylated first methionine as such is removed from the polypeptide after protein synthesis.
24. Life span of mRNA:- In prokaryotes it is short, few seconds to few minutes. While in eukaryotes
life span of mRNA is long, few hours to a day or sometimes more.25. Prevention of ribosomal association:- In prokaryotes IF3 prevents the association of ribosomal subunitsin the absence of initiation complex. While in eukaryotes eIF3 prevents the association of ribosomal subunits in the absence of initiation complex.
26. Post translational modifications:- In prokaryotes post translational modifications of proteins takes place in the cytoplasm. While in eukaryotes post translational modifications usually takes place in the endoplasmic reticulum or Golgi bodies or in the cytoplasm.