Structure and Role of tRNA

Structure and Role of tRNA

It is also called soluble or sRNA.
There are over 100 types of tRNAs.
Transfer RNA constitutes about 15% of the total RNA.
tRNA is the smallest RNA with 70-85 nucleotides and sedimentation coefficient of 4S.
The nitrogen bases of several of its nucleotides get modified e.g., pseudouridine (ψ), dihydrouridine (DHU), inosine (I).
This causes coiling of tRNA into L-shaped form (3D Klug, 1974) or clover-like form (2D, Holley, 1965).
About half of the nucleotides are base paired to produce paired stems.
Five regions are unpaired or single stranded— AA-binding site, T ψ С loop, DHU loop, extra arm and anticodon loop.

1. Primary Structure of tRNA

The primary structure refers to the linear sequence of ribonucleotides in the tRNA molecule, running from the 5′ (five prime) end to the 3′ (three prime) end.
The tRNA molecule typically ranges from 60 to 90 ribonucleotides in length.
The most commonly found length of tRNA is 76 nucleotides.
About 20% of these nucleotides are chemically modified, making tRNA distinct from other types of RNA (like mRNA or rRNA).
These modifications include methylations, deaminations, and isomerizations of standard bases (A, U, C, G), which result in unusual or modified bases such as:
- Inosine (I) – derived from Adenine (A)
- Pseudouridine (Ψ) – derived from Uracil (U)
- Dihydrouridine (D) – a reduced form of Uracil (U)
- Lysidine – derived from Cytosine (C)

These modifications are critical for the structural stability, proper folding, and functional interactions of tRNA during translation.

Even in the linear (primary) structure, certain domains (or arms) of tRNA are functionally distinguishable due to the presence of modified bases:

1. D arm (Dihydrouridine arm):

Contains Dihydrouridine, hence the name.
Serves as a recognition site for the enzyme aminoacyl-tRNA synthetase, which attaches a specific amino acid to the tRNA.

2. Anticodon arm:

Includes a stretch of nucleotides forming the anticodon, a triplet that is complementary to a codon on the mRNA.
Often contains unusual bases, such as Inosine, which allows wobble base pairing (flexibility in recognizing more than one codon).
Critical for codon recognition and translation specificity during protein synthesis.

3. TΨC arm (Thymine-Pseudouridine-Cytosine arm):

Characterized by the modified base Pseudouridine (Ψ).
Plays a major role in binding the tRNA to the ribosome, specifically to the ribosomal RNA (rRNA) of the large ribosomal subunit.

4. Acceptor stem (or Acceptor arm):

Located at the 3′ terminal of the tRNA.
Always ends in a CCA sequence, which is not encoded by DNA, but added post-transcriptionally by a special enzyme (tRNA nucleotidyltransferase).
The adenine (A) in the terminal CCA has a free 3′-OH group, which acts as the attachment site for an amino acid, forming aminoacyl-tRNA during protein synthesis.
Plays a central role in translation, as this is where the amino acid is linked by an ester bond.

🌿 Secondary Structure of tRNA (Cloverleaf Model)

The secondary structure of tRNA forms when its linear chain of nucleotides folds upon itself by intramolecular base pairing, resulting in a cloverleaf-like structure. This structure is essential for the function of tRNA in translation, enabling it to interact with ribosomes, aminoacyl-tRNA synthetases, and mRNA.

🔹 Length and Folding

The tRNA sequence is typically 74 to 95 nucleotides long.
It folds into a cloverleaf shape consisting of four main arms (sometimes five in longer tRNAs):
1. Acceptor Arm
2. D Arm (Dihydrouridine Arm)
3. Anticodon Arm
4. TΨC Arm (Thymine-Pseudouridine-Cytosine Arm)
5. Variable Arm (present in some tRNAs)

🌱 Structural Components

1. Acceptor Arm

Formed by base pairing between the 5′ and 3′ ends of the tRNA.
It is 7–9 base pairs long.
Does not form a loop.
Ends in a free 3′-OH group of adenine (A) in the terminal CCA sequence.
The carboxylic acid group of the amino acid forms an ester bond with this 3′-OH, a process called charging, catalyzed by aminoacyl-tRNA synthetase.
Located opposite the anticodon arm.

