1. Protein Synthesis Biology 12 Mr. McIntyre

2. Translation: From messenger RNA to protein: The information encoded in the DNA is transferred to messenger RNA and then decoded by the ribosome to produce proteins.

3. 5’-ATGCCTAGGTACCTATGA-3’ 3’-TACGGATCCATGGATACT-5’ 5’-AUGCCUAGGUACCUAUGA-3’ N-MET-PRO-ARG-TYR-LEU-C DNA Transcription decoded as Translation mRNA Protein

6. Alanine tRNA

7. Generalized tRNA

8. = UH 2 Modified Bases Found in tRNAs

11. tRNAs are activated by amino-acyl tRNA synthetases

13. Structure of an amino acyl-tRNA synthetase bound to a tRNA

14. One mechanism for maintaining high fidelity of protein synthesis is the high fidelity of aa-tRNA synthetases

15. Amino-acyl tRNA synthetases: One synthetase for each amino acid a single synthetase may recognize multiple tRNAs for the same amino acid Two classes of synthetase. Different 3-dimensional structures Differ in which side of the tRNA they recognize and how they bind ATP Class I - monomeric, acylates the 2’OH on the terminal ribose Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp Tyr, Val Class II - dimeric, acylate the 3’OH on the terminal ribose Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

16. Two levels of control to ensure that the proper amino acid is incorporated into protein: 1) Charging of the proper tRNA

17. 2) Matching the cognate tRNA to the messenger RNA

18. Incorporation of amino acids into polypeptide chains I

19. Incorporation of amino acids into polypeptide chains II

20. Protein synthesis occurs on ribosomes

22. and mitochondria

23. Ribosome Assembly The proteins of each ribosomal subunit are organized around rRNA molecules 16S rRNA

24. Ribosome Assembly: takes place largely in a specialized domain of the nucleus, the nucleolus

26. In the nucleolus, RNA polymerase I transcribes the rDNA repeats to produce a 45S RNA precursor The 45S precursor is processed and cleaved into mature rRNAs and ribosomal proteins then bind to generate the large and small ribosomal subunits

27. 23S rRNA secondary structure

28. 3D organization of the eukaryotic large subunit rRNA

29. Ribosomal Proteins decorate the surface of the ribosome Large subunit. Grey = rRNA Gold = ribosomal proteins

30. Ribosomal proteins often have extensions that snake into the core of the rRNA structure Crystal structure of L19 L15 (yellow) positioned in a fragment of the rRNA (red)

31. The ribosomal proteins are important for maintaining the stability and integrity of the ribosome, but NOT for catalysis ie. the ribosomal RNA acts as a ribozyme

32. Mitochondrial or Prokaryotic Eukaryotic 60S subunit 80S ribosome 40S subunit The large and small subunits come together to form the ribosome

33. The association of the large and small subunits creates the structural features on the ribosome that are essential for protein synthesis Three tRNA binding sites: A site = amino-acyl tRNA binding site P site = peptidyl-tRNA binding site E site = exit site

34. In addition to the APE sites there is an mRNA binding groove that holds onto the message being translated

35. There is a tunnel through the large subunit that allows the growing polypeptide chain to pass out of the ribosome

37. Peptide bond formation is catalyzed by the large subunit rRNA

41. Incorporation of the correct amino acyl-tRNA is determined by base-pairing interactions between the anticodon of the tRNA and the messenger RNA

42. Proper reading of the anticodon is the second important quality control step ensuring accurate protein synthesis =EF-1 Elongation factors Introduce a two-step “Kinetic proofreading”

43. A second elongation factor EF-G or EF-2, drives the translocation of the ribosome along the mRNA Together GTP hydrolysis by EF-1 and EF-2 help drive protein synthesis forward

44. Termination of translation is triggered by stop codons Release factor enters the A site and triggers hydrolysis the peptidyl-tRNA bond leading to release of the protein.

