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Molecular biology of protein expression

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Molecular biology of protein expression

  1. 1. Molecular Biology of Protein Expression Protein Synthesis (Central Dogma) DNA → mRNA → Protein By Dr.Surendra Nadh Benerji.D M.Sc, M.Phil, Ph.D. (Biotechnology), Acharya Nagarjuna University E Mail: dsnbenerji@gmail.com, Mobile: 9247407189
  2. 2. Protein Synthesis-Pathway
  3. 3. Most biological activities are carried out by proteins, and their synthesis is at the heart of cellular function RNA plays 3 Distinct & Important Roles: tRNA – the key or adapter to the genetic code. rRNA – Provides scaffold & catalysis. mRNA – The intermediary between gene and protein
  4. 4. Protein SynthesisProtein Synthesis • The production (synthesis) of proteinsproteins. • 3 phases3 phases: 1.1. TranscriptionTranscription 2.2. RNA processingRNA processing 3.3. TranslationTranslation • Remember:Remember: DNADNA →→ RNARNA →→ ProteinProtein
  5. 5. TranscriptionTranscription TranslationTranslation DNA mRNA Ribosome Protein Prokaryotic CellProkaryotic Cell DNADNA →→ RNARNA →→ ProteinProtein
  6. 6. DNADNA →→ RNARNA →→ ProteinProtein Nuclear membrane TranscriptionTranscription RNA ProcessingRNA Processing TranslationTranslation DNA Pre-mRNA mRNA Ribosome Protein EukaryoticEukaryotic CellCell
  7. 7. RNARNA differs from DNADNA 1. RNARNA has a sugar ribosesugar ribose DNADNA has a sugar deoxyribosesugar deoxyribose 2. RNARNA contains uracil (U)uracil (U) DNADNA has thymine (T)thymine (T) 3. RNARNA molecule is single-strandedsingle-stranded DNADNA is double-strandeddouble-stranded
  8. 8. Types of RNATypes of RNA • Three types ofThree types of RNARNA: A.A. messenger RNA (mRNA)messenger RNA (mRNA) B.B. transfer RNA (tRNA)transfer RNA (tRNA) C.C. ribosome RNA (rRNA)ribosome RNA (rRNA) • Remember: all produced in theRemember: all produced in the NucleusNucleus
  9. 9. Messenger RNA (mRNA)Messenger RNA (mRNA) • Carries the information for a specific ProteinProtein. • Made up of 500 to 1000 NucleotidesNucleotides long. • Made up of codonscodons (sequence of three bases: AUG - methionine). • Each codoncodon, is specific for an Amino acidAmino acid.
  10. 10. Messenger RNAMessenger RNA (mRNA)(mRNA) methionine glycine serine isoleucine glycine alanine stop codon proteinprotein A U G G G C U C C A U C G G C G C A U A AmRNAmRNA start codon Primary structure of a proteinPrimary structure of a protein aa1 aa2 aa3 aa4 aa5 aa6 peptide bonds codon 2 codon 3 codon 4 codon 5 codon 6 codon 7codon 1
  11. 11. Transfer RNA (tRNA)Transfer RNA (tRNA) • Made up of 75 to 80 nucleotides long. • Picks up the appropriate amino acidamino acid floating in the cytoplasm (amino acid activating enzymeamino acid activating enzyme) • Transports amino acidsamino acids to the mRNAmRNA. • Have anticodonsanticodons that are complementary to mRNAmRNA codonscodons. • Recognizes the appropriate codonscodons on the mRNAmRNA and bonds to them with H-bonds.
  12. 12. tRNA Secondary Structure ‘pseudo uridine’ Added post- transcriptionally - many modified bases (7-15/molecule) - modifications happen to standard bases AFTER transcription Why the modifications? - may stabilize 3D structure - may help recognition by synthetase - ‘Wobble’ pairing aa Anticodon loop
  13. 13. tRNA Synthetases 1. Associates correct amino acid  tRNA pair 2. Generates a high energy “aminoacyl” covalent bond between the two (cleavage of this bond releases energy and drives protein synthesis) - Typically one tRNA synthetase exists for each tRNA Function: - if an aa has a unique side group, the enzymes specificity can be achieved by a binding pocket alone. (i.e., Methionine) - however, with a.a.’s with similar side-chains (i.e., Valine & Isoleucine) the synthetases have editing function tRNA synthetases are highly selective:
  14. 14. General strategy for Synthetase’s editing function 1. aa binding site excludes amino acids which are too large 2. Editing Site is hydrolytic (cleaves) - excludes amino acids of the correct size - hydrolyzes aa-tRNA linkage of amino acids small enough to enter “Double Sieve” mechanism
  15. 15. Figure 27.10 Formation of aminoacyl tRNA (“charged tRNA”) by the aminoacyl tRNA synthetases Formation of aminoacyl tRNA (“charged tRNA”) by the aminoacyl tRNA synthetases
  16. 16. Ribosomal RNA (rRNA)Ribosomal RNA (rRNA) • Made up of rRNArRNA is 100 to 3000 nucleotides long. • Important structural component of a ribosome.ribosome. • Associates with proteinsproteins to form ribosomes.ribosomes.
