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  • 1. Biochemistry Chen Yonggang Zhejiang University Schools of Medicine
  • 2. Translation, making protein following nucleic acid directions
  • 3. Bodega Bay, Sonoma County
  • 4. Breakfast at The Tides, Bodega Bay
  • 5. The process of using base pairing language to create a protein is termed Translation
    • Any process requires:
      • A mechanism Ribosome
      • Information-directions mRNA
      • Raw materials amino acids / tRNA
      • Energy ATP
    • Any process has stages :
      • Beginning Initiation
      • Middle Elongation
      • End Termination
  • 6. Translation requires a Dictionary
    • The dictionary of Translation is called the Genetic Code [Table 6.1]
    • Correlates mRNA with Protein
      • 3 nucleotides = 1 amino acid 4 3 = 64
        • 4 possible nts 20 possible aa
    • 3 nucleotides read 5’-3’ are called a codon
      • Codes for 1 amino acid
  • 7. The Genetic Code
  • 8. The Genetic Code
    • Triplet made of codons
    • Non-overlapping read sequentially
    • Unpunctuated once started, set frame
    • Degenerate > than one codon/AA
    • Nearly universal mitochondrial code
    • Start signals AUG[met]
    • Stop signals UAG, UAA, UGA
  • 9. Players in Translation
    • Ribosome the machinery
    • mRNA the information
    • Aminoacyl-tRNA the translator!
      • Amino Acids/tRNA
      • ATP
  • 10. Ribosomes are ribonucleoprotein complexes table 6.7 Small subunit Large Subunit PROCARYOTIC EUCARYOTIC 70 S 30S 50S RNA 5S, 16S, 23S PROTEINS 55 80 S 40S 60S RNA 5S, 5.8S,18S,28S PROTEINS 84
  • 11. Ribosomes must be assembled with an mRNA
    • The initiation process requires protein factors
    • A mRNA must be recognized and reading frame must be set
    • Aminoacyl-tRNAs must be available
    5’ 3’
  • 12. Since the Translator is the Aminoacyl-tRNA, it must be important
    • Cells have 30+ tRNAs
    • tRNAs are redundant for some amino acids
    • Cells have 20 Aminoacyl-tRNA Synthetases
    • Aminoacyl-tRNA synthetases recognize 1 amino acid and 1 or more tRNAs
    • Aminoacylation is very precise
  • 13. Aminoacyl-tRNA Synthetases are critical to Translation
    • 1 Aminoacyl-tRNA Synthetase recognizes 1 Amino Acid and binds it
    • 1 Aminoacyl-tRNA Synthetase recognizes 1 or more tRNAs specific for 1 amino acid
    • The aminoacyl-tRNA Synthetase catalyzes a two step reaction which overall is
    • AA x + tRNA x + ATP AA x -tRNA x + AMP + PPi
    • Page 239
  • 14. The first step involves forming an enzyme-bound aminoacyl adenylate The hydrolysis of the PPi makes the process irriversible
  • 15. The second step transfers the amino acid to the 3’OH of the tRNA, retaining the energy of the adenylate
  • 16. tRNAs fold into L-shaped structures Figure 2.59
  • 17. Functional Sites of tRNAs Figure 2.58
    • CCA OH 3’ Acceptor Sequence
    • Amino acid acceptor stem
    • D stem and loop
    • Extra loop
    • Anticodon stem and loop
    • Anticodon
    • T  C stem and loop
    • 5’ Terminus
  • 18. The anticodon forms antiparallel base pairs with a codon in the mRNA
    • Each tRNA has a unique anticodon
    • There are 61 codons which base pair with tRNA anticodons, most pairing is Watson-Crick but Wobble in the 5’ base of the anticodon allows degeneracy
    • 3 codons do not normally base pair with anticodons-UAA, UAG, UGA. The lack of a complementary anticodon- Termination Codons
  • 19. Wobble allows one codon to base pair with up to three anticodons Base stacking in the anticodon assures that bases 2 and 3 of the anticodon will follow Watson-Crick rules. Base 1 can wobble
  • 20. Depending on base 1 it can pair with 1,2 or 3 bases
    • If the wobble base is U, it can H bond to A (expected) or G (unexpected).
    • If the wobble base is G, it can H bond to C (expected) or U (unexpected).
    • A and C form only the expected base pairs.
    • Inosine in the wobble position can H bond to A, C, and U.