45. Release of the protein causes the disassociation of the ribosome into its constituent subunits.

46. Release Factor is a molecular mimic of a tRNA eRF1tRNA

47. Initiation of Translation Initiation is controlled differently in prokaryotic and eukaryotic ribosomes In prokaryotes a single transcript can give rise to multiple proteins

48. In prokaryotes, specific sequences in the mRNA around the AUG codon, called Shine-Delgarno sequences, are recognized by an intiation complex consisting of a Met amino-acyl tRNA, Initiation Factors (IFs) and the small ribosomal subunit

49. GTP hydrolysis by IF2 coincident with release of the IFs and binding of the large ribosomal subunit leads to formation of a complete ribosome,on the mRNA and ready to translate.

50. Eukaryotic mRNAs have a distinct structure at the 5’ end

51. Structure of the 7-methyl guanosine cap The 7me-G cap is required for an mRNA to be translated

52. In contrast, Eukaryotes use a scanning mechanism to intiate translation. Recognition of the AUG triggers GTP hydrolysis by eIF-2

53. GTP hydrolysis by eIF2 is a signal for binding of the large subunit and beginning of translation

54. Messenger RNAs are translated on polyribosomes

55. Protein synthesis is often regulated at the level of translation initiation

56. An example of control of specific mRNAs: regulation by iron (Fe): Ferritin is a cytosolic iron binding protein expressed when iron is abundant in the cell. Transferrin receptor is a plasma membrane receptor important for the import of iron into the cytosol. They are coordinately regulated, in opposite directions, by control of protein synthesis.

57. Regulation by iron (Fe):

58. There is also general control of translational initiation. ie. all transcripts of the cell are effected (though the relative effect differs between specific mRNAs) Global downregulation or upregulation can occur in response to various stimuli the most common are 1) Nutrient availability low nutrient (amino acids/carbohydrate) downregulates translation 2) Growth factor signals. stimulation of cell division upregulates translation

59. General control of translational initiation is exerted through two primary mechanisms. Control of the phosphorylation of eIF2 Control of the phosphorylation of eIF4 binding proteins

60. Control of translation by eIF2 phosphorylation Stimulated by Amino acid deprivation

61. Control of translation by eIF4E availability The 7ME G cap binding subunit of eIF4, eIF4E, is sequestered by eIF4E binding protiens (4E-BPs). The binding of these proteins is regulated by their phosphorylation state Growth Factors Nutrient Limitation

62. Nutritional signals can control both the recognition of the mRNA and loading of the 40S subunit. Nutritional controls 2

63. Modification of the translation machinery is a common feature of viral life cycles e.g. Picornaviruses Polio virus Encephalomyocarditis virus Picornaviruses have single stranded RNA genomes.

64. Poliovirus Life Cycle

65. The poliovirus genome is translated into a single, large polyprotein that then auto-proteolyzes itself into smaller proteins. One of these proteins, viral protease 2A cleaves the translation initiation factor eIF4G so that it can no longer function as a bridge between the methyl cap binding subunit and the 40S subunit

66. The consequence of this cleavage is that translation of cellular mRNAs stops But…the viral RNA is still translated due to the presence of an internal ribosomal entry site (IRES). This acts like a bacterial initiation site to allow Cap-independent initiation from internal AUG codons. What is X?

67. “X” is not a protein, as suggested by the textbook model at right, rather it is a structure in the mRNA itself that can bind to the remaining fragment of eIF4G

68. Some cellular mRNAs are also translated using IRESs During G2/M phase of the cell cycle, translation is generally downregulated by activation of 4E-BPs. Many proteins expressed during this period bypass this control by using IRES elements

69. Ribosomal Frameshifting Because translation uses a triplet code, there are three potential reading frames in each mRNA

70. As the ribosome translocates, it moves in three nucleotide steps, ensuring that the frame defined by the AUG is used throughout translation If the ribosome moves 1 or 2 (or 4 or 5) nucleotides this produces a frameshift

71. Many retroviruses induce ribosomal frameshifting in the synthesis of viral proteins e.g. HIV

72. Translation Inhibitors are important antibiotics