  17. 17. Structure of 16S rRNA
  18. 18. Structure of 23 S rRNA
  19. 19. Secondary structure of ribosomal RNA (rRNA) - very large & complex -various ribosomal proteins are associated with discrete areas of the RNA - initially determined by a variety of biochemical methods
  20. 20. RibosomesRibosomes • Large and small subunits.Large and small subunits. • Composed of rRNA (40%)rRNA (40%) and proteins (60%).proteins (60%). • Both units come together and help bind the mRNAmRNA and tRNA.tRNA. • Two sites forTwo sites for tRNAtRNA a. P siteP site (first and last tRNA will attachtRNA will attach) b. A siteA site
  21. 21. The Ribosome – Structure and Assembly “Large” 50S subunit “Small” 30S subunit tRNA (3 bound) Electron density map of a prokaryotic ribosome
  22. 22. Ribosome Structure Prokaryotic Ribosome Eukaryotic Ribosome 70S = 2.8 million daltons 80S = 4.5 million daltons 23S rRNA 50 S 5 S rRNA 34 proteins 30 S 16 S rRNA 21 proteins 28S rRNA 5.8S rRNA 60S 5S rRNA 50 proteins 40S 18S rRNA 35 proteins
  23. 23. Overview of the Ribosome Structure
  24. 24. Overview of Protein Synthesis 1. Components required mRNA tRNAs for 20 AA’s Ribosomes -surface for binding - peptidyl transferase - control of error rate Bacterial 5’ AUG---------stop AUG -----stop 3’ Eukaryotic Cap AUG--------stop 3’ AAAAAAA
  25. 25. TranslationTranslation • Synthesis of proteinsproteins in the cytoplasmcytoplasm • Involves the following:Involves the following: 1. mRNA (codons)mRNA (codons) 2. tRNA (anticodons)tRNA (anticodons) 3. rRNArRNA 4. ribosomesribosomes 5. Amino acidsAmino acids
  26. 26. TranslationTranslation • Translation has three parts: 1. InitiationInitiation: Start Codon (AUG) 2. ElongationElongation: mRNA, tRNA, EF’s 3. TerminationTermination: Stop Codon (UAG)
  27. 27. TranslationTranslation P Site A Site Large subunit Small subunit mRNAmRNA A U G C U A C U U C G
  28. 28. Role of initiation factors in bacterial system • Initiation factors required for correct selection of reading frame • All three initiation factors are required: IF1, IF2, IF3 • These are soluble proteins that interact transiently with ribosome • Binding of fmet - tRNA requires initiation factor 2 and GTP Properties of bacterial initiation factors MW Binds GTP Abundance relative to ribosome IF1 9,000 No 1/7 IF2 120,000 Yes 1/7 IF3 32,000 No 1/7
  29. 29. An empty ribosome is shown on the left and a loaded ribosome on the right. Three major RNA-binding sites on a ribosome
  30. 30. Initiation Figure 27.20 3 key proteins involved, these are known as Initiation Factors (IFs) IF 1 & 3 aid in the disassociation of the 30S & 50S subunits. IF 2 - a GTPase which ‘presents’ the ‘initiator tRNA’
  31. 31. Most proteins begin on AUG (GUG & UUG less frequently) The first amino acid incorporated (in prokaryotes) is Methionine. - Methionine of an initiator tRNA is modified: Formylated HC-NH-CH-COOH O R (amino acid side chain) How does the ribosome know where to begin protein synthesis? - Note: f-Met looks like a peptide! - Normal tRNAmet only recognizes AUG while tRNAf-Met will recognize AUG, GUG & UUG codons - f-Met only used at initiation
  32. 32. Shine-Delgarno Sequence
  33. 33. InitiationInitiation mRNAmRNA A U G C U A C U U C G 2-tRNA G aa2 A U A 1-tRNA U A C aa1 anticodon hydrogen bonds codon
  34. 34. Initiation of Translation in Eukaryotes AAAAAAAAACap AUG----------------Stop 5’UTR 3’UTR Important points: • No direct binding between mRNA and rRNA • Small ribosome subunit binds directly to cap – requires specific initiation factor – eIF4e • Other initiation factors can unwind double stranded regions in the mRNA – eIF4 group • Small subunit scans mRNA till it finds correct AUG • Correct AUG is embedded in preferred sequence GccAccAUGG
  35. 35. Eukaryotic Initiation Factors Factor MW Subunits Function eIF1 15,000 Pleiotropic effects eIF2 130,000 36,000 38,000 55,000 Binding met-tRNA & GTP eIF2B 270,000 26,000 39,000 58,000 GTP-exchange on EIF2 67,000 82,000 eIF3 550,000 Ribosome dissoc, mRNA binding eIF4A 46,000 ATP-dependent unwinding eIF4B 80,000 mRNA binding eIF4C 17,000 Ribosome dissociation eIF4D 15,000 Subunit joining eIF4 F 290,000 24,000 46,000 220,000 Cap-recognition, mRNA binding eIF5 150,000 Release of bound factors eIF6 25,000 Ribosome dissociation
  36. 36. Initiation phase in Eukaryotes
  37. 37. mRNAmRNA A U G C U A C U U C G 1-tRNA 2-tRNA U A C G aa1 aa2 A U A anticