Mechanism of Translation, Initiation, Elongation and Termination

The synthesis of protein from mRNA involves translation of the language of nucleic acids into language of proteins.
For initiation and elongation of a polypeptide, the formation of aminoacyl transfer RNAs is a prerequisite,

Formation of Aminoacyl tRNA

a) Activation of amino acid

This reaction is brought about by the binding of an amino acid with ATP and is mediated by specific activating enzymes known as aminoacyl tRNA synthetases or aaRs.
As a result of this reaction between amino acid and adenosine triphosphate, mediated by specific enzyme, a complex (amino acyl-AMP- enzyme complex) is formed.
Amino acyl-RNA synthetases are specific with respect to amino acids.
For different amino acids, different enzymes would be required.

AA + ATP –ENZYME- (aa1-AMP) -Enz1 + PPi

B) The transfer of amino acid to tRNA

The aminoacyl-AMP-enzyme complex, formed during the step outlined above, reacts with a particular tRNA and transfers the amino acid to the tRNA.
A particular amino acid would require a particular enzyme and a particular species of tRNA.
This would mean that for 20 amino acids, at least 20 different enzymes and also at least 20 different t-RNA species would be required.

(aa1-AMP) Enz1 + t-RNA1 —— aa1- t-RNA1 + AMP + Enz1

Initiation of Polypeptide

The initiation of polypeptide chain is always brought about by the amino acid methionine, which is regularly coded by the codon AUG,
In eukaryotes, formulation of initiating methionine is not brought about due to the absence of tRNAfmet in plants and animals.
Initiation in higher organisms will therefore take place without formylation.

Initiation in eukaryotes

Initiation of the polypeptide chain in eukaryotes is similar to that of prokaryotes, except the following minor differences.
(i) In eukaryotes there are more initiation factors.
They are named by putting a prefix ‘e’ to signify their eukaryotic origin.
These factors are eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF4D, eIF4F, eIF5 and eIF6.
(ii) In eukaryotes, formylation of methionine does not take place.
(iii) In eukaryotes, smaller subunit associates with initiator tRNA-imet, without the help of mRNA, while in prokaryotes, generally the 30S-mRNA complex is first formed which then associates with f-met-tRNAfmet.

Kozak’s ribosome scanning hypothesis for translation in eukaryotes

In 1983, Marilyn Kozak proposed a hypothesis for initiation of translation by eukaryotic ribosome.
According to this hypothesis, the 40S smaller subunit of a eukaryotic ribosome with its associated met-tRNA moves down the mRNA from the 5′ end, until it encounters the first AUG.
At this point, the 60S subunits join and the translation begins.
The 80S ribosome, after reaching termination, releases protein and dissociates in two subunits.

Protein Synthesis Process

The overall mechanism of protein synthesis in eukaryotes is basically the same as in prokaryotes.

However, there are some significant differences:

Whereas a prokaryotic ribosome has a sedimentation coefficient of the 70S and subunits of 30S and 50S, a eukaryotic ribosome has a sedimentation coefficient of 80S with subunits of 40S and 60S.
The composition of eukaryotic ribosomal subunits is also more complex than prokaryotic subunits but the function of each subunit is essentially the same as in prokaryotes.
In eukaryotes, each mRNA is monocistronic, that is, discounting any subsequent post-translational cleavage reactions that may occur; the mRNA encodes a single protein. In prokaryotes, many mRNAs are polycistronic, that is they encode several proteins. Each coding sequence in a prokaryotic mRNA has its own initiation and termination codons.
Initiation of protein synthesis in eukaryotes requires at least nine distinct eukaryotic initiation factors (eIFs) compared with the three initiation factors (IFs) in prokaryotes.
In eukaryotes, the initiating amino acid is methionine, not N-formylmethionine as in prokaryotes.
As in prokaryotes, a special initiator tRNA is required for initiation and is distinct from the tRNA that recognizes and binds to codons for methionine at internal positions in the mRNA. When charged with methionine ready to begin initiation, this is known as Met-tRNAimet
The main difference between initiation of translation in prokaryotes and eukaryotes is that in bacteria, a Shine–Dalgarno sequence lies 5’ to the AUG initiation codon and is the binding site for the 30S ribosomal subunit.
In contrast, most eukaryotic mRNAs do not contain Shine–Dalgarno sequences. Instead, a 40S ribosomal subunit attaches at the 5’ end of the mRNA and moves downstream (i.e. in a 5’ to 3’ direction) until it finds the AUG initiation codon. This process is called scanning.
Prokaryotic translation requires no helicase, presumably because protein synthesis in bacteria can start even as the mRNA is still being synthesized whereas, in eukaryotes, transcription in the nucleus and translation in the cytoplasm are separate events that allow time for mRNA secondary structure to form.

