translation RBS RBS: ribosome binding site Ribosome(r RNA + r protein)
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Transcript of 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.
• 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.