DNA Translation

2. DNA Translation

3. Learning Objectives • Student will be able to • Discuss DNA Translation • Explain Post Translational Modifications • Solve clinical problem

4. Translation • The process of Protein synthesis is called translation • In this process the language of nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. • Translation requires a genetic code through which the information contained in the nucleic acid sequence is expressed to produce a specific sequence of amino acids.

5. The Genetic code • Is a dictionary that identify the correspondence between a sequence of nucleotide bases and a sequence of amino acids. • Each individual word in the code is composed of three nucleotide bases. These genetic words are called codons.

6. Codons • Codons are presented in the mRNA language of adenine (A), guanine (G), cytosine (C), and uracil(U). Their nucleotide sequences • are always written from the 5'-end to the 3'-end. The four nucleotide bases are used to produce the three-base codons. • Therefore, 64 different combinations of bases, taken three at a time (a triplet code) as shown in Figure 31.2.

8. This table (or “dictionary”) can be used to translate any codon and, thus, to determine which amino acids are coded for by an mRNA sequence. • For example, the codon 5'-AUG-3' codes for methioninethe • [ AUG is the initiation (start) codon for translation.] • Sixty-one of the 64 codonscode for the 20 common amino acids. • Termination (“stop” or “nonsense”) codons: Three of the codons, UAG, UGA, and UAA, do not code for amino acids, but rather are termination codons. When one of these codons appears in an mRNA sequence, synthesis of the polypeptide will stops.

9. Figure 17.4 DNAtemplatestrand DNA 5 3 molecule A A A A A T C C C C G G T T T T A G G G G C T C Gene 1 3 5 TRANSCRIPTION Gene 2 U G G U U U G G C U C A 5 3 mRNA Codon TRANSLATION Gly Phe Trp Protein Ser Gene 3 Amino acid

10. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C Codons 5 3 mRNA Translation • Translation is the RNA-directed synthesis of a polypeptide • Translation involves • mRNA • Ribosomes - Ribosomal RNA • Transfer RNA • Genetic coding - codons

11. 3 A Amino acid attachment site C C 5 A C G C G C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C A G * U G * * G A G C Hydrogen bonds (a) G C U A G * A * A C * U A G A Anticodon Transfer RNA • Consists of a single RNA strand that is only about 80 nucleotides long • Each carries a specific amino acid on one end and has an anticodon on the other end • A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids. • tRNA brings the amino acids to the ribosomes, The “anticodon” is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule

12. Amino acid attachment site 5 3 Hydrogen bonds A A G 3 5 Anticodon Anticodon (c) Symbol used in the book (b) Three-dimensional structure Transfer RNA • 3 dimensional tRNA molecule is roughly “L” shaped

13. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Exit tunnel Growing polypeptide tRNA molecules Large subunit E P A Small subunit 5 3 mRNA Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. (a) Ribosomes • Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis • The 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA

14. P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) Large subunit E P A mRNA binding site Small subunit Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. (b) Ribosome • The ribosome has three binding sites for tRNA • The P site • The A site • The E site

15. Growing polypeptide Amino end Next amino acid to be added to polypeptide chain tRNA 3 mRNA Codons 5 (c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. Building a Polypeptide

16. ATP loses two P groups and joins amino acid as AMP. 2 Appropriate tRNA covalently Bonds to amino Acid, displacing AMP. 3 4 Activated amino acid is released by the enzyme. Building a Molecule of tRNA • A specific enzyme called an aminoacyl-tRNA synthetase joins each amino acid to the correct tRNA Amino acid Aminoacyl-tRNA synthetase (enzyme) Active site binds the amino acid and ATP. 1 Adenosine P P P ATP Adenosine P Pyrophosphate P Pi Pi Pi Phosphates tRNA Adenosine P AMP Aminoacyl tRNA (an “activated amino acid”) Figure 17.15

17. Building a Polypeptide • We can divide translation into three stages • Initiation • Elongation • Termination • The AUG start codon is recognized by methionyl-tRNA or Met • Once the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chain • RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain • Translation ends when a stop codon (UAA, UAG, UGA) is reached

18. Translation: Initiation • mRNA binds to a ribosome, and the transfer RNA corresponding to the START codon binds to this complex. Ribosomes are composed of 2 subunits (large and small), which come together when the messenger RNA attaches during the initiation process.

19. Translation: Elongation • Elongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. • The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site.

20. Translation: Elongation • The ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid. • At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it.

21. Translation: Termination • The elongation cycle repeats as the ribosome moves down the messenger RNA, translating it one codon and one amino acid at a time. • The process repeats until a STOP codon is reached.

22. Post Translational Modifications of Polypeptide Chain • Many polypeptide chains are covalently modified either while they are attached to the ribosome or after their synthesis.