  • 21. Thus 31 tRNAs can read 61 codons
  • 22. Translation takes place in three stages
    • Initiation- once per protein it gets the system in motion
    • Elongation-repeated for each codon in the mRNA making a peptide bond
    • Termination-finishes and releases the newly synthesized protein
  • 23. Initiation A common mechanism
  • 24. Procaryotic initiation assembles the pre-translational complex
    • Mechanism is similar for eucaryotes and procaryotes [differences are important]
    • Components:
      • Small subunit containing a specific mRNA sequence(Shine-Dalgarno) which guides the mRNA into correct position for reading frame relative to the 16S rRNA
      • Proteinaceous initiation factors
      • Initiator AA-tRNA
      • mRNA(monocistronic for eucaryotes, polycistronic for procaryotes)
  • 25. Differences in the process provide the basis for specific antibiotic action
    • Procaryotes
    • 30S ribosomal subunit
    • IF-1, IF-2, IF-3
    • fMet-tRNA MetF
    • GTP
    • Eucaryotes
    • 40S ribosomal subunit
    • eIF-2a, eIF-3, eIF-4a, eIF-4c, eIF-4e, eIF-4g, eIF-5, eIF-6
    • Met-tRNA Meti
    • GTP
  • 26. Initiation Factors have Specific Roles
    • Procaryotes
    • IF-3 binds 30S
    • IF-2 binds initiator AA-tRNA
    • IF-1 GTP hydrolysis
    • RNA:RNA base pairing indexes mRNA
    • Eucaryotes
    • eIF-2 itRNA Binding
    • eIF-3 40S anti-association
    • eIF-4g binds mRNA
    • eIF-4e cap binding
    • eIF-4a mRNA indexing
    • eIF-4c ribosomal i AA-tRNA
    • eIF-5 GTP hydrolysis
    • eIF-6 60S anti-association
  • 27. In procaryotes IFs 1,2 and 3 are needed to begin IF-3 is an 30S anti-association factor IF-2 binds and preps initiator AA-tRNA IF-1 is a GTP binding hydrolase These allow the association of the 30S, Met-tRNA metF and factors to bind in preparation for mRNA and 50S binding
  • 28.  
  • 29. Initiation is similar for pro- and eucaryotes Devlin 6.7
  • 30. Intiation occurs once per translational cycle
    • The preinitiation complex is formed on the small subunit
    • GTP is bound to initiation factors. GTP hydrolysis carries out a process and drives a conformational change which leads to the next activity
    • The mRNA is indexed to appropriate AUG codon
    • The mRNA is locked into the cleft between small and large subunits
    • Addition of the large subunit creates A , P and E sites on the ribosome
    • The initiator AA-tRNA is locked into the P site
  • 31. Devlin 6.7 Eucaryotic initiation is similar
  • 32. Eucaryotic initiation has differences
    • The mRNA is not indexed by the ribosomal rRNA
    • Cap binding is essential for initiation
    • The initiation complex does not use formylated methionine but does use a specific initiator Methionine-specific aminoacyl-tRNA for initiation
    • Protein synthesis occurs at the first AUG
  • 33. The association of all initation components creates a 70S ribosome with initiator tRNA in the P site
  • 34. Elongation A repeated experience
  • 35. Once initiation is complete the ribosome is ready for elongation
    • Elongation is the process of addition of amino acids to the C-terminus of the growing polypeptide
    • Synthesis of each peptide bond requires energy derived from the cleavage of the AA-tRNA ester bond. The ribosomal enzyme doing this is called Peptidyl Transferase
    • Elongation is repeated as many times as there are codons in the mRNA
  • 36. As is the case for initiator tRNA all aminoacyl-RNAs must be present for protein synthesis
    • Good nutrition requires that all amino acids must be available in the diet
    • For procaryotes most can be synthesized at an expense of energy
    • Eucaryotes are able to form some but not all amino acids, thus some are essential in the diet
  • 37. Pools of AA-tRNAs are formed by the Aminoacyl-tRNA Synthetases
    • AA-tRNA synthetases recognize 2 o and 3 o structure near the T  C,D, and extra loop and the acceptor stem on the L-shaped tRNA molecules
    • AA-tRNA synthetases recognize 3-dimensional structure and functional groups of the amino acids
    • As we saw earlier, AA-tRNA synthetases use ATP to form a high-energy ester bond at the 3’OH on the tRNA
  • 38. Once an AA x -tRNA x is formed, the Amino Acid becomes Invisible
    • The ribosome mediates the association between codons on the mRNA and anticodons on the tRNA
    • Specificity of AA incorporation depends upon the anticodon of the tRNA
    • Whatever is on the tRNA will be incorporated into the protein at the site
    • The tRNA adapts the AA to the specified site
  • 39. Following Initiation the Ribosome has 3 functional sites
    • A site-aminoacyl-tRNA binding site [incoming AA-tRNA]
    • P site-peptidyl-tRNA binding site[attachment of growing polypeptide site
    • E site-spent tRNA exit site
    A P E
  • 40. Each elongation cycle requires elongation factors
    • Procaryotes
    • EF-T AA-tRNA binding to A site, GTP binding/hydrolysis
    • EF-G GTP hydrolysis, ribosomal conformational change, index peptidyl-tRNA to P site, expulsion of spent tRNA from E site
    • Eucaryotes
    • EF-1 AA-tRNA binding to A site, GTP binding/hydrolysis