2. D Arm

Contains a stem of 4–5 base pairs and a loop with modified pyrimidines, especially Dihydrouridine (D).
Functions as the recognition site for aminoacyl-tRNA synthetase.

3. Anticodon Arm

Composed of a stem of 6 base pairs and a loop of 7 unpaired nucleotides.
Contains the anticodon triplet, which base-pairs with the mRNA codon during translation.
Modified bases like Inosine, Pseudouridine, or Lysidine may be found in or near the anticodon, enabling wobble base pairing.

4. TΨC Arm

Contains a stem of 5 base pairs and a loop of 7 unpaired nucleotides.
The loop includes the conserved sequence Thymine (T) – Pseudouridine (Ψ) – Cytosine (C).
Plays a crucial role in ribosome binding during translation.

5. Variable Arm (Optional)

Present in some tRNAs between the anticodon and TΨC arms.
Length ranges from 3 to 21 nucleotides.
Does not participate in extensive base pairing, appears as a flexible loop.
Contributes to the structural stability and can influence the identity and function of tRNA.

Tertiary Structure of tRNA (L-shaped 3D Conformation)

The tertiary structure of tRNA represents its most stable and functional three-dimensional (3D) configuration, which is essential for its biological activity during translation in protein synthesis.

🔹 L-shaped Structure

In the 3D conformation, the tRNA folds into a compact L-shaped molecule.
This L-shape arises when:
- The acceptor stem and TΨC stem align and stack on one side.
- The anticodon stem and D-stem stack on the other side.
These two helices (acceptor-TΨC and anticodon-D) are oriented perpendicularly, forming an extended elbow-like shape.

🔹 Loop Interactions and Stabilization

The D-loop and TΨC-loop interact closely through tertiary hydrogen bonds, especially involving modified bases, such as dihydrouridine and pseudouridine.
This D-loop/TΨC-loop interaction is a critical point of tertiary contact that stabilizes the elbow bend of the L-shape.

🔹 Stabilization by Hydrogen Bonding

The 3D structure is further stabilized by:
- Hydrogen bonds between nitrogenous bases, such as G–U, G–C, and A–U.
- Hydrogen bonds between bases and the ribose-phosphate backbone.
- Stacking interactions of base-paired regions and non-canonical base pairs.
Modified nucleotides in tRNA also contribute to structural flexibility and precise folding.

🔹 Functional Importance

This tertiary configuration positions the anticodon loop and amino acid acceptor site at opposite ends, allowing:
- Accurate codon-anticodon recognition with mRNA during translation.
- Proper amino acid attachment at the 3′ CCA end.
The L-shape is universally conserved across all domains of life, highlighting its evolutionary importance.

🔹 Tertiary Structure is Essential for:

Binding to ribosomes.
Interacting with aminoacyl-tRNA synthetase enzymes.
Fitting into the A, P, and E sites of the ribosome during translation.
High-fidelity decoding of genetic information.

🎯 Functional Role of tRNA

tRNA is more than a passive carrier — it’s central to the fidelity, regulation, and efficiency of protein synthesis.

🔸 1. Aminoacylation by tRNA Synthetases

Enzymes called aminoacyl-tRNA synthetases (one per amino acid, 20 total types) link the correct amino acid to its corresponding tRNA.
Two-step process:
1. Activation: Amino acid + ATP → aminoacyl-AMP + PPi
2. Transfer: Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
Some synthetases have proofreading (editing) domains to correct mischarged tRNAs, preventing translation errors.

🔸 2. Decoding mRNA in the Ribosome

The anticodon loop recognizes and pairs with the complementary mRNA codon in the ribosome A-site.
Codon-anticodon interaction is scrutinized by rRNA of the ribosome to ensure accurate base-pairing.

