Protein translation and post-translational modification

Question 1

Mechanism of translation: summarise the mechanisms which ribosomes use to translate a mRNA sequence into a protein sequence, ensuring the fidelity of the genetic code

Answer 1

Protein Synthesis: Translation

• Mature mRNA (spliced, capped and polyadenylated) is now free in the cytoplasm, ready to be translated by ribosomes
• 7MeG = Methylated RNA Cap, and is the ENTRY POINT for ribosome
• AAAAAn = Poly A Tail
• UTR = Untranslated region (involved in regulation of translation instead)

3 materials needed for translation:

1. Mature mRNA
2. Charged tRNA
3. Ribosome

Codons

• The genetic code is a TRIPLET code, and each set of 3 bases (called a CODON) codes for 1 amino acid
• There is only 1 “start” codon: Methionine (AUG)
• There are 3 “stop” codons: UAA, UAG, UGA
• There is some redundancy built into the code; 64 combinations of bases for 20 amino acids
• This gives some protection against mutations for some codons

tRNA:

• Structure: Clover-shaped
• Carries amino acid on its 3’ end
• Has ANTICODON to match with codon on mRNA
• Antiparallel binding, as in DNA
• 64 tRNAs, one for each codon
• So, an amino acid can have several tRNA
• When tRNA holds amino acid, it is called CHARGED

Charging tRNA:

1. Free amino acid binds to AMINOACYL tRNA SYNTHASE
2. Aminoacyl tRNA synthase cleaves pyrophosphate from ATP and binds remaining AMP to amino acid
3. Amino acid becomes ADENYLATED
4. Adenylated amino acid is then attached to tRNA
5. Aminoacyl tRNA SYNTHASE and AMP then detach, leaving the charged tRNA

Translation (Initiation)

• In eukaryotes, ribosomal subunits dissociate (40S and 60S)
• Initiation Factors 4G and 4E bind to the methylated RNA cap
• Charged Met-tRNA, eIF-2 (carrying GTP) and 40S recognise the eIF4G-4E-7MeG structure as the 5’ end
• Begins reading until start codon AUG, which starts the FRAME of translation
• Pre-initiation complex forms
• GTP hydrolysed to GDP + Pi to bind 60S subunit to complex (reaction only possible with Met-tRNA, so translation only ever starts here)
• Met-tRNA binds to P-site (peptidyl) on ribosome

Elongation and Termination

ELONGATION:

1. The next tRNA binds to A-site (amino acyl) on ribosome
2. PEPTIDYL TRANSFERASE creates peptide bond between the two amino acids on the 60S Subunit
3. ELONGATION FACTORS then move the ribosome along mRNA using GTP, called TRANSLOCATION
4. In the time taken for GTP to hydrolyse, there is opportunity for incorrect base pairs to dissociate
5. So, the pauses given by GTP hydrolysis increases accuracy of translation

TERMINATION:

1. Stop codon attracts RELEASE FACTORS (NOT tRNA, there are no tRNA for stop codons)
2. Bind to empty A site on ribosome
3. Peptidyl Transferase bonds water to final amino acid, creating carboxyl group
4. Translation complex dissociates into cytoplasm

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Question 2

Prokaryotes versus eukaryotes: explain why some antibiotics inhibit protein synthesis in prokaryotes but not eukaryotes

Answer 2

In eukaryotes, ribosomal subunits dissociate (40S and 60S)

• Prokaryotes have 30S and 50S subunits
• Antibiotics target prokaryote subunits specifically, hence why antibiotics have no effect on humans

Question 3

Secretory pathway: explain the features of a newly-synthesised protein that are required for it to enter the secretory pathway

Question 4

Post-translation modification: summarise the ways in which newly-synthesised proteins can be post-translationally modified

Answer 4

Modification

1. First 20-24 amino acids are a SIGNAL RECOGNITION PARTICLE (SRP), containing the SIGNAL SEQUENCE
   
   • Rich in hydrophobic amino acids
2. SRP detected by SRP RECEPTOR
   
   • SRP Receptor located on Rough Endoplasmic Reticulum (RER) surface
   • Translation temporarily stopped
3. SRP peptide binds to SRP Receptor
   
   • Translation resumes
   • Polypeptide is fed into lumen of RER as it is synthesised
4. After translation, polypeptide is within RER and SRP is degraded
5. Polypeptide is then cleaved and folded as necessary
   
   • If polypeptide is destined to be transmembrane protein or requires further modification, hydrophobic groups added onto the end to anchor it into the RER membrane

• Example of Modification: Insulin
  
  • Undergoes Disulphide bond formation in ER and Golgi Body
  • Undergoes proteolytic cleavage in secretory vesicle
• Note: Chain C is released into cytoplasm and then into blood as a waste product
  
  • It is difficult to detect insulin levels in blood
  • So instead, we can measure Chain C levels as an indirect measure insulin