Highlights Lecture #20 Spring 2017

Highlights Translation 2

1. Prokaryotes use a modified form of methionine for the first amino acid in a protein. It is known as fmet (formyl-methionine). fmet is used ONLY as the first amino acid of a protein. When AUG appears in the middle of a coding region of an mRNA, methionine (not fmet) is put into the protein. The formyl group is put onto methionine after the methionine is attached to the tRNA.

2. Translation initiation requires 1) an mRNA; 2) a start codon in the mRNA – AUG; 3) the 30S ribosomal subunit; 4) the 50S ribosomal subunit; 5) proteins called initiation factors; and 6) GTP.

3. During initiation of translation, the first tRNA (linked to fmet) forms base pairing between its anti-codon and the codon of the mRNA. A sequence known as the Shine-Dalgarno sequence in the mRNA (in prokaryotes only) helps the ribosome to properly align the mRNA in the ribosome. The Shine-Dalgarno sequence forms a base paired region with the 16S rRNA of the 30S subunit of the ribosome. During initiation, the initiator tRNA (fmet in prokaryotes) is placed in the P site of the ribosome. It is the only tRNA that starts in the P site. All others start in the A site.

4. Protein synthesis uses GTP as an energy source (not ATP).

5. Elongation of translation occurs by binding of an incoming tRNA (which will base pair with the next codon) in a region of the ribosome known as the ‘A’ site. The previous tRNA is found on the adjacent ‘P site’ of the ribosome. A third site of the ribosome where tRNAs are found is the ‘E site’, which is where tRNAs exit the ribosome after they have been used.

6. Translation involves linking amino acids properly together via peptide bonds. Note that the growing polypeptide chain is held on a tRNA. After the peptide bond forms, the tRNA with the polypeptide chain moves to the P site and a new tRNA with an amino acid comes into the A site. This process also involves the mRNA sliding one codon down the chain to put a new codon in the A site.

7. After the tRNA properly binds in the A site, a peptide bond is formed between the peptide on the tRNA in the P site and the amino acid on the tRNA in the A site. The catalyst for the peptide bond is the 23S rRNAs in the ribosome. Thus, peptide bonds are formed by ribozymes (catalytic RNAs). The result is a peptide linked to the tRNA in the A site.

8. The next step in elongation occurs when the tRNA with the polypeptide in the A site moves over to the P site (displacing the tRNA previously there to the E site, from which it exits the ribosome). The A site is thus left open. The ribosome slides three nucleotides along the mRNA, bringing a new complementary sequence into the A site. Movement of the ribosome along the mRNA is facilitated by EF-G and requires GTP.

9. Two other EF proteins are required for elongation. EF-Tu is the most abundant E. coli protein and it functions to carry the tRNA to the A site of the ribosome. EF-Tu makes sure that the properly base-paired tRNA is in the A site. EF-Tu also protects the bond between the amino acid and the tRNA from water.

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