Cell-Free Protein Synthesis Experiment

Deciphering the Genetic Code

• The genetic code was deciphered using synthetic mRNA and evaluating which amino acids were incorporated
• Amino acids are encoded by triplets of nucleotide bases (codons)
• The code is degenerate – most amino acids are coded for by more than one codon
• One start codon (AUG) and three stop codons
• Where you begin to read the code will determine which triplets are produced
• Each possible grouping of triplets is called a ‘reading frame’

Translation: Requirements and Components

• RNA required for translation:
  • mRNA (encodes proteins)
  • tRNA (delivers amino acids to the ribosome)
  • rRNA (structural and enzymatic component of the ribosome)
• Ribosomes are required for translation

Translation: tRNA Molecule

• tRNA is the adaptor molecule that links an mRNA codon with a specific amino acid
• tRNA molecule:
  • 75-90 nucleotides
  • Extensive internal base pairing
  • Clover leaf structure
  • Contains unusual bases
  • CCA-OH sequence at 3’ end
  • Anticodon on central loop
• Anticodon:
  • A triplet of bases that are complementary to the codon
  • These bases are unpaired and available for hydrogen bonding

Translation: Wobble Mechanism

• Wobble pairing allows a single tRNA anticodon to recognize more than one codon
• Often the first two letters in the codon are the same, and the third base can vary
• Third position less critical and can follow non-Watson-Crick base pairing between mRNA and tRNA

Translation: Initiation, Elongation, and Termination

• Initiation:
  • Requires initiation factors (IFs)
  • fMet-tRNAfMet in complex with IF2-GTP
  • Hydrolysis of GTP accompanied by the binding of the large 50S subunit
  • 16S rRNA binds the Shine-Dalgarno sequence in the mRNA to line up fMet-tRNAfMet with AUG start codon
• Elongation:
  • Next aminoacyl tRNA binds to elongation factor EF-Tu GTP and enters A site in the ribosome
  • If the anticodon of the incoming tRNA is complementary to the codon, then hydrolysis of GTP takes place, and EF-Tu GDP + Pi are released
  • Peptidyl transferase process: the wall is built by lifting the incomplete wall, and placing the new brick underneath
• Termination:
  • Requires a release factor that mimics the shape of a tRNA

Differences between Prokaryotic and Eukaryotic Translation

• Key differences between prokaryotic and eukaryotic translation

Learning Outcomes

• Recap of learning outcomes:
  • Outline how the genetic code was deciphered
  • Explain how amino acids are encoded by groups of three nucleotide bases
  • Describe the components required for translation
  • Outline the process of activation of an amino acid to form an amino acyl tRNA
  • Describe the 3 stages of translation in prokaryotic cells
  • Identify ways in which eukaryotic translation differs from prokaryotic translation

This quiz assesses understanding of a cell-free system experiment involving ribosomes, amino acids, and filter paper to measure protein synthesis. It tests knowledge of how to identify radio-labelled amino acids in a tube.