Genetics 5 - Protein metabolism

• Codons - Triplet of bases on mRNA that encode for a specific amino acid or stop codon
• Genetic code:

  • 64 different combinations (4 x 4 x 4 = 64) of bases
  • Code for 20 amino acids and 3 stop codons
  • Redundancy” in the genetic code
  • Highly conserved
• What are the 3 stop codons?

  Amber (UAG), Opal (UGA), Ochre (UAA)
• Wobble base pairing:

  • occurs at 5 prime Wobble position on tRNA
  • type of non-Watson-Crick base pairing in which 3rd base within a codon can be variable
  • can lead to redundancy in genetic code, where more than one codon can code for a single amino acid
• Variable wobble base pairing

  A) A, G or I

  B) G or I

  C) U

  D) C

  
• What is a silent mutation?

  When there is a change in a single base but no change in an amino acid
• What is a missense mutation?

  When there is a change in a single base that results in a change in an amino acid
• What is a nonsense mutation?

  When there is a change in a single base that results in the formation of a stop codon
• What is a Frameshift mutation?

  When a base is inserted or deleted within a sequence that results in a change in all consequent codons.
• How do Aminoacyl-tRNA synthetases work?

  They activate amino acids, allowing them to bind to tRNA on 3' OH using ATP. Proofreading then occurs, where tRNA and amino acid are read at synthesis site. If amino acid is wrong it is removed at editing site and aminoacyl-tRNA bond is hydrolysed.
• The process of translation in proteins is catalysed by ribosomes.
• What is the E site in protein translation?

  Exit site
• What is the P-site in protein translation?

  Peptidyl site
• What is the A site in protein translation?

  Aminoacyl site
• Initiation of eukaryotic Translation in proteins

  1. Eukaryotic initiation factor type 4 (elF4E) binds to 5' cap on mRNA at start codon AUG (Methionine)
  2. Small ribosomal subunit (40s) and initiation factors ( eIF2 and eIF4G) bind to mRNA
  3. eIF2 binds initiator tRNA with start codon in mRNA
  4. GTP bound to eIF2 is hydrolysed into eIF2-GDP + Pi allowing energy to be released which allows large ribosomal subunit (60s) to bind to initiation complex
• Elongation in eukaryotic translations of proteins STEPS 1&2 :

  1. Initiator tRNA with amino acid is in P-site of 60s bound to start codon of mRNA
  2. The A-site with specific codon is open, so tRNA with complementary anticodon binds, using energy from hydrolysis of eIF1-GTP -> eIF1-GDP + Pi
• Elongation in eukaryotic translation of proteins STEPS 3-5

  1. N-terminus of amino acid on tRNA in A site exhibits a nucleophilic attack on the C-terminus of amino acid on tRNA in P site, joining amino acid from previous tRNA molecule to amino acid on new tRNA, using enzyme peptidyl transferase
  2. tRNA without amino acid in P-site will get translocated to the E- site and eventually out of ribosome and the tRNA, using energy from hydrolysis of eIF2-GTP
  3. process continues until stop codon is reached
• Termination in eukaryotic translation of proteins

  1. Once a stop codon has been reached on mRNA such as UGA, UAA or UAG in the A-site of the ribosomal subunit translation ends
  2. Eukaryotic release factor type 1 (eRF1) with GTP bound attaches to stop codon
  3. GTP on eRF1 is hydrolysed to eRF1-GDP and Pi which triggers hydrolysis of polypeptidyl-tRNA
  4. Completed Protein is released along with 60s ribosomal subunit, release factors and tRNA molecule
• What is eIF2?


  eukaryotic initiation factor type 2, binds to GTP, part of translation initiation ,
• eIF2B - guanine nucleotide exchange factor
• eIF2 Phosphorylation

  • protein synthesis decreases
  • eIF2 (P) binds to eIF2B tightly causing it to become inactive preventing initiation of protein translation
• What causes eIF2 Phosphorylation?

  Cell cycle regulation, cell stress ( viruses, temperature, nutrients, lack of growth factors)
• What is the Shine-Dalgarno Sequence (SDS)?

  A clump of purine nucleotides near the 5’ end of mRNA about 8 nucleotides upstream from start codon
• Initiation in prokaryotic translation of proteins

  1. Start codon on mRNA is AUG , codes for N-formyl methionine
  2. Small ribosomal subunits (30s) will bind to SDS with the help of IF3
  3. 30s bound to SDS slide down till they reach start codon at P site
  4. IF1 is recruited to aid the binding of tRNA with attached N-formyl methionine and complementary anti-codon to start codon at p-site
  5. hydrolysis of IF2-GTP released energy allowing the binding of large ribosomal sub-unit (50s) and IF1, IF2 and IF3 dissociate
• Elongation in prokaryotic translation of proteins

  • EF-Tu - allows proof reading
  • EF-G - drives ribosome movement, GTP hydrolysis release
  • incorrect tRNAs dissociate
• N-formylmethionine (fMet)

  • Additional formyl group
  • First N-terminal residue of many bacterial proteins
  • often cleaved
  • 50% cleaved when sequence is 10aa long
  • can be detected by our immune system
• Translation inhibitors that act on bacteria

  • Tetracycline - blocks aminoacyl-tRNA binding to ribosome at A-site
  • Streptomycin - prevents transition from translation initiation to elongation and causes miscoding
  • Chloramphenicol - locks peptidyl transferase on ribosomes
  • Erythromycin - binds ribosome exit channel preventing elongation
• What are the 4 inhibitors of protein translation that only act on bacteria?

  Tetracycline, streptomycin, chloramphenicol and erythromycin
• What is the translation inhibitor that acts on bacteria and eukaryotes?

  Puromycin - causes release of nascent polypeptide chains by its addition to the growing chain end
• Translation inhibitors that only act on eukaryotes:

  Cycloheximide - blocks ribosome translocation

  Anisomycin - blocks ribosomes peptidyl transferase
• Nonsense-mediated decay (NMD)

  • reduces errors in gene expression
  • degrades mRNA with premature stop codons
  • When there are premature stop codons, exon junction complexes (EJCs) persist and recruit Upf proteins (UPF3A and UPF3B) which trigger mRNA degradation
• Energy required for translation

  • tRNA synthetase - 2 x ATP / amino acid & ATP -> AMP
  • initiation - 1 x ATP/ protein (GTP hydrolysis)
  • Elongation - 2 x ATP/aa & 2 x GTP hydrolysis
  • termination - 1 x ATP/ protein (GTP hydrolysis)
• What are the two protein folding chaperones?

  Hsp70 and Hsp60 chaperones - hydrolyse ATP
• Western blot:

  • Detect specific proteins
  • transfer protein from polyacrylamide gel to Nitrocellulose
  • Probe nitrocellulose with specific antibodies
  • Detect antibodies