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 + PP i + 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.