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.

AUGCUUGAAUAA

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.

Figure 8.17

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.

Folding up the polypeptide chain

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.