2. Chapter 12. Protein biosynthesis  Protein biosynthesis is a process to express genetic information in living cells, which is called translation. The genetic information flows as: DNA RNA Protein TranscriptionTranslation Reverse transcription

3. 1. Components of Protein Biosynthesis Protein biosynthesis requires: amino acids, mRNA, tRNA, ribosomes, protein factors, and synthetic enzymes. 1) Messenger RNA: a template for protein biosynthesis, which is read in a 5’  3’ direction. Each three nucleotides form a codon representing for a specific amino acid. Thus, the base sequence of an mRNA molecule determines the amino acid sequence of the protein.

4. Codons in mRNA UCAG U Phe Leu Ser Tyr Stop Cys Stop Trp UCAGUCAG C Leu Pro His Gln Arg UCAGUCAG A Ile Met Thr Asn Lys Ser Arg UCAGUCAG G Val Ala Asp Glu Gly UCAGUCAG

5.  mRNA in eukaryotes is usually monocistronic: one mRNA encodes only a single polypeptide chain.  mRNA in prokaryotes usually encodes more than one polypeptide chain. This is called polycistronic.

6. A)Degeneracy of codons: refers to the fact that an amino acid has more than one codon.  one of the consequences of degeneracy is that a mutation which produces a base change in DNA may not result in an amino acid change in the encoded protein.  Synonyms: refers to the codons for the same amino acid. e.g. GUU, GUC, GUA, GUG represent for Val.

7. B) Universility of codons: this genetic code system is used by all living organisms except in some cases: in cytosol in mitochondria AUAIleMet UGAStopTrp AGAArgStop (animal) CGGArgTrp (plant) CUNLeuThr (yeast)

8. C) Reading frames: refer to the different combinations for each three nucleotides that are read as a codon: each mRNA sequence can be read in three possible reading frames. Reading frame 1: UUA UGA GCG CUA AAU Leu Stop Ala Leu Asn Reading frame 2: U UAU GAG CGC UAA AU Tyr Glu Arg Stop Reading frame 3: UU AUG AGC GCU AAA U Met Ser Ala Lys

9. D) Open reading frames: refer to the runs of codons that start with ATG and end with TGA, TAA, or TAG. The open reading frames can be used to predict the protein sequence encoded.

10. 2) Transfer RNAs: the fidelity of protein biosynthesis requires tRNAs to serve as adapters that can recognize the correspondent codons and carry amino acids to the right positions in translation.  Each tRNA only brings with it an amino acid, and recognizes and binds to a specific codon.

11. Secondary structure of tRNA 3 C A U A C A A A A A A A A A A A A A A A G A G A A A A A A A A C A G A T A 鴠 A C A U A 5 T  C loop DHU loop extra arm Anticodon loop

12. Tertiary structure of tRNA

13. Codon-anticodon interaction by base pairing tRNA

14.  Wobble base pairing: base pairing between the 3’ position of the codon and 5’ position of the anticodon may occur by a non- standard way. This allows one tRNA to recognize more than one codon. Examples of wobble base pairing Anticodon wobble position baseC A G U I Codon wobble position baseG U C A C U G U A

15. Wobble base pairing of inosine with three nucleosides

16. 3) rRNAs and ribosomes: As the site of protein biosynthesis, ribosome is made up of two subunits, one is large and another is small.

17. Eukaryotic ribosome (80S) Prokaryotic ribosome (70S) large subunit small subunit Subunit size 60S 40S 50S 30S rRNAs 5S, 5.8S, 28S 18S 5S, 23S 16S Proteins 49 33 35 21 Composition of ribosomes in eukaryotes and prokaryotes

18. A) Polysomes: several ribosomes bind to and translate a single mRNA molecule simultaneously B) Free ribosomes: ribosomes occur free in the cytosol, usually synthesizing proteins of cytosol, nucleus, mitochondria or other organelles C) Membrane bound ribosomes: ribosomes bind to the membrane of rough endoplasmic reticulum, usually synthesizing secretory proteins or membrane proteins.

19. Polysomes

20. 4) Aminoacyl-tRNA synthetase: is also called amino acid activating enzyme, which catalyzes the following reactions.

21. 2. Steps of Protein Biosynthesis The steps of protein biosynthesis include: initiation, elongation, and termination or release. 1) Initiation: Translation begins with the assembly of an initiation complex consisting of an mRNA, a ribosome, and the initiator tRNA (fMet-tRNAi Met or Met- tRNAi Met ). The process requires a number of protein factors, known as initiation factors.

22. Formation of the initiation complex in eukaryotic translation

23.  In prokaryotes, initiation factors IF1 and IF3 bind to the 30S subunit while IF2 binds to GTP·fMet-tRNAi Met. The two complexes and mRNA combine to form a pre-initiation complex, releasing IF3. The 50S subunit binds with this complex, with hydrolyzation of the bound GTP to GDP and Pi, and release of IF1 and IF2, to form a completed initiation complex.

24. Formation of the initiation complex in prokaryotic translation

25. Prokaryotic and eukaryotic initiation factors Prokaryotic eukaryotic Function IF 1 eIF 1 IF 1 binds to small subunit before mRNA binding. eIF 1 assists mRNA binding. IF 2 eIF 2a eIF 2b eIF 2c Bind initiator tRNA, stabilize ternary complex, cause GTP/GDP exchange. IF 3 eIF3 Bind to the small subunit, assist mRNA binding, cause dissociation of subunits after translation. eIF 4a eIF 4b eIF 4c Recognize and bind the mRNA cap, assist mRNA binding, eIF 4d eIF 4e eIF 4f hydrolyze ATP to drive scanning for the initiator codon. eIF 5 Promotes GTP hydrolysis and release of other initiator factors. eIF 6 Assists subunit dissociation.