23. Trimming • Many proteins destined for secretion from the cell are initially made as large, precursor molecules that are not functionally active. Portions of the protein chain must be removed by specialized endoproteases, resulting in the release of an active molecule. The cellular site of the cleavage reaction depends on the protein to be modified. Some precursor proteins are cleaved in the endoplasmic reticulum or the Golgi apparatus, others are cleaved in developing secretory vesicles (for example, insulin, and still others, such as collagen , are cleaved after secretion. • Zymogens are inactive precursors of secreted enzymes (including the proteases required for digestion). They become activated through cleavage when they reach their proper sites of action. For example, the pancreatic zymogen, trypsinogen, becomes activated to trypsin in the small intestine (see Figure 19.5, p. 249). The synthesis of proteases as zymogens protects the cell from being digested by its own products.

24. Covalent Modification

25. Covalent Modification • Phosphorylation • Glycosylation • Hydroxylation • Carboxylation • Biotinylation • Acetylation

26. Covalent Modification • Methylation • Alkylation • Glutamylation • Lipoylation • Sulfation

27. Covalent Modification • Phosphorylation • The addition of a phosphate (PO4) group to a protein or a small molecule. • Can occur on Serine, Threonine, Tyrosine.

29. Covalent Modification • Glycosylation • The addition of saccharide to a protein or a lipid molecule. • N-Linked Glycosylation • Amide nitrogen of Asparagine • O-Linked Glycosylation • Hydroxyl oxygen of Serine and Therionine.

31. Covalent Modification • Hydroxylation • The addition of hydroxyl group to proline of protein. • Carboxylation • The addition of carboxyl group to glutamate.

34. Covalent Modification • Biotinylation • The addition of biotin to protein or nucleic acid. • Acetylation • The addition of an acetyl group, usually at the N-terminus of the protein.

36. Covalent Modification • Methylation • The addition of a methyl group, usually at lysine or arginine residues. • Alkylation • The addition of an alkyl group (e.g. methyl, ethyl).

37. Covalent Modification • Glutamylation • Covalent linkage of glutamic acid residues to tubulin and some other • Lipoylation • The attachment of a lipoate functionality • Sulfation • The addition of a sulfate group to a tyrosine.

38. Effect of Different Antibiotics on Translation Sterptomycinbinds to the 30S subunit and distort its structure, Interfereing with the initiation of the Protein Synthesis. Tetracyclinesinteract with 30S subunit, blocking access of the aminoacyl-tRNA to the A-site thereby inhibiting elongation.

39. Puromycinbears a structural resemblance to aminoacyl tRNA and accepts peptide from the P site, causing inhibition of elongation. • ChloramphenicolInhibits prokaryotic peptidyltransferase. High levels may also inhibit mitochondrial protein synthesis.

40. Erythromycinbinds irreversibly to a site on the 50S subunit and blocks the tunnel by which the peptide leaves the ribosome. Thereby inhibiting translocation. • Diptheria toxinInactivates the eukaryotic elongation factor EF2 hereby preventing translocation.

41. Practice Questions

42. Q1. During translation, Catalyzing bonding between adjacent amino acids is through which of the following enzyme: helps in a. peptidyl transferase b. aminoacyl-tRNA synthetase c. Shifting of ribosome from one codon to other on mRNA d. Removal of tRNA after formation of peptide bond

43. Ans: A a. The peptidyl transferase is an aminoacyltransferase (EC 2.3.2.12) as well as the primary enzymatic function of the ribosome, which forms peptide bonds between adjacent amino acids using tRNAs during the translation process of protein biosynthesis. b. An enzyme called aminoacyl-tRNA synthetase adds the correct amino acid to its tRNA. The correct amino acid is added to its tRNA by a specific enzyme called an aminoacyl-tRNA synthetase. c. Shifting of ribosome from one codon to other on mRNA d. Removal of tRNA after formation of peptide bond

44. 2. In Prokaryotes, first amino acid in polypeptide chain is a. Methionine b. N-formyl methionine c. N-methyl methionine d. Methyl methionine

45. Ans B • Methionine is used as a first amino acid in eukaryotes. • The first amino acid in a polypeptide chain of prokaryotes is carried by the initiator tRNA, which always carries formylmethionine.

46. 3. During translation, proteins are synthesized by: a. ribosomes using the information on DNA b. lysosomes using the information on DNA c. ribosomes using the information on mRNA d. lysosomes using the information on mRNA

47. Ans C • Translation is the final step on the way from DNA to protein. It is the synthesis of proteins directed by a mRNA template. The information contained in the nucleotide sequence of the mRNA is read as three letter words (triplets), called codons. Each word stands for one amino acid.

48. 4. Tetracycline blocks protein synthesis by inhibiting a. binding of aminoacyl tRNA b.  DNA-dependent RNA polymerase  c. peptidyl transferase d. translocase enzyme

49. Ans A • Tetracycline antibiotics are protein synthesis inhibitors, inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex. • Rifamycin inhibits prokaryotic DNA transcription into mRNA by inhibiting DNA-dependent RNA polymerase by binding its beta-subunit • Chloramphenicol, Macrolides Quinupristin/dalfopristin and Geneticin are inhibiting peptidyl transferase enzyme. • Macrolides,clindamycin and aminoglycosides (with all these three having other potential mechanisms of action as well), have evidence of inhibition of ribosomal translocation.

50. Which of the following process is involved in post-transcription modification? • Acetylation • addition of a 5' cap • Glycocylation • Hair pin loop formation