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Biochemistry

Biochemistry. Chen Yonggang Zhejiang University Schools of Medicine. Translation, making protein following nucleic acid directions. Bodega Bay, Sonoma County. Breakfast at The Tides, Bodega Bay. The process of using base pairing language to create a protein is termed Translation.

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Biochemistry

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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 43= 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 PROCARYOTIC EUCARYOTIC 80 S 40S 60S RNA 5S, 5.8S,18S,28S PROTEINS 84 70 S 30S 50S RNA 5S, 16S, 23S PROTEINS 55 Small subunit Large Subunit

  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 3’ 5’

  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 AAx + tRNAx + ATP AAx-tRNAx + 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 structuresFigure 2.59

  17. Functional Sites of tRNAsFigure 2.58 • CCAOH 3’ Acceptor Sequence • Amino acid acceptor stem • D stem and loop • Extra loop • Anticodon stem and loop • Anticodon • TyC 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. Procaryotes 30S ribosomal subunit IF-1, IF-2, IF-3 fMet-tRNAMetF GTP Eucaryotes 40S ribosomal subunit eIF-2a, eIF-3, eIF-4a, eIF-4c, eIF-4e, eIF-4g, eIF-5, eIF-6 Met-tRNAMeti GTP Differences in the process provide the basis for specific antibiotic action

  26. 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 Initiation Factors have Specific Roles

  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. Initiation is similar for pro- and eucaryotesDevlin 6.7

  29. 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

  30. Eucaryotic initiation is similar Devlin 6.7

  31. Eucaryotic initiation has differences • The mRNA is not indexed by the ribosomal rRNA (eukaryotic mRNAs do not have Shine-Dalgarno sequence) • 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

  32. The association of all initiation components creates a 70S ribosome with initiator tRNA in the P site

  33. Elongation A repeated experience

  34. 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

  35. 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

  36. Pools of AA-tRNAs are formed by the Aminoacyl-tRNA Synthetases • AA-tRNA synthetases recognize 2o and 3o structure near the TyC,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

  37. Once an AAx-tRNAx 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

  38. Following Initiation the Ribosome has 3 functional sites • A site-aminoacyl-tRNA binding site [incoming AA-tRNA, only initiator AA-tRNA goes to the P site] • P site-peptidyl-tRNA binding site[attachment of growing polypeptide site • E site-spent tRNA exit site E P A

  39. 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 Each elongation cycle requires elongation factors

  40. In procaryotes, under the control of EF-T, a second aminoacyl-tRNA is bound in the A site

  41. In eucaryotes similar events occur Devlin 6.8

  42. 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

  43. 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

  44. Peptidyl transferase synthesizes a peptide bond forming a dipeptide

  45. 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

  46. 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(codon):AC(anticodon) from A to P site by the action of a translocase • Time to find AA-tRNA is important to fidelity

  47. EF-G mediated GTP hydrolysis translocates the mRNA and peptidyl-tRNA expelling the spent tRNA

  48. Eucaryotic translocation is similar Devlin 6.8

  49. This elongation cycle is repeated as many times as there are codons

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