Genetic Code and tRNA Overview

The Wobble

• The wobble occurs at the 3’ end of the codon and 5’ end of the anti-codon.
• The wobble is a result of the arrangement of hydrogen bonds of base pairs at the third position of the codon.
• The degeneracy of the genetic code allows for the wobble to always result in the translation of the same amino acid.
• A complete set of codons can be read by fewer than 61 tRNAs.

tRNA

• tRNA molecules carry specific amino acids to the ribosome.
• tRNA molecules are encoded by different genes.
• There are 20 aminoacyl-tRNA synthases which "charge" the tRNA molecules.
• All tRNA molecules possess specific aa-tRNA synthetase recognition sites.
• tRNA molecules have a cloverleaf structure with an acceptor stem and an anticodon loop that binds to the mRNA codon.

Ribosomes

• The overall ribosome shape is determined by rRNA.
• Ribosomes are discrete in size.
• Ribosomes have two sites:
  • P site (peptidyl site)
  • A site (aminoacyl site)
• Ribosomes are essential for polypeptide synthesis.

Step 1- Charging of tRNA (aminoacylation)

• Amino acids are attached to tRNAs by aminoacyl-tRNA synthetase.
• There are 20 aminoacyl-tRNA synthases capable of charging all tRNAs.
• tRNA molecules possess specific aa-tRNA synthetase recognition sites.
• The process uses energy derived from ATP hydrolysis.
• This results in a charged tRNA called aminoacyl-tRNA.

Initiation, Elongation and Termination

• Initiation:
  • This stage involves assembly of a functional ribosome in the correct location on the mRNA molecule.
  • The assembled ribosome is ready to begin protein synthesis.
• Elongation:
  • tRNA molecules bring the correct amino acid to the ribosome.
  • The amino acid is joined to the growing polypeptide chain.
  • The ribosome moves to the next codon on the mRNA.
• Termination:
  • This stage is triggered by the ribosome reaching a stop codon.
  • The entire complex disassembles, releasing the newly synthesized polypeptide.

Prokaryotic vs Eukaryotic Translation

• Prokaryotes:
  • Translation occurs in the cytoplasm.
  • Transcription and translation occur simultaneously.
  • Translation begins at Shine-Dalgarno sequences.
  • The initiating tRNA is formyl-methionine.
• Eukaryotes:
  • Translation occurs in the cytoplasm.
  • Transcription and translation occur separately.
  • Translation begins at the AUG codon, which is embedded in a short initiation sequence called the Kozak sequence.
  • The initiating tRNA is methionine.

Initiation (Prokaryotes)

• Binding of 30S subunit and initiation factors:
  • Initiation factor 1 (IF1) associates with the 30S subunit in the A site, blocking tRNA entry.
  • Initiation factor 3 (IF3) allows the 30S subunit to bind to the specific site on the mRNA.
  • Initiation factor 2 (IF2) binds to the 30S P site, facilitating the binding of formylmethionine tRNA (fMet-tRNA) to IF2.
  • IF2 moves fMet-tRNA to the P site.
  • Hydrolysis of GTP occurs following the arrival of the 50S subunit, leading to a conformational change and forming a 70S ribosome.

Step 2- Initiation Steps (Prokaryotes)

• The Shine-Dalgarno sequence is complementary to the 3’ end of the 16S rRNA.
• The initiator tRNA (fMet tRNA) binds to the AUG start codon on the mRNA.
• IF3 is removed and recycled.
• IF1 and IF2 are released, and GTP is hydrolyzed, leading to the binding of the 50S rRNA subunit.
• This results in a 70S initiation complex comprising of mRNA, 70S ribosome, and fMet-tRNA.

Elongation

• The A site is empty.
• The P site contains the peptidyl tRNA.
• The E site contains an uncharged tRNA.
• Elongation requires elongation factors (EF-Tu, EF-Ts, and EF-G).
• EF-Tu-GTP: Recognizes and transports aminoacyl-tRNA to the A site of the ribosome.
• EF-Tu is released from the ribosome after GTP hydrolysis to GDP.