translation

• Genetic code


  The correspondence between the linear sequence of 4 types of nucleic acid bases and the 20 types of amino acids in protein

  
• Decoding the genetic code

  • Read in triplicate (CODON)-discovered by Crick and Brenner
  • Non-overlapping
  • Degenerate
  • Nearly universal
  • 20 amino acids encoded by 4 nucleotides

  
• 64 codons – only 20 amino acids. Therefore there is redundancy and codons that do not code for amino acids (stop codons)

  
• Amino acids


  • Humans only produce 10 AAs, the rest is supplied by our diet
  • Proteins are essential for all cellular processes
  • AAs charge, size, shape, reactivity, hydrophobicity, determines protein folding and stability
• Mutations


  • Permanent change in the DNA sequence
  • Mutations range from single base polymorphisms (SNPs) to large segments of chromosomes being lost or inserted
  • Can be inherited (germline) or acquired through a lifetime (somatic)
  • Can be found in coding and non-coding regions (promoters, enhancers, UTR)

  
• Classes of mutations


  • Point mutation
  • Chromosomal mutation

  
• Point mutations


  • Most common type of mutation
  • If point mutation occurs in the first base of the codon-missense mutation
  • If point mutation occurs in the second base of the codon-missense mutation
  • If point mutation occurs in the third base of the codon-silent mutation-degeneracy

  
• Transition and transversion mutations

  • Transition: A purine base is substituted for a purine base, A pyrimidine base is substituted for a pyrimidine base
  • Transversion: A purine base is substituted for a pyrimidine base, A pyrimidine base is substituted for a purine base
• Insertion/deletion frameshift mutations


  A point mutation insertion/deletion will induce a frameshift. However, insertion or deletion of 3 nucleotides will not induce a frameshift. Instead either amino acids are introduced or lost

  
• How is gene expression regulated?

  • The structure of chromatin and promoter accessibility
  • Transcriptional control-promoter regions
  • mRNA processing
  • mRNA stability
  • Translation control
  • post-translational modification
• Translation


  The process of forming a protein molecule at a ribosomal site of protein synthesis from information contained in messenger RNA
• Challenges in translation


  • Decode the nucleotide sequence into corresponding amino acids
  • Join amino acids in a specific manner
  • Complete the process with accuracy and speed
  • When does the molecular machinery know when to start/stop?
• Amino acid side chains

  Have little or no specific affinity for purine and pyrimidine bases of RNA – need an adaptor molecule: tRNA
• Genes can be expressed with different efficiencies
• Translational machinery


  • Protein 'factors' (eg IF, EF)
  • mRNA: codons
  • tRNA: anti-codon
  • Amino-acyl-tRNA synthetases: couple specific tRNA to specific amino acids
  • Ribosome (rRNA and protein): co-ordinates correct mRNA - tRNA recognition and catalyzes peptide bond formation
• mRNA in prokaryotes


  • A single mRNA molecule can encode many proteins (polycistronic mRNA)
  • RBS (ribosome binding sequence- contains the Shine-Dalgarno sequence) 3-9 bp upstream of start codon, complementary to sequence in 16S rRNA in ribosome, aligns ribosome with beginning of ORF (open reading frame). Spacing between RBS and start codon influences translational activity of particular ORF
• Translation initiation recap


  • Translational machinery
  • mRNA (mature in eukaryotes with 5'cap and polyA tail)
  • tRNA
  • Ribosomes
  • Protein factors
  • Amino-acyl-tRNA synthetases
• mRNA in eukaryotes


  • mRNA encodes a single protein (monocistronic)
  • 5' cap – methylated guanine jointed to 5' end by unusual 5'-5' linkage; used to recruit ribosome; ribosome then scans along mRNA to find start codon
  • Kozak sequence – 5'ACCATGG 3', increases efficiency of translation; thought to interact with initiator tRNA (not ribosome)
  • 3' poly-A tail – promotes efficient cycling of ribosomes
• The Crick wobble hypothesis


  • tRNA has an anti-codon that binds through complementary base pairing with the codon found on mRNA
  • Strands are anti-parallel 3' third position in the codon binds to the 5' first position in the anti-codon
  • In theory, need a different anti-codon to bind to each codon- therefore one tRNA is needed per codon
  • Approx 40 tRNAs in cells-less tRNA due to the wobble concept
• Prokaryotic initiation


  1. Shine-Dalgarno sequence on mRNA binds to 16s rRNA

  2. Charged tRNA placed in P site

  3. AA = fMet-tRNAi

  4. 30S preinitiation complex

  5. Large subunit binds – 70s initiation complex

  6. Protein factors involved are IF1,2,3
• Eukaryotic initiation


  1. Uses a cap and scan model-scans across the 5' UTR until start codon

  2. mRNA is modified (via kokaz sequence)

