My protein biosynthesis


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My protein biosynthesis

  1. 1. Protein Synthesis Dr. Gangadhar Chatterjee
  2. 2. Protein synthesis or Translation
  3. 3. RNA Eukaryotic cells have four major classes of RNA: ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA). • The first three are involved in protein synthesis • snRNA is involved in mRNA splicing.
  4. 4. Generalized tRNA
  5. 5. Modified Bases Found in tRNAs = UH2
  6. 6. The anticodon region typically consists of a sequence of seven nucleotides: variable (N), modified purine ((Pu*), X, Y, Z, and two pyrimidines (Py) in the 3′ to 5′ direction.
  7. 7. mRNA
  8. 8. Ribosome & rRNA
  9. 9. THE GENETIC CODE • dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. • usually presented in the messenger RNA language of adenine (A), guanine (G), cytosine (C), and uracil (U).
  10. 10. How to translate a codon:
  11. 11. Termination ("stop" or "nonsense") codons: • UAG • UGA • UAA • signals that synthesis of the peptide chain coded for by that mRNA is completed
  12. 12. Characteristics of the genetic code 1. Specificity:(unambiguous) specific codon always codes for the same amino acid. 2. Universality: specificity of the genetic code has been conserved from very early stages of evolution. 3. Redundancy or Degeneracy: a given amino acid may have more than one
  13. 13. 4. Nonoverlapping and commaless: read from a fixed starting point as a continuous sequence of bases, taken three at a time.
  15. 15. COMPONENTS REQUIRED FOR TRANSLATION A. Amino acids B. Transfer RNA (tRNA) 1. Amino acid attachment site:When a tRNA has a Covalently attached amino acid, it is said to be charged. 2. Anticodon:
  16. 16. C. Aminoacyl-tRNA synthetases • The charging reactions have an error rate of less than 10–4 and so are extremely accurate. • The synthetases have a "proofreading" or "editing" activity that can remove mischarged amino acids from the tRNA molecule
  17. 17. D. Messenger RNA (mRNA) E. Functionally competent ribosomes 1. Ribosomal RNA (rRNA) 2. Ribosomal proteins
  18. 18. 3. A, P, and E sites on the ribosome
  19. 19. In addition to the APE sites there is an mRNA binding groove that holds onto the message being translated
  20. 20. 4. Cellular location of ribosomes: • In eukaryotic cells, the ribosomes either "free" in the cytosol or are in close association with the endoplasmic reticulum F. Protein factors: Initiation, elongation, and termination (or release) factors G. ATP and GTP are required as sources of energy • Cleavage of four high-energy bonds is required for the addition of one amino
  21. 21. CODON RECOGNITION BY tRNA A. Antiparallel binding between codon and anticodon B. Wobble hypothesis: "wobble" allows a single tRNA to recognize more
  22. 22. STEPS IN PROTEIN SYNTHESIS • The mRNA is translated from its 5'-end to its 3'-end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. • Prokaryotic mRNA: POLYCISTRONIC • Eukaryotic mRNA: MONOCISTRONIC • three separate steps: Initiation Elongation
  24. 24. Initiation • Assembly of components of translation system before peptide bond formation. • Includes two ribosomal subunit mRNA to be translated aminoacyl-tRNA specified by 1st codon of mRNA GTP initiation factors ( IF-1,IF-2, IF-3)
  25. 25. 1. Shine-Dalgarno sequence: 2. Initiation codon:
  26. 26. Elongation • ribosome moves from the 5'-end to the 3'-end of the mRNA • Delivery of the aminoacyltRNA whose codon appears next on the mRNA template in the ribosomal A site is facilitated by elongation factors EF-Tu and EF-Ts and requires GTP • formation of the peptide bonds is catalyzed by peptidyltransferase,
  27. 27. Termination • occurs when one of the three termination codons moves into the A site. • recognized by release factors: • RF-1, which recognizes the termination codons UAA and UAG • RF-2, which recognizes UGA and UAA RF-3,which binds GTP and stimulates the activity of RF-1 and RF-2.
  30. 30. Initiation • Involves Several Protein-RNA Complexes tRNA, rRNA, mRNA, and at least ten eukaryotic initiation factors (eIFs), some of which have multiple (three to eight)subunits, GTP, ATP, and amino acids. • four steps: (1) Dissociation of the ribosome into its 40S and 60S subunits; (2) 40S preinitiation complex formation (3) Formation of 43S initiation complex and (4) combination with the 60S ribosomal subunit to form the 80S initiation complex.
  31. 31. RIBOSOMAL DISSOCIATION initiation factors, eIF-3 and eIF-1A, bind to the newly dissociated 40S ribosomal subunit delays reassociation with the 60S subunit and allows other translation initiation factors to associate with the 40S subunit.
  32. 32. FORMATION OF THE 43S PREINITIATION COMPLEX • first step involves the binding of GTP by eIF-2. • binary complex then binds to mettRNAi, a tRNA specifically involved in binding to the initiation codon AUG. • ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex. • stabilized by association with eIF-3 and eIF-1A.