🔸 3. Participation in Translation Stages

🟩 Initiation:

tRNA^Met (or tRNA^fMet in prokaryotes) starts translation.
Binds to the P-site of the ribosome with the help of initiation factors.

🟨 Elongation:

Charged tRNA enters the A-site, guided by elongation factors (EF-Tu in prokaryotes, eEF1A in eukaryotes).
If codon-anticodon pairing is correct, peptidyl transferase activity in the ribosome catalyzes the peptide bond.
The ribosome then shifts (translocates), moving the tRNA to the P-site, and ejects the old one from the E-site.

🟥 Termination:

When a stop codon is encountered, no tRNA pairs.
Instead, a release factor binds, and the last tRNA releases the polypeptide.

🔸 4. Wobble Hypothesis

Describes flexibility at the third codon base.
Allows fewer tRNA species to recognize multiple codons:
- Example: tRNA with anticodon GAI can pair with UCU, UCC, and UCA (for serine).

🔸 5. tRNA Turnover and Quality Control

Misfolded or uncharged tRNAs can be degraded.
Rapid tRNA decay (RTD) pathways remove defective tRNAs.
tRNA modifications (m⁷G, Ψ, I, m¹A) are monitored — defects here are linked to diseases and stress responses.

🧬 Regulatory & Cellular Roles Beyond Translation

Though traditionally known for translation, tRNAs have other surprising roles:

➤ tRNA Fragments (tRFs):

Generated by cleavage during stress or apoptosis.
Function in gene regulation, RNA silencing, or cell signaling.

➤ tRNA-Derived Stress Response:

Cells under oxidative or heat stress cleave tRNAs to suppress translation and protect resources.

➤ Mitochondrial tRNAs:

Mitochondria have their own set of tRNAs encoded by mtDNA.

Mutations in mitochondrial tRNAs cause diseases like MELAS and MERRF.

Which end of tRNA carries amino acids?
A. 3′ end
B. 5′ end
C. Anticodon end
D. D-arm
[Answer: A] (PYQ: CSIR-NET 2019)

The anticodon is present on which arm of the tRNA?
A. D-arm
B. TΨC arm
C. Anticodon arm
D. Acceptor arm
[Answer: C] (PYQ: GATE-BT 2020)

The enzyme aminoacyl-tRNA synthetase binds to which arm of the tRNA?
A. Anticodon arm
B. TΨC arm
C. D-arm
D. Variable arm
[Answer: C] (PYQ: DBT-JRF 2021)

The loop containing modified base dihydrouridine is known as:
A. TΨC loop
B. D-loop
C. Variable loop
D. Anticodon loop
[Answer: B] (PYQ: ICAR-NET 2018)

Which modified base is present in TΨC loop of tRNA?
A. Pseudouridine
B. Dihydrouridine
C. Inosine
D. Thymidine
[Answer: A] (PYQ: CSIR-NET 2018)

Charging of tRNA refers to:
A. Transcription
B. Addition of amino acid
C. Codon recognition
D. Base modification
[Answer: B] (PYQ: CSIR-NET 2017)

What is the shape of tRNA tertiary structure?
A. Cloverleaf
B. Circular
C. L-shaped
D. Helical
[Answer: C] (PYQ: CSIR-NET 2020)

In eukaryotic cells, the 3′ end of tRNA terminates with which sequence?
A. GCU
B. AGC
C. CCA
D. AAA
[Answer: C] (PYQ: DU MSc Entrance 2019)

The function of anticodon in tRNA is to:
A. Bind ribosome
B. Attach amino acid
C. Recognize mRNA codon
D. Provide energy for translation
[Answer: C] (PYQ: JAM 2021)

Inosine, found in tRNA, is derived from which base?
A. Cytosine
B. Uracil
C. Guanine
D. Adenine
[Answer: D] (PYQ: CSIR-NET 2021)

The number of nucleotides in a typical tRNA is:
A. 20-30
B. 60-90
C. 100-150
D. 150-200
[Answer: B]