  3. 43S preinitiation complex + modified mRNA = 48S initiation complex

  4. 48S initiation complex + large subunit = 80S initiation complex

  5. eIF protein factors + large SU = 80S initiation complex

  6. 48S complex
• Translation elongation


  1. Elongation can only take place once the initiation complex is made

  2. The correctly positioned tRNAi at the P site can now allow the stepwise addition of amino acids

  3. Elongation factors are needed to help this process
• Elongation in eukaryotes


  1. Entry of next aa-tRNA at A site

  2. GTP hydrolysis, ribosome conformation change

  3. Peptide bond formation

  4. Ribosome translocation
• Transfer RNA (tRNA)


  • Small RNA adaptor molecule participates in protein synthesis
  • Two important regions: anti-codon and amino acid attachment site
  • Many types tRNA molecule (40-60 depending on the species) - each type attached to different amino acid (via aminoacyl-tRNA synthetases) each type recognizes specific codon (or codons)-not always the case due to the wobble theory
• Elongation in prokaryotes


  1. EF-Tu-GTP recruits tRNA with 2nd AA attached

  2. EF-Tu masks the AA, preventing a peptide bond forming until correct codon-anti-codon base pairing results in GTP hydrolysis

  3. GTP hydrolysis releases EF-Tu-GDP and unmasks the AA

  4. Peptidyltransferase reaction catalysed by 23S rRNA

  5. EF-G-GTP complex binds to ribosome in A site

  6. Factor binding centre stimulates GTP hydrolysis

  7. Causes conformation change that triggers translocation, and release EF-G-GDP
• Charging tRNA


  1. AA is charged by the addition of AMP from ATP via enzyme

  2. Uncharged tRNA binds to the enzyme

  3. Enzyme transfers the AA to tRNA

  4. Release of tRNA with AA

  5. AA is bound to tRNA via a covalent bond
• Elongation


  Loading + peptidyl transferase reaction + translocation
• Large SU


  80S initiation complex
• Ribosomes


  • Complexes of rRNA and proteins
  • Recognition of mRNA by tRNA and catalyzes peptide bond formation
  • 2 ribosome subunits: large subunit contains peptidyl transferase centre, small subunit contain decoding center
  • 3 tRNA molecules bind simultaneously (A, P, E sites)
  • Ribosome 'tunnels' allow mRNA to thread through, and exit in the form of a polypeptide chain
• Despite differences in size and number, ribosomes in prokaryotes vs eukaryotes have a similar structure/function
• Factors in elongation


  Eukaryotic: EF1α.GTP

  Prokaryotic: EF-Tu.GTP

  Eukaryotic: Catalysed by larger 28s rRNA

  Prokaryotic: Catalysed by 23S ribosome

  Both rRNA mediated not protein

  Eukaryotic: EF2.GTP

  Prokaryotic: EF-G-GTP
• Small ribosomal subunit


  Framework for tRNAs to match the codons in mRNA
• Eukaryotic initiation (1st stage)

  1. 48S complex

  2. mRNA preparation

  3. eIF used to help position tRNAi into P site

  4. eIFs associate with 5'cap of mRNA

  5. Ribosome is recruited to 5' cap of mRNA

  6. Ribosome/tRNAi scans for AUG start codon

  7. Correct positioning of tRNAi / start codon in P site
• Translation termination


  1. Recognition of a stop codon

  2. Release of a polypeptide chain

  3. Recycling of ribosomes
• Large ribosomal subunit


  Catalyse the formation of the peptide bonds that link the AAs together
• Multiple ribosomes translate mRNA simultaneously, but a ribosome can only bind 1 mRNA at a time
• Overview of translation


  The three stages of translation: initiation, elongation, termination
• Recognition of a stop codon

  No tRNA can recognise a stop codon - no role of tRNA in termination

  Protein release factors (RF) mimic the tRNA structure and anti-codon

  Release factors catalyse the release of the polypeptide chain from the peptidyl tRNA

  Two classes of RFs: Class I and class II

  Class I: Recognise stop codons and catalyse hydrolysis leading to polypeptide chain release

  Class II: Triggers release of class I RFs
• Scanning model of eukaryotic initiation

  • In prokaryotes, annealing between the 16S sequence in the small subunit and the Shine-Dalgarno sequence –places the AUG codon in the P site
  • This mechanism is lacking in eukaryotes-use a scanning method by the pre-initiation complex
  • Scans across the 5'UTR until start codon is found by small subunit –aided by the Kozak sequence
  • Helped by eIFs
  • Cap binding eIF4F complex binds to activate mRNA ready for the pre-initiation complex to bind
• Translation initiation


  The formation of an initiation complex in which the mRNA start codon is correctly positioned on the ribosome, and the initiator tRNA is annealed to the start codon and bound to the ribosome