Protein synthesis (or translation) takes place in three stages:

Initiation
Elongation and
Termination

Initiation of Protein Synthesis

The first step is the formation of a pre-initiation complex consisting of the 40S small ribosomal subunit, Met-tRNAimet, eIF-2, and GTP.
The pre-initiation complex binds to the 5’ end of the eukaryotic mRNA, a step that requires eIF-4F (also called cap-binding complex) and eIF-3.
The eIF-4F complex consists of eIF-4A, eIF-4E, and eIF-4G; eIF-4E binds to the 5’ cap on the mRNA whilst eIF-4G interacts with the poly (A) binding protein on the poly (A) tail.
The eIF-4A is an ATP-dependent RNA helicase that unwinds any secondary structures in the mRNA, preparing it for translation.
The complex then moves along the mRNA in a 5’ to 3’ direction until it locates the AUG initiation codon (i.e. scanning).
The 5’ untranslated regions of eukaryotic mRNAs vary in length but can be several hundred nucleotides long and may contain secondary structures such as hairpin loops. These secondary structures are probably removed by initiation factors of the scanning complex.
The initiation codon is usually recognizable because it is often (but not always) contained in a short sequence called the Kozak consensus (5’-ACCAUGG-3’).
Once the complex is positioned over the initiation codon, the 60S large ribosomal subunit binds to form an 80S initiation complex, a step that requires the hydrolysis of GTP and leads to the release of several initiation factors.

Elongation of Polypeptide

Elongation depends on eukaryotic elongation factors.
Three elongation factors, eEF-1A, eEF-IB, and eEF-2, are involved which have similar functions to their prokaryotic counterparts EF-Tu, EF-Ts and EF-G.
At the end of the initiation step, the mRNA is positioned so that the next codon can be translated during the elongation stage of protein synthesis.
The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an aminoacyl-tRNA.
During chain elongation, each additional amino acid is added to the nascent polypeptide chain in a three-step microcycle.
The steps in this microcycle are:

Positioning the correct aminoacyl-tRNA in the A site of the ribosome,
Forming the peptide bond and
Shifting the mRNA by one codon relative to the ribosome.

Although most codons encode the same amino acids in both prokaryotes and eukaryotes, the mRNAs synthesized within the organelles of some eukaryotes use a variant of the genetic code.
During elongation in bacteria, the deacylated tRNA in the P site moves to the E site prior to leaving the ribosome. In contrast, although the situation is still not completely clear, in eukaryotes the deacylated tRNA appears to be ejected directly from the ribosome.

Formation of peptide bond

This is a catalytic reaction during which a peptide bond is formed between the free carboxyl group of the peptidyl tRNA at the ‘P’ site and the free amino group present with amino acyl tRNA, which is available at the A site.
The 50S rRNA has peptidyl transferase activity, so that the ribosome is described as a ribozyme.
According to this displacement model, the peptidyl chain remains in a constant position relative to the ribosome, while the tRNA moves during the peptide reaction.
After the formation of peptide bonds, the tRNA at ‘P’ site is deacylated and the tRNA at ‘A’ site now carries the polypeptide.

Translocation of peptidyl tRNA From ‘A’ to ‘P’ site.