    • EF-2 GTP hydrolysis, ribosomal conformational change, index peptidyl-tRNA to P site, expulsion of tRNA from E site
  • 41. In procaryotes, under the control of EF-T, a second aminoacyl-tRNA is bound in the A site
  • 42. Devlin 6.8 In eucaryotes similar events occur
  • 43. Hydrolysis of bound GTP changes the conformation of the Ribosome
    • The conformational change locks the aminoacyl-tRNA into the A site
    • Brings the anticodon in close approximation with the codon
    • Prepares the ribosome for binding of another GTP binding hydrolase EF-G
  • 44. The energy for peptide bond formation derives from the aminoacyl-tRNA ester bond
    • Cleaving the ester bond provides energy for the formation of a peptide bond
    • Catalysis is most likely provided by an integral 50/60S ribozyme, the peptidyl transferase, an RNA-containing enzyme(parts of the 23s rRNA) in the ribosome
    • Upon synthesis of the peptide bond, the growing polypeptide chain is linked to the tRNA on the P site
  • 45. Peptidyl transferase synthesizes a peptide bond forming a dipeptide
  • 46. The peptide bond is formed using the energy derived from the aminoacyl ester bond and moves the peptide to the A site-bound Aminoacyl-tRNA
  • 47. Following peptide bond formation a new factor drives translocation of the peptide
    • Specificity provided by antiparallel codon-anticodon pairing between A site-bound AA-tRNA and mRNA
    • Translocation driven by EF-G/2 catalyzed GTP hydrolysis-derived conformational change
    • mRNA ratchets 5’-3’ through the ribosome moving the C:AC from A to P site by the action of a translocase
    • Time to find AA-tRNA is important to fidelity
  • 48. EF-G mediated GTP hydrolysis translocates the mRNA and peptidyl-tRNA expelling the spent tRNA
  • 49. Devlin 6.8 Eucaryotic translocation is similar
  • 50. This elongation cycle is repeated as many times as there are codons
  • 51. EF-T/1 mediated binding is followed peptide bond formation and EF-G/2 mediated peptidyl transfer
  • 52. Eucaryotic elongation is similar to the procaryotic process
  • 53. The growing polypeptide chain remains attached to the last tRNA added The next codon is UAG
  • 54. When a termination codon occupies the the A site no AA-tRNA will bind
    • Termination codons work because no tRNA has a complementary anticodon
    • When the site is occupied by UAA, UAG or UGA time passes without A site occupancy by an AA-tRNA
    • This allows binding of release or termination factors, proteins[size and shape of tRNAs] that change the activity of peptidyl transferase to a peptidyl hydrolase and thus mediate release of the polypeptide from the ribosome
  • 55. Termination requires proteinaceous termination factors
    • Procaryotes
    • Release Factor GTP binding, GTP hydrolysis, conformational change, cleavage of 3’-peptidyl- CCA OH ester linkage, expulsion of polpeptide, dissociation of 30S and 50S subunits
    • Eucaryotes
    • eRF GTP binding, GTP hydrolysis, conformational change, cleavage of 3’-peptidyl-CCA OH ester linkage, expulsion of polypeptide, dissociation of 40S and 60S subunits
  • 56. Devlin 6.10
  • 57. Posttranslational modification
    • Some newly made proteins, both prokaryotic and eukaryotic, do not attain their final biologically active conformation until they have been altered by one or more processing reactions called posttranslational modification
  • 58. Different ways of modification
    • Amino-Terminal and Carboxyl-Terminal Modification
    • Loss of Signal Sequence: the 15 to 30 residues at the amino-terminal end of some proteins play a role in directing the protein to its ultimate destination in the cell. Such signal sequences are ultimately removed by peptidase
    • Modification of Individual Amino Acids:
    • The hydroxyl groups of Ser, Thr, and Tyr can be phosphorylated , some others can be carboxylated and methylated.
  • 59.
    • Attachment of Carbohydrate Side Chains: such as glycoproteins, N-linked oligosaccharides (e.g. Asn), O-linked-oligosaccharides(e.g. Ser or Thr)
    • Addition of Isoprenyl Groups
    • Addition of Prosthetic Groups:Two examples are the biotin molecule of acetyl-CoA carboxylase and the heme group of hemoglobin or cytochrome c.
  • 60.
    • Proteolytic Processing: proinsulin and proteases such as chymotrypsinogen and trypsinogen(zymogen activation)
    • Formation of Disulfide Cross-link: intrachain or interchain disulfide bridges between Cys residues
  • 61. Because of differences in translation bacterial growth can be inhibited by antibiotics Devlin 6.8
  • 62. Eucaryotes can be targeted by microorganisms
    • Diptheria toxin carries out its effects by mediating a covalent modification of EF-2
    • NAD + + EF-2 ADP-Ribose-EF2 + Nicotinamide
    • ADP-ribosylated EF-2 is ineffective, thus interrupting polypeptide synthesis
    • An effective anti-peoplotic
  • 63. What’s Next?
    • Once made can proteins be modified?
    • How is protein folding effected?
    • How are proteins exported after synthesis?
    • How is protein turnover controlled?
    I can hardly wait!