26. 2) Elongation: Elongation of polypeptide chain consists of a series of cycles, called ribosomal cycles, each of which forms a new peptide bond. Three steps: entry, peptide bond formation, and translocation.

27. A) Entry of aminoacyl-tRNA to the A site of ribosome ( A. in prokaryotes, B. in eukaryotes. AA = aminoacyl )

28. B) Peptide bond formation dipeptidyl-tRNA Peptidyl- transferase

29. C) Translocation: Translocation is a process involves the shift of the newly formed peptidyl(n+1)-tRNA from the A site to the P site, with release of the deacylated tRNA from the ribosome. This process is mediated by another elongation factor, EF-G in prokaryotes, or eEF2 in eukaryotes.

30. The translocation step in protein biosynthesis

31. D) Termination: when a ribosome moves onto the stop codon of mRNA, the stop codon in the A site cannot be recognized by any aminoacyl-tRNA molecules. Instead, release factors interact with the mRNA- ribosome complex, leading to discharge of the newly synthesized polypeptide from the complex.

32. Termination of protein biosynthesis

33. Elongation and termination factors in prokaryotes and eukaryotes

34. (Eukaryotic)

35. 3. Posttranslational Processing Newly synthesized polypeptides usually undergo structural changes called posttranslational processing.  The most important posttranslational processing: modification and folding.

36. 1)Posttranslational modification: A) Modification of protein primary structures  Removal of the N-terminal Met residue

37.  Posttranslational processing of human preproinsulin

38. B) Glycosylation: occurs in most membrane and secretary proteins, such as glycoproteins. Two types of glycoproteins in humans: O-linked and N-linked. Formed in endoplasmic reticulum and Golgi apparatus. N-linked O-linked

39. C) Modification of protein on higher-level structures Acetylation of the amino terminus: Acetyl-SCoA + H 2 N-protein Acetyl-NH-protein + HSCoA Phosphorylation: ATP ADP Protein kinase Protein Phosphoprotein Phosphatase Pi H 2 O

40. 2) Folding of newly synthesized polypeptides Newly synthesized polypeptide chains usually undergo folding, a process that requires protein factors called molecular chaperones. Two types of molecular chaperones: chaperones and chaperonins. The major function of molecular chaperones is to assist the correct folding of nascent polypeptide chains by blocking their hopeless entangling or insignificant intermolecular interactions.

41. Molecular chaperones belong to the “heat- shock protein (HSP) family”. A)Chaperone proteins: include HSP70, HSP40, and GrpE.  The binding-release cycle of chaperone proteins with a nascent polypeptide earns time for the proper folding of the unfolded polypeptide chain. The cycle continues until the polypeptide chain is folded to a native conformation.

42. The binding-release cycle of a chaperon-polypeptide complex

43. B) Chaperonins: are also heat-shock proteins. They participate in the folding of a variety of proteins by forming a cylindrical structure (a ring) enclosing a central cavity.  The target polypeptide chain enters the central cavity of the folding machine, where it is properly folded and is then released. The entering-folding process repeats until a native 3D structure of the protein is formed.

44. A folding cycle of a polypeptide by GroEL-GroES chaperonins in E. coli cell

45. 4. Protein targeting Protein targeting is a process in which a newly synthesized protein is delivered to a specific extracellular or intracellular location.  Secretory proteins are first synthesized by ribosomes bound to the rough ER (RER), with a signal sequence (or called signal peptide) at the N-terminal end, which directs the protein to be delivered to its functioning place.

46. (A)Signal peptides of secretory proteins. (B) Type III integral membrane proteins with signal-peptide, internal signal-peptide, and stop-transfer sequences. N- Hydrophobic area Secretory protein Signal peptidase cleavage site Stop-transfer sequence Type III integral membrane protein Signal peptide Internal signal peptide Signal peptide N- A. B.

47. The signal peptide directs a newly synthesized secretary protein to enter into the RER lumen SRP: signal recognition particle

48. 5. Clinical correlation of protein biosynthesis Protein biosynthesis is the means to express the genes that control metabolisms in cells. Any mistake occurs in protein biosynthesis may result in severe consequences in metabolism.

49. 1)Molecular Diseases: refer to those resulting from abnormal protein structures due to mutation of genes.  Sickle-cell anemia: a result from the replacement of an amino acid residue at position 6 of the  - chain, glutamate, by another one, valine. Position of  -chain 1 2 3 4 5 6 7 8 Hemoglobin A Val-His-Leu-Thr-Pro-Glu-Glu-Lys- Hemoglobin S Val-His-Leu-Thr-Pro-Val-Glu-Lys-

50. 2) Action of some antibiotics: they carry out antimicrobial activities via inhibition of protein synthesis in the microorganism, such as tetracyclines, streptomycin, chloramphenicol, and so on.

51. Some antibiotic inhibitors of protein biosynthesis

52. 3) Effect of some biological molecules : Interferons (IFNs) are cytokines produced during immune response to antigens, especially to viral infections. Two functions of IFNs: cause viral RNA degradation and inhibit protein biosynthesis in cells. IFN protein kinase phosphorylation of eIF-2a inhibition of protein biosynthesis in cells inhibition of the viral replication.