The peptidyl tRNA present at ‘A’ site is now Translocated to ‘P’ site.
For translocation of peptidyl tRNA from ‘A’ site to P site, there are two models available:
(i) According to two sites model, deacylated tRNA is liberated from ‘P’ site, and with the help of one GTP molecule and an elongation factor EF-G, the peptidyl tRNA is translocated from ‘A’ to ’P’ site.
Thus according to this model, tRNA is either entirely in the A site or entirely in the P Site.
The requirement of EF-G and GTP for translocation was revealed by the use of antibiotics.
The elongation factor EF-G binds to the ribosome and is released on hydrolysis of GTP, which is a ribosomal function. EF-G and EF-Tu cannot bind to ribosome simultaneously, so that the entry of a fresh aa-tRNA on ‘A’ site and the translocation of peptidyl tRNA from ‘A’ to ‘P’ site has to follow each other and cannot occur simultaneously.
In eukaryotes, the elongation factor needed for translocation is called eEF-2, for the formation of one peptide bond.
One ATP molecule and two GTP molecules (one for transfer of aa-tRNA to ‘A’ site and the other for translocation of peptidyl tRNA from ‘A’ to ‘P’ site) are required.

Termination of Polypeptide

Terminations in mRNA with stop codon

Termination of the polypeptide chain is brought about by the presence of any one of the three combination codons, namely UAA,UAG and UGA.
These termination codons are recognized by one of the two release factors RF1 and RF2.
The release factors to act on ‘A’ site, since suppressor rRNA is capable of recognizing by entry at ‘A’ site.
A third release factor RF3 stimulate the action of RF1 and RF2 in a GTP-dependent and condon independent manner GTP molecule is hydrolysed during release of a polypeptide, when RF3 stimulates RF1 and RF2. .
For release reaction, the poly peptidyl tRNA must be present on ‘P’ site and the release factors help in splitting of the carboxyl group between the polypeptide and the last tRNA carrying this chain.
Polypeptide is thus released and the ribosome dissociates into two subunits with the help of ribosome release factor or RRF.
It has been shown that the translation apparatus in chloroplasts and mitochondria differs from that in cytoplasm in eukaryotes in the following respects.
(i) Ribosomes in these organelles are smaller in size than these in cytoplasm.
(ii) The tRNAs are specific and differ, the number of tRNAs in mitochondria being 22 as against 55 in cytoplasm.
(iii) Initiation of translation takes place by formyl-methionyl tRNA both in chloroplasts and mitochondria, although no formylation takes place in cytoplasm.
(iv) Translation in chloroplasts and mitochondria can be inhibited by chloramphenicol.

1. [PYQ – CSIR NET, 2018]
What is the role of the Kozak sequence in eukaryotic translation?
a) Termination of translation
b) Enhancing ribosome recognition of the start codon
c) Inhibition of translation
d) Binding of release factor
Answer: b

2. Which of the following is the correct order of translation stages in eukaryotes?
a) Termination → Initiation → Elongation
b) Elongation → Termination → Initiation
c) Initiation → Elongation → Termination
d) Initiation → Termination → Elongation
Answer: c

3. [PYQ – GATE BT, 2021]
Which of the following initiation factors is responsible for binding Met-tRNAiMet to the 40S ribosomal subunit in eukaryotes?
a) eIF-4F
b) eIF-2
c) eIF-6
d) eIF-3
Answer: b

4. Which of the following is NOT true about eukaryotic translation?
a) It requires a 5′ cap and poly(A) tail.
b) It begins with formyl-methionine.
c) It requires eIFs (eukaryotic initiation factors).
d) It occurs in the cytoplasm.
Answer: b

5. [PYQ – DBT JRF, 2017]
What is the function of eEF-2 in eukaryotic translation?
a) Delivers aminoacyl-tRNA to ribosome
b) Initiates translation by binding to the 5’ cap
c) Helps translocate the ribosome along mRNA
d) Catalyzes peptide bond formation
Answer: c

6. In eukaryotic translation, the ribosome scans the mRNA from the 5’ end to locate:
a) Stop codon
b) Poly(A) tail
c) AUG start codon
d) Shine-Dalgarno sequence
Answer: c