Which arm of tRNA is not a looped structure?
A. D-arm
B. TΨC arm
C. Acceptor arm
D. Anticodon arm
[Answer: C]

Which of the following is NOT a part of the cloverleaf model of tRNA?
A. D-arm
B. TΨC arm
C. Leader sequence
D. Anticodon arm
[Answer: C]

Aminoacyl tRNA synthetase attaches amino acid to which base of the CCA end?
A. Cytosine
B. Guanine
C. Adenine
D. Uracil
[Answer: C]

Which of the following is NOT found in the D-loop?
A. Dihydrouridine
B. Unusual purines
C. Ribosome binding site
D. Pyrimidine modifications
[Answer: C]

15. The anticodon GAA in tRNA pairs with which mRNA codon?
A. CUU
B. UUC
C. TTC
D. GAA
[Answer: B]

16. In the tertiary structure, D-loop and T-loop interact via:
A. Ionic bonding
B. Phosphodiester bonds
C. Hydrogen bonding
D. Covalent bonding
[Answer: C]

17. Modified bases in tRNA contribute to:
A. Degradation
B. Stability and folding
C. DNA replication
D. Transcription
[Answer: B]

18. The fifth arm found in longer tRNAs is called:
A. Acceptor arm
B. TΨC arm
C. Variable arm
D. Leader arm
[Answer: C]

19. Wobble base pairing is facilitated by:
A. Dihydrouridine
B. Thymidine
C. Inosine
D. Cytosine
[Answer: C]

20.Which of the following unusual bases is commonly found in the D-arm of tRNA?
A. Pseudouridine
B. Inosine
C. Dihydrouridine
D. Lysidine
Answer: C. Dihydrouridine

Q22. (PYQ – CSIR-NET 2021) Which loop in the tRNA contains the anticodon that base-pairs with the codon on mRNA?
A. D loop
B. Anticodon loop
C. TΨC loop
D. Variable loop
Answer: B. Anticodon loop

Q23. The enzyme that charges tRNA with an amino acid is:
A. Ribosomal synthetase
B. Aminoacyl tRNA synthetase
C. Peptidyl transferase
D. RNA polymerase
Answer: B. Aminoacyl tRNA synthetase

Q24. The cloverleaf model of tRNA represents its:
A. Primary structure
B. Secondary structure
C. Tertiary structure
D. Quaternary structure
Answer: B. Secondary structure

Q25. (PYQ – DBT JRF 2020) The 3′ end of all functional tRNAs has the sequence:
A. CCA
B. UAA
C. AUG
D. GCU
Answer: A. CCA

Q26. Which structural feature is essential for the attachment of amino acids to tRNA?
A. Anticodon loop
B. D loop
C. TΨC loop
D. Acceptor arm
Answer: D. Acceptor arm

Q27. Which of the following bases is derived from uracil and found in tRNA?
A. Inosine
B. Pseudouridine
C. Thymine
D. Cytosine
Answer: B. Pseudouridine

Q28. (PYQ – GATE 2019) The L-shaped tertiary structure of tRNA results from interactions between:
A. Acceptor and D arms
B. D and TΨC loops
C. Codon and anticodon
D. tRNA and rRNA
Answer: B. D and TΨC loops

Q29. Variable arm in tRNA is located between:
A. Acceptor arm and D-arm
B. D-arm and Anticodon arm
C. Anticodon arm and TΨC arm
D. TΨC arm and Acceptor arm
Answer: C. Anticodon arm and TΨC arm

Q30. Which structural arm of tRNA plays a role in ribosome binding?
A. Acceptor arm
B. Anticodon arm
C. TΨC arm
D. D arm
Answer: C. TΨC arm

Q31. Which one of the following forms the most stable structure of tRNA?
A. Primary structure
B. Secondary structure
C. Tertiary structure
D. Quaternary structure
Answer: C. Tertiary structure

