DNA Translation

Question 1

What are the components needed for translation?

Answer 1

1. mRNA
2. tRNA
3. Free amino acids
4. rRNA
5. Ribosomes

Question 2

What are the key regions on ribosomes that are essential for translation?

Answer 2

1. Small subunit:
   
   • 40S
   • 1 binding site for mRNA
   • 2 binding sites for tRNA
2. Large subunit:
   
   • 60S
   • “P” (Peptidyl-tRNA binding) site: holds tRNA carrying growing polypeptide chain
   • “A” (Aminoacyl-tRNA binding) site: holds tRNA carrying next amino acid to be added to the chain
   • “E” (Exit) site for tRNA: discharged tRNAs leave ribosomes

Question 3

What are the steps involved in translation?

Answer 3

1. Amino acid activation
2. Initiation of translation
3. Elongation of translation
4. Termination of translation

Question 4

What happens during amino acid activation?

Answer 4

1. The attachment of amino acid to its specific tRNA is mediated by aminoacyl tRNA synthetases.
2. There are at least 20 aminoacyl tRNA synthetases, one for each amino acid and it is able to bind all the different tRNAs that code for its specific amino acid.
3. Aminoacyl tRNA synthetases has an active sit which specifically binds one type of amino acid with the appropriate tRNA molecule.
4. Synthetases catalyses the covalent attachment of the specific amino acid to its tRNA (reaction is driven by hydrolysis of ATP)
   
   • Results in formation of aminoacyl-tRNA complex
   • Amino acid is joined by its carboxyl group to the 3’ OH of the tRNA by an ester bond
   • “charged”: tRNA with amino acid linked to it
5. Formation of initiator tRNAmet (tRNA + methionine amino acid) —> used for recognition of start codon AUG

**The first level of specificity: anticodon on tRNA is matched appropriately with a specific amino acid -> fidelity of the code is ensured by the specificity of the aminoacyl-tRNA synthetase enzyme

Question 5

What happens during initiation of translation?

Answer 5

1. Small ribosomal subunit binds to the 5’ end of the mRNA and a initiator tRNA, which has the anticodon UAC (code for AUG), and carries the amino acid methionine
2. Small subunit moves downstream (5’ -> 3’) along the mRNA till it reaches the start codon AUG, which signals the start of translation
3. Initial tRNA binds with start codon throug hydrogen bonding
4. Large ribosomal subunit then attaches to complex with the help of initiation factors
5. Translation initiation complex is made up of:
   
   • Large and small ribosomal subunit
   • Initiator tRNA (with anticodon UAC)
   • Initiation factors
6. Initiator tRNA is positioned at “P” site of large ribosomal subunit
7. Energy provided by hydrolysis of large ribosomal subunit

**the matching of a specific anticodon (tRNA) to codon (mRNA) confers a second level of specificity to ensure fidelity in information transfer

Question 6

What happens during elongation of translation?

Answer 6

1. Codon recognition:
   
   • Anticodon of incoming aminoacyl-tRNA basepairs with complementary mRNA codon in the “A” site of large ribosomal subunit, assisted by elongation factors
   • Hydrolysis of GTP required
2. Peptide Bond Formation:
   
   • Large ribosomal subunit catalyses a chemical reaction that releases methionine from the initiator tRNA at the “P” site, and attaches it instead by a peptide bond to the 2nd amino acid which is attached to the aminoacyl-tRNA at the “A” site
   • catalysed by peptide transferase present on the large ribosomal subunit
3. Translocation:
   
   • Ribosome translocates three more nucleotides along mRNA molecule in the 5’ to 3’ direction (Hydrolysis of GTP required)
   • This movement relocates the initial tRNA to “E” and ejects it from the ribosome, repositions the growing polypeptide chain to the “P” site, exposes the next codon on the mRNA at the “A” site
   • The now empty “A” site receives another aminoacyl-tRNA which has an anticodon complementary to the third codon on the mRNA

Question 7

What happens during chain termination of translation?

Answer 7

1. Elongation continues until stop codon (UAA/ UAG/ UGA) in mRNA reaches the “A” site.
2. Release factor (a protein: eRF) binds directly to the stop codon in the “A” site
3. This causes the addition of a water molecule instead of an amino acid to the polypeptide chain, hydrolysing the polypeptide chain from the tRNA in the P site
4. Polypeptide is then released from the ribosome
5. Ribosome then disassembles into its subunits

Question 8

What happens to the released polypeptide chain?

Answer 8

Protein folding and post-translational modifications

1. During its synthesis, the polypeptide chain begins to assume its secondary and tertiary structures automatically
2. The polypeptide may also undergo modification at the Golgi body (eg glycosylation)