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translation

translation. RBS. RBS: ribosome binding site. Ribosome(r RNA + r protein). Introductory remarks. Ribosomes are the sites of protein synthesis. They occur in the cytoplasm, mitochondria and chloroplasts.

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translation

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  1. translation RBS RBS: ribosome binding site Ribosome(r RNA + r protein)

  2. 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.

  3. AUGCUUGAAUAA

  4. 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.

  5. The wobble mechanism(2) • As an example GCC and GCU both code for alanine. Wobble pairing permits the anticodon sequence 3´CGG 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Figure 8.17

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Folding up the polypeptide chain

  18. 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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