7. [PYQ – ICMR JRF, 2019]
Which of the following is the initiation codon for eukaryotic translation?
a) GUG
b) AUG
c) UAA
d) UGA
Answer: b

8. Which initiation factor recognizes the 5’ cap structure of eukaryotic mRNA?
a) eIF-4E
b) eIF-3
c) eIF-2
d) eIF-6
Answer: a

9. Which of the following enzymes catalyzes the formation of peptide bonds during elongation?
a) Peptidyl transferase
b) Aminoacyl tRNA synthetase
c) eIF-2
d) RF1
Answer: a

10. [PYQ – CSIR NET, 2020]
Which of the following release factors is involved in eukaryotic translation termination?
a) RF1 and RF2
b) eRF1 and eRF3
c) RF3 only
d) EF-G
Answer: b

11. What is the sedimentation coefficient of the eukaryotic ribosome?
a) 70S (30S + 50S)
b) 80S (40S + 60S)
c) 60S (20S + 40S)
d) 100S (50S + 50S)
Answer: b

12. The aminoacyl tRNA synthetase:
a) Catalyzes the peptide bond formation
b) Helps in ribosome assembly
c) Activates amino acids for attachment to tRNA
d) Degrades mRNA
Answer: c

13. [PYQ – GATE XL, 2016]
Which of the following components of the ribosome has peptidyl transferase activity?
a) 40S
b) 60S
c) 50S
d) 30S
Answer: b

14. The process of scanning the mRNA by the ribosome is proposed by:
a) Watson and Crick
b) Rosalind Franklin
c) Marilyn Kozak
d) Sydney Brenner
Answer: c

15. What is the role of eIF-4A in the translation process?
a) Cap binding
b) Helicase to unwind mRNA secondary structure
c) Binding to 60S subunit
d) Release factor
Answer: b

Q11. Which one of the following is the correct function of eIF-4A in eukaryotic translation?
a) Cap recognition
b) Poly-A tail binding
c) RNA helicase activity
d) Ribosome binding
Answer: c
[PYQ – CSIR NET June 2017]

Q12. In eukaryotic cells, the initiation complex formation requires which of the following components?
a) 70S ribosome, fMet-tRNA
b) 40S ribosome, Met-tRNAi, eIF2-GTP
c) 30S ribosome, IF2, fMet-tRNA
d) 50S ribosome, eEF1
Answer: b
[PYQ – GATE Life Sciences 2016]

Q13. The 5’ cap of eukaryotic mRNA is recognized by which eukaryotic initiation factor?
a) eIF4E
b) eIF3
c) eIF4A
d) eIF2
Answer: a
[PYQ – CSIR NET Dec 2019]

Q14. In eukaryotic translation, Met-tRNAi binds directly to which site of the ribosome during initiation?
a) A-site
b) E-site
c) P-site
d) T-site
Answer: c
[PYQ – DBT-JRF 2020]

Q15. Which one of the following statements about eukaryotic translation is incorrect?
a) It begins with methionine as the first amino acid.
b) Formylation of methionine occurs as in prokaryotes.
c) Initiation factors are more complex than in prokaryotes.
d) Scanning model is followed for locating start codon.
Answer: b
[PYQ – CSIR NET June 2015]

Q16. Which of the following elongation factors is responsible for the translocation step in eukaryotic translation?
a) eIF-2
b) eEF-2
c) eIF-4G
d) EF-G
Answer: b
[PYQ – GATE BT 2018]

Q17. What is the main difference between translation initiation in eukaryotes and prokaryotes?
a) Use of methionine vs. fMet
b) Polycistronic vs. monocistronic mRNA
c) Presence vs. absence of Shine-Dalgarno sequence
d) All of the above
Answer: d
[PYQ – CSIR NET Dec 2020]

Q18. The Kozak consensus sequence in eukaryotic mRNA is important for:
a) Termination of translation
b) Splicing of introns
c) Initiation codon recognition
d) Addition of 5’ cap
Answer: c
[PYQ – ICMR JRF 2019]