  33. 33. eIF-2 is one of two control points for protein synthesis initiation • eIF-2 consists of α, β, and γ subunits. • eIF-2α is phosphorylated (on serine 51) by at least four different protein kinases (HCR, PKR, PERK, and GCN2) • Kinases are activated under stress and lo energy level of cell. • Such conditions include amino acid and glucose starvation, virus infection, misfolded proteins, serum deprivation, hyperosmolality, and heat
  34. 34. FORMATION OF THE 43S INITIATION COMPLEX • methyl-guanosyl triphosphate cap facilitates the binding of mRNA to the 43S preinitiation complex. • A cap binding protein complex, eIF-4F (4F) binds to the cap through the 4E protein. • association of the 43S preinitiation complex with the mRNA cap, the complex scans the mRNA for a suitable initiation codon. • this is the 5′-most AUG, but the precise initiation codon is determined by so-called Kozak consensus sequences that surround the AUG:
  35. 35. The Regulation of eIF-4E Controls the Rate of Initiation • 4E is responsible for recognition of the mRNA cap structure, which is a ratelimiting step in translation • Insulin and mitogenic growth factors result in the phosphorylation of 4E on ser 209 (or thr 210). • Phosphorylated 4E binds to the cap much more avidly than does th nonphosphorylated form, thus
  36. 36. Activation of eIF-4E by insulin and formation of the cap binding eIF-4F complex
  37. 37. There is also general control of translational initiatio ie. all transcripts of the cell are effected (though the relativ effect differs between specific mRNAs) Global downregulation or upregulation can occur in respo to various stimuli .Most common are 1) Nutrient availability low nutrient (amino acids/carbohydrate) downregulates translation 2) Growth factor signals. stimulation of cell division upregulates translation
  38. 38. ROLE OF THE POLY(A) TAIL IN INITIATION • stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions • Pab1p, bound to the poly(A) tail, interacts with eIF-4G which in turn binds to eIF-4E that is bound to the cap structure. • possible that a circular structure is formed and that this helps direct the
  39. 39. FORMATION OF THE 80S INITIATION COMPLEX • binding of the 60S ribosomal subunit to the 48S initiation complex involves hydrolysis of the GTP • results in release of the initiation factors bound to the 48S initiation complex (these factors then are recycled) and the rapid association of the 40S and 60S subunits to form the 80S ribosome. • ready for the elongation cycle to
  40. 40. Elongation • steps are (1) binding of aminoacyl-tRNA to the A site (2) peptide bond formation (3) translocation
  41. 41. BINDING OF AMINOACYLTRNA TO THE A SITE • Elongation factor EF1A forms a ternary complex with GTP and the entering aminoacyl-tRNA • complex then allows the aminoacyltRNA to enterthe A site with the release of EF1A•GDP and phosphate
  42. 42. PEPTIDE BOND FORMATION • α-amino group of the new aminoacyltRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site • reaction is catalyzed by peptidyltransferase • The reaction results in attachment of the growing peptide chain to the tRNA in
  43. 43. TRANSLOCATION • elongation factor 2 (EF2) binds to and displaces the peptidyl tRNA from the A site to the P site • the deacylated tRNA is on the E site, from which it leaves the ribosome • effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA-EF1AGTP.
  44. 44. peptide elongation process
  45. 45. Termination Occurs When a Stop Codon Is Recognized • terminating codon of mRNA (UAA, UAG, UGA) appears in the A site • there is no tRNA with an anticodon capable of recognizing such a termination signal • Releasing factor RF1 recognizes that a stop codon resides in the A site
  46. 46. Release Factor is a molecular mimic of a tRNA eRF1 tRNA
  47. 47. Polysomes Are Assemblies of Ribosomes • Multiple ribosomes on the same mRNA molecule form a polyribosome or “polysome.”
  48. 48. Translation - animation
  49. 49. The Machinery of Protein Synthesis Can Respond to Environmental Threats • 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.
  50. 50. Regulation by iron (Fe):
  51. 51. Modification of the translation machinery is a commo feature of viral life cycles e.g. Picornaviruses Polio virus Encephalomyocarditis virus Picornaviruses have single stranded RNA genomes.
  52. 52. Poliovirus Life Cycle
  53. 53. 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
  54. 54. The consequence of this cleavage is that translation of cellular mRNAs stops translated due to the presence of But…the viral RNA is still an internal ribosomal entry site (IRES). This acts like a bacterial initiation site to allow Cap-independent initiation from internal AUG codons a reaction that requires 4G but not 4E. What is X?
  55. 55. “X” is not a protein, rather it is a structure in the mRNA itself that can bind to the remaining fragment of eIF4G
  56. 56. 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 expresse during this period bypass this control by using IRES elements
  57. 57. ENERGY requirement for Translation ATP  One ATP in formation of initiation complex  One ATP in formation of 48S initiation complexbinding of eIF-4A and eIF-4B GTP  Binding with eIF-2 for forming binary complex  For formation of 80S initiation complex  Binding of aminoacyl tRNA at A site  EF2-GTP complex hydrolyzed to give energy for translocation  RF-3 with bound GTP required for termination TOTAL= 4 ATP + 5 GTP
  58. 58. POSTTRANSLATIONAL MODIFICATION A. Trimming Portions of the protein chain must be removed by specialized endoproteases, resulting in the release of an active molecule B. Covalent alterations 1. Phosphorylation • Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less
  59. 59. 2. Glycosylation proteins that are destined to become part of a plasma membrane or lysosome or to be secreted from the cell have carbohydrate chains attached to serine or threonine hydroxyl groups (O-linked) or the amide nitrogen of asparagine (N-linked). • 3. Hydroxylation: Proline and lysine residues of the a-chains of collagen are extensively hydroxylated in the endoplasmic reticulum Other covalent modifications:
  60. 60. THANK YOU