Q32. (PYQ – CSIR NET 2020) What is the significance of the anticodon in tRNA?
A. Recognition by ribosome
B. Attachment of amino acid
C. Base pairing with mRNA codon
D. Transport of ribosomes
Answer: C. Base pairing with mRNA codon

Q33. Inosine is derived from which purine base?
A. Guanine
B. Cytosine
C. Adenine
D. Uracil
Answer: C. Adenine

Q34. Which enzyme adds CCA sequence to the 3′ end of tRNA during processing?
A. RNA polymerase III
B. CCA transferase
C. Nuclease
D. Ligase
Answer: B. CCA transferase

Q35. Which of the following is not a modified base found in tRNA?
A. Pseudouridine
B. Inosine
C. Methylguanine
D. Uridine
Answer: D. Uridine

Q36. (PYQ – ICMR JRF 2021) The acceptor stem of tRNA is formed by base pairing between:
A. D arm and TΨC arm
B. 5′ and 3′ ends
C. Anticodon and D arm
D. Variable arm and TΨC arm
Answer: B. 5′ and 3′ ends

Q37. What is the length (in nucleotides) of the anticodon loop in a typical tRNA?
A. 3
B. 5
C. 7
D. 9
Answer: C. 7

Q38. Which of the following is a function of aminoacyl tRNA synthetase?
A. Peptide bond formation
B. tRNA splicing
C. tRNA charging
D. tRNA transport
Answer: C. tRNA charging

Q39. (PYQ – GATE 2022) How does the tRNA ensure the correct amino acid is delivered during translation?
A. Through anticodon loop
B. By ribosome scanning
C. By matching with rRNA
D. Through enzyme specificity
Answer: D. Through enzyme specificity

Q40. The rare base ‘lysidine’ is derived from which nitrogenous base?
A. Adenine
B. Cytosine
C. Thymine
D. Guanine
Answer: B. Cytosine

Q41. The loop in tRNA that contains the recognition site for aminoacyl tRNA synthetase is:
A. Anticodon loop
B. D loop
C. TΨC loop
D. Variable loop
Answer: B. D loop

Q42. In the tRNA cloverleaf model, how many total loops are usually present?
A. Two
B. Three
C. Four
D. Five
Answer: D. Five (includes variable loop)

Q43. Which structural feature is essential for ribosome binding in tRNA?
A. D loop
B. TΨC loop
C. Variable arm
D. Acceptor stem
Answer: B. TΨC loop

Q44. What is the function of the variable arm in tRNA?
A. Codon recognition
B. Ribosome binding
C. Provides structural stability
D. Binding with mRNA
Answer: C. Provides structural stability

Q45. Which of the following does not play a role in tRNA tertiary structure stability?
A. Hydrogen bonding
B. Base stacking
C. Peptide bonds
D. Base-sugar interactions
Answer: C. Peptide bonds

Q46. (PYQ – CSIR NET 2022) What ensures high fidelity during translation of genetic code?
A. Ribosome accuracy
B. Codon-anticodon base pairing
C. tRNA structure
D. Aminoacyl tRNA synthetase proofreading
Answer: D. Aminoacyl tRNA synthetase proofreading

Q47. The stem of the TΨC arm typically contains:
A. 3 base pairs
B. 5 base pairs
C. 7 base pairs
D. 9 base pairs
Answer: B. 5 base pairs

Q48. The anticodon region of tRNA binds to:
A. Ribosomal RNA
B. DNA
C. mRNA codon
D. Enzyme site
Answer: C. mRNA codon

Q49. Which base pairing is allowed at the wobble position of the tRNA anticodon?
A. Strict Watson-Crick only
B. G-U and I-U only
C. Flexible pairing including inosine
D. A-U only
Answer: C. Flexible pairing including inosine

Q50. The L-shaped 3D structure of tRNA is primarily stabilized by:
A. DNA interactions
B. rRNA binding
C. Hydrogen bonds and base stacking
D. Peptide bonding
Answer: C. Hydrogen bonds and base stacking