translation

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translation
RBS
RBS ribosome binding site
Ribosome(r RNA r protein)
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Introductory remarks

• Ribosomes are the sites of protein synthesis.
  They occur in the cytoplasm, mitochondria and
  chloroplasts.
• Ribosomes translate the information contained in
  mRNA into amino acid sequences with a high degree
  of accuracy. The actual peptide bond forming step
  is actually carried out by rRNA rather than an
  enzyme.

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AUGCUUGAAUAA
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The wobble mechanism (1)

• Although the Watson-Crick base pairing rules are
  always obeyed in DNA they are not absolute in
  codon-anti-codon pairing.
• Specifically the recognition between first base
  of the anticodon and the third base of the codon
  has slightly more flexibility.
• This is known as wobble pairing.

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The wobble mechanism (2)

• As an example GCC and GCU both code for alanine.
  Wobble pairing permits the anticodon sequence
  3CGG 5 to recognise both these sequences.
• Another variation is that the base I
  (hypoxanthine) in the first anticodon position
  can recognise C, U and A in the third anticodon
  position.

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How are amino acids attached to tRNA molecules?
(1)

• It is essential that the same amino acid is
  attached to a tRNA as is coded for by the
  anticodon in that tRNA.
• The enzymes, which attach amino acids to tRNAs,
  are aminoacyl tRNA synthetases.
• There must be at least one such enzyme for each
  amino acid.
• The way in which an aminoacyl tRNA synthetase
  recognises a tRNA varies from one tRNA to another.

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How are amino acids attached to tRNA molecules?
(2)

• Overall the reaction is
• Amino acid tRNA ATP ? Aminoacyl-tRNA PPi
  AMP
• This is called amino acid activation.
• The pyrophosphate is hydrolysed to inorganic
  phosphate, which pulls the reaction to the right.
• Once an amino acid is attached to a tRNA there is
  no further recognition.

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How are amino acids attached to tRNA molecules?
(3)

• The activation reaction occurs in two stages.
• In the first an aminoacyl-AMP complex forms and
  remains attached to the enzyme.
• In the second aminoacyl-tRNA forms.

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Initiation of translation

• It is essential that translation begins at
  exactly the correct point in the mRNA.
• This is particularly important since the triplet
  code gives rise to three possible reading frames
  and only one of these codes for the desired
  protein.
• An error of one or two bases gives rise to a
  frameshift.

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Initiation of translation in E. coli

• Each mRNA contains a sequence of 4-9 bases that
  is complementary to a sequence in the 16 S rRNA.
  This positions the small subunit, with the first
  and second codons aligned with the P and A sites.
  
• Two different tRNAs recognise AUG, one
  exclusively for initiation the other exclusively
  for internal AUG codons.
• In E. coli initiator tRNA is not delivered as
  methionine but as N-formylmethionine attached to
  the initiator tRNA.
• Following completion of the protein the formyl
  group is usually removed, as is also often the
  case for the methionine.

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Cytoplasmic elongation factors

• Two soluble proteins (EF-Tu and EF-G) are used
  during chain elongation.
• Both bind GTP and hydrolyze it during their
  action on the ribosome, yielding GDP and Pi.
• The GDP forms are released from the ribosome and
  reconverted to the GTP forms in the cytoplasm.
• EF-Tu deliver aminoacyl-tRNA to the ribosome,
  EF-G is concerned with ribosome movement along
  the mRNA.

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Figure 8.17
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What is a polysome?

• Once a ribosome has moved about 30 codons along a
  mRNA another ribosome can attach.
• This results in several ribosomes reading the
  same mRNA. This structure is called a
  polyribosome or a polysome.

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Protein synthesis in eukaryotes (1)

• Ribosomes in eukaryotes are larger (80 S, with 60
  S and 40 S subunits).
• The methionine attached to initiator tRNA is not
  formylated.
• There is no Shine-Dalgarno sequence in eukaryotic
  mRNA. Instead a number of proteins attach to the
  cap of the mRNA and bind the 40 S subunit.
• There are three initiation factors eIF1, 2 and 3.

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Protein synthesis in eukaryotes (2)

• The 40 S subunit then moves along the mRNA in an
  ATP requiring reaction until it finds the first
  AUG.
• The 60 S subunit then joins the complex and
  initiation occurs. GTP is hydrolysed at this
  point.
• The various protein factors found in E. coli have
  equivalents in eukaryotes.
• Eukaryotic mRNAs are monocistronic, which is a
  necessary consequence of this method of
  initiation.

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Protein synthesis in mitochondria

• Ribosomes in mitochondria resemble those in
  prokaryotes.
• Initiator tRNA has N-formylmethionine attached to
  it.
• The genetic code is slightly different.
• Codon-anticodon interactions are also different
  with the result that mitochondria only need 22
  tRNAs.
• Most mitochondrial proteins are coded for by
  nuclear genes.

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• Folding up the polypeptide chain

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Chaperones (heat shock proteins)

• Newly synthesised polypeptide chains are unfolded
  and will associate with other chains randomly via
  their hydrophobic groups unless this is
  prevented.
• Chaperones have this role.
• They were initially discovered as heat shock
  proteins because they stabilise proteins that are
  unfolded by heat as well as newly synthesised
  proteins.

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