Highlights Translation 2

1. Amino acid activation can be thought of as the first step in the process of translation. Amino acids are activated by covalently linking them to the 3' end of specific tRNAs. Enzymes called aminoacyl-tRNA-synthetases catalyze the covalent linkage of amino acids to tRNAs. There is one aminoacyl-tRNA synthetase for each amino acid.

2. Amino acids are not linked randomly to tRNAs. Rather, aminacyl-tRNA-synthetases "look" at the anti-codon of a tRNA to decide which amino acid to put on the 3' end of each tRNA. In this way, each tRNA with a particular anti-codon always has the same amino acid on the end of it. Therefore when a tRNA base pairs with a messenger RNA in translation, it is always bringing the right amino acid to be incorporated into the growing protein chain.

3. The first amino acid put into proteins is methionine, since that is the amino acid specified in the genetic code for the first codon of a protein - AUG. Prokaryotes use a modified form of methionine for the first amino acid. 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.

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

5. 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.

6. 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.

7. 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.

8. 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 in slide 26 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.

9. 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.

10. 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.

11. 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.

12. Translation is a target for action of antibiotics. This works because the phenomenon of translation in prokaryotes is different enough from that of eukaryotes that specific inhibitors of prokaryotic translation can be found that have no effect on eukaryotic translation. As a result, prokaryotes can be killed without any effect on eukaryotes taking said antibiotics. One translational inhibitor of prokaryotic translation is the antibiotic puromycin, which acts to prematurely terminate prokaryotic translation at the elongation phase. This happens because puromycin looks like a tRNA and fits into the A (or P) site of the ribosome. In the A site, it can be attached to the growing polypeptid chain, but it is soon released, causing premature termination. In addition, puromycin can bind to the P site of ribosomes and prevents anything from binding there.

13. Proper termination of translation is the last step of the process of making a protein. The process occurs when a stop codon appears in the 'A' site of the ribosome. Since there are no tRNAs with a complementary anti-codon to the stop codons, release factors bind to the stop codon. They cause the bond between the polypeptide and the tRNA in the P site to be broken because they carry water to it and this breaks the bond. Thus, the final polypeptide is released from the ribosome and the entire complex then comes apart.

14. Eukaryotic mRNAs have a 5' cap and a 3' polyA tail. Eukaryotic translation occurs much like prokaryotic translation, but the factors are differently named. One important difference of eukaryotic translation is that the initiation complex in eukaryotes involves both the 5' cap and the 3' polyA sequence in a looped structure. The 5' cap and 3' poly-A sequence both play roles in helping to increase the stability of eukaryotic mRNAs.