Q19. Which of the following statements is true regarding eukaryotic ribosomes?
a) They consist of 30S and 50S subunits
b) They consist of 40S and 60S subunits
c) They function in the nucleus
d) They are sensitive to chloramphenicol
Answer: b
[PYQ – BHU PET 2016]

Q20. Which of the following does not participate in the formation of the eukaryotic pre-initiation complex?
a) 40S subunit
b) eIF2-GTP
c) 60S subunit
d) Met-tRNAi
Answer: c
[PYQ – CSIR NET June 2018]

Q21. In eukaryotes, which elongation factor is responsible for bringing aminoacyl-tRNA to the A site of the ribosome?
a) eIF2
b) eEF1α
c) eEF2
d) eIF4E
Answer: b
[PYQ – CSIR NET Dec 2017]

Q22. Termination of translation in eukaryotes involves which release factor?
a) RF1
b) RF2
c) eRF1
d) eIF5
Answer: c
[PYQ – CSIR NET June 2019]

Q23. Which of the following statements about eukaryotic termination is correct?
a) Stop codons are recognized by tRNAs.
b) eRF1 recognizes all three stop codons.
c) eRF3 binds to the poly-A tail.
d) Peptidyl transferase catalyzes peptide release during termination.
Answer: b
[PYQ – ICMR JRF 2021]

Q24. The role of eEF2 in eukaryotic translation is:
a) Initiation codon recognition
b) Peptide bond formation
c) Translocation of ribosome
d) Release of polypeptide chain
Answer: c
[PYQ – DBT-JRF 2020]

Q25. In mitochondrial translation, which of the following is not true?
a) Uses 70S-type ribosomes
b) mRNA lacks Shine-Dalgarno sequence
c) Uses N-formylmethionine as the initiator amino acid
d) Is inhibited by cycloheximide
Answer: d
[PYQ – CSIR NET Dec 2016]

Q26. Which antibiotic inhibits eukaryotic cytoplasmic translation but not mitochondrial translation?
a) Chloramphenicol
b) Puromycin
c) Cycloheximide
d) Rifampicin
Answer: c
[PYQ – GATE Life Sciences 2020]

Q27. Circularization of eukaryotic mRNA involves interaction between:
a) eIF4E and eIF2
b) eIF4E and poly(A) binding protein
c) eIF3 and 60S subunit
d) eIF2 and ribosome
Answer: b
[PYQ – CSIR NET June 2021]

Q28. Which of the following represents the correct order of events in eukaryotic translation initiation?
a) Binding of 60S → tRNA loading → mRNA binding
b) Binding of 40S → Met-tRNAi → mRNA → 60S joining
c) mRNA binding → 60S binding → tRNA charging
d) 80S formation → scanning → start codon recognition
Answer: b
[PYQ – BHU PET 2020]

Q29. The initiation complex scans the mRNA for the start codon. Which feature helps in the correct identification of AUG?
a) Shine-Dalgarno sequence
b) Kozak sequence
c) GC-rich box
d) TATA box
Answer: b
[PYQ – CSIR NET Dec 2020]

Q30. The translation machinery of chloroplasts resembles:
a) Nuclear machinery
b) Mitochondrial machinery
c) Prokaryotic machinery
d) Eukaryotic cytoplasmic machinery
Answer: c
[PYQ – CUET PG 2022]

Q31. The hydrolysis of GTP during translocation in eukaryotes is catalyzed by:
a) eIF2
b) eIF4A
c) eEF2
d) eRF1
Answer: c
[PYQ – CSIR NET June 2015]

Q32. Which one of the following release factors is GTP-dependent?
a) RF1
b) eRF1
c) eRF3
d) RF3
Answer: c
[PYQ – GATE 2018]

Q33. In eukaryotic cells, translation and transcription are:
a) Coupled
b) Simultaneous in the nucleus
c) Separated both spatially and temporally
d) Coupled in the mitochondria
Answer: c
[PYQ – DBT BET 2021]

Q34. Which of the following statements about eukaryotic translation elongation is correct?
a) Peptidyl transferase activity is a function of 40S subunit.
b) Peptidyl-tRNA occupies the E site during elongation.
c) Aminoacyl-tRNA enters at the A site.
d) mRNA is degraded during elongation.
Answer: c
[PYQ – ICMR JRF 2019]

Q35. During eukaryotic translation, the first amino acid incorporated is:
a) N-formylmethionine
b) Alanine
c) Methionine
d) Valine
Answer: c
[PYQ – CSIR NET June 2018]

Q36. Which of the following is a unique feature of mitochondrial translation in humans?
a) Use of 80S ribosomes
b) Use of circular mRNA
c) Use of a non-universal genetic code
d) Presence of polycistronic mRNAs only
Answer: c
[PYQ – CSIR NET Dec 2019]

Q37. The termination of translation in eukaryotes is triggered when:
a) Ribosome reaches 3′ poly-A tail
b) Ribosome encounters a stop codon
c) tRNA is depleted
d) Exonuclease removes the ribosome
Answer: b
[PYQ – GATE Life Sciences 2022]

Q38. Which of the following factors binds to the 5′ cap of eukaryotic mRNA?
a) eIF4A
b) eIF4B
c) eIF4E
d) eIF3
Answer: c
[PYQ – CSIR NET June 2016]

Q39. Which of the following correctly matches a translation inhibitor with its target?
a) Chloramphenicol – 80S ribosome
b) Cycloheximide – mitochondrial ribosome
c) Puromycin – causes premature chain termination
d) Rifampicin – inhibits ribosomal translocation
Answer: c
[PYQ – DBT-JRF 2020]

Q40. What is the function of eIF2 in eukaryotic translation?
a) Cap recognition
b) Scanning of mRNA
c) Binding of initiator tRNA to 40S subunit
d) Joining of 60S subunit
Answer: c
[PYQ – CSIR NET Dec 2015]

Q41. Which of the following steps in translation does eEF1A facilitate?
a) Ribosome assembly
b) Translocation
c) Delivery of aminoacyl-tRNA to A site
d) Peptide bond formation
Answer: c
[PYQ – CSIR NET Dec 2018]

Q42. The energy for peptide bond formation in translation is directly derived from:
a) ATP
b) GTP
c) Hydrolysis of aminoacyl-tRNA
d) Ribosomal movement
Answer: c

Q43. Which RNA molecule has enzymatic (ribozyme) activity during translation?
a) tRNA
b) mRNA
c) 28S rRNA
d) 5S rRNA
Answer: c
[PYQ – GATE 2019]

Q44. The function of eEF2 is:
a) Delivery of tRNA
b) Ribosome translocation
c) Release of mRNA
d) Charging of tRNA
Answer: b
[PYQ – CSIR NET June 2020]

Q45. Which of the following is NOT a component of the pre-initiation complex in eukaryotes?
a) eIF2
b) eIF3
c) 60S ribosomal subunit
d) Met-tRNAi
Answer: c

Q46. Which one of the following best describes the Kozak sequence?
a) A termination signal
b) A translation enhancer
c) A start codon motif in eukaryotic mRNA
d) A splice junction signal
Answer: c
[PYQ – DBT-JRF 2019]

Q47. Which factor is required for ribosome recycling in eukaryotes?
a) eIF4G
b) ABCE1
c) eRF2
d) eIF5
Answer: b
[PYQ – CSIR NET Dec 2022]

Q48. Which of the following is true about the eukaryotic mRNA structure involved in translation?
a) Lacks poly(A) tail
b) Translation starts before transcription ends
c) 5’ cap is essential for initiation
d) Introns are always present
Answer: c

Q49. Which translation factor helps in proofreading the correct codon-anticodon interaction during elongation?
a) eIF1
b) eEF1B
c) eEF1A
d) eIF2
Answer: c

Q50. In eukaryotic translation, the formation of a 43S preinitiation complex involves:
a) 60S subunit, mRNA, and eIF4G
b) 40S subunit, eIF2-GTP-Met-tRNAi, and eIF3
c) Full 80S ribosome and eIF1
d) eIF4E, eIF4A, and poly-A tail
Answer: b
[PYQ – GATE Life Sciences 2020]