Translation

translation

1. TRANSLATION Prepared by A. Nandakumar, M.Tech

2. Translation The process involves in converting the nucleic acid “language,” the genetic code, to protein “language,” and is therefore known as translation. This was first formulated by Sir Francis Crick.

3. Amino Acid Coding There are 20 amino acids in proteins but only four different bases in the mRNA. During translation,the bases of mRNA are read off in groups of three, which are known as codons . Four different bases gives 64 possible groups of three bases; that is, 64 different codons in the genetic code.

4. In addition, three of the codons are used for punctuation. Those are the stop codons that signal the end of a polypeptide chain. To read the codons,a set of adapter molecules that recognize the codon on the mRNA at one end and carry the corresponding amino acid attached to their other end is needed. These adapters are small RNA molecules, or transfer RNA ( tRNA ).

5. This adapter contains anticodon at one end which are complementary to the three bases of the codon on the mRNA. The codon and anticodon recognize each other by base pairing and are held together by hydrogen bonds.

6. Components of ribosome & difference Prokaryotes Eukaryotes 70s Ribosome composed of 30s (small subunit) & 50s (large subunit). Small subunit - 16s rRNA contains 21 different proteins. Large subunit – 5s & 23s rRNA contains 32 different proteins. T he initiator tRNA is first charged with methionine by methionyl-tRNA synthetase . The methionine residue is then converted to N- formylmethionine by transformylase . Multiple start sites. I nitiation factors, IF1, IF2 and IF3. 80s Ribosome composed of 60s (large subunit) & 40s (small subunit). 5’ r cap instead of Shine- Dalgarno sequence. In eukaryotes, the methionine on the initiator tRNA is not modified. The initiating codon in eukaryotes is always AUG. I nitiation factors, eIF1, eIF2, eIF3, eIF4, eIF5 and eIF6.

7. Overview of prokaryotic translation

8. Translation: The Process of Protein Synthesis 1. Ribosomes translate the genetic message of mRNA into proteins. 2. The mRNA is translated 5’  3’, producing a corresponding N-terminal  C-terminal polypeptide. 3. Amino acids bound to tRNAs are inserted in the proper sequence due to: a. Specific binding of each amino acid to its tRNA . b. Specific base pairing between the mRNA codon and tRNA anticodon .

9. Charging t RNA 1. Aminoacyl-tRNA synthetase attaches amino acids to their specific tRNA molecules. The charging process ( aminoacylation ) produces a charged tRNA ( aminoacyl-tRNA ), using energy from ATP hydrolysis. 2. There are 20 different aminoacyl-tRNA synthetase enzymes, one for each amino acid. Some of these enzymes recognize tRNAs by their anticodon regions. 3. The amino acid and ATP bind to the specific aminoacyl-tRNA synthetase enzyme. ATP loses two phosphates and the resulting AMP is bound to the amino acid, forming aminoacyl -AMP. 4. The tRNA binds to the enzyme, and the amino acid is transferred onto it, displacing the AMP. The aminoacyl-tRNA is released from the enzyme.

10. Charging of a t RNA molecule by aminoacyl- t RNA synthetase

11. 5. The amino acid is now covalently attached by its carboxyl group to the 3’r end of the tRNA . Every tRNA has a 3’r adenine, and the amino acid is attached to the 3’r–OH or 2’r–OH of this nucleotide.

12. Charged tRNA

13. Formylation of tRNA

14. Initiation In prokaryotes, initiation requires the large and small ribosome subunits, the mRNA, the initiator tRNA , three initiation factor(IFs) and GTP. IF1 and IF3 bind to the 30S subunit and prevent the large subunit binding. IF2+GTP can then bind and will help the initiator tRNA to bind later. This small subunit complex can now attach to an mRNA via its ribosome-binding site. The initiator tRNA can then base-pair with the AUG initiation codon which releases IF3 thus creating the 30S initiation complex. The large subunit then binds, displacing IF1 and IF2+GTP, giving the 70S initiation comple which is the fully assembled ribosome at the correct position on the mRNA.

15. Shine- dalgarno sequence Shine- Dalgarno sequence is an ribosomal binding site in prokaryotic mRNA, generally located around 8 bases upstream of the start codon AUG. Shine- Dalgarno sequence exists both in bacteria & archaea and also in some chloroplast & mitochondria. Shine- Dalgarno sequence helps to make ribosome available to the mRNA to initiate protein synthesis by aligning it with the start codon .

16. elongation Elongation involves the three factors( Efs ), EF- Tu , EF-Ts and EF -G, GTP, charged tRNA and the 70S initiation complex(or its equivalent). It takes place in three steps. A charged tRNA is delivered as a complex with EF- Tu and GTP. The GTP is hydrolyzed and EF-Tu.GTP is released which can be re-used with the help of EF-Ts and GTP(via the EF- Tu -EF-Ts exchange cycle). Peptidyl transferase makes a peptide bond by joining the two adjacent amino acid without the input of more energy. Translaocase(EF-G), with energy from GTP, move the ribosome one codon along the mRNA, ejecting the uncharged tRNA and transferring the growing peptide chain to the P-site.

17. Action of peptidyl transferase The two aminoacyl-tRNAs are positioned by the ribosome for peptide bond formation, which occurs in two steps : a. In the P site, the bond between the amino acid and its tRNA is cleaved. b. Peptidyl transferase forms a peptide bond between the now-free amino acid in the P site and the amino acid attached to the tRNA in the A site. Experiments indicate that the 23S rRNA is most likely the catalyst for peptide bond formation. c. The tRNA in the A site now has the growing polypeptide chain attached to it.

18. termination Release factors(RF1 or RF2) recognize the stop codon and, helped by RF3, make peptidyl transferase join the polypeptide chain to a water molecule, thus releasing it. Ribosome release factor helps to dissociate the ribosome subunit from the mRNA.

19. Translation factors

20. Overview of eukaryotic translation

21. initiation This is the major point of difference between prokaryotic and eukaryotic protein synthesis, there being at least nine eIF involved. Functionally, these factors can be grouped. They either bind to the ribosome subunit or to the mRNA, deliver the initiator tRNA or displace other factors. In contrast to the events in prokaryotes, initiation involves the initiator tRNA binding to the 40S subunit before it can bind to the mRNA. Phosphorylation of eIF2, which delivers the initiator tRNA , is an important control point.

22. Elongation & termination This stage of protein synthesis is essentially identical to that described for prokaryotes. The factors EF- Tu , EF-Ts and equivalents called eEF1α, eEF1βγ and eEF2 respectively, which carry out the same roles. EF-G have direct eukaryotic activity. Eukaryotes use only one release factor ( eRF ), which requires GTP, for termination of protein synthesis. It can recognize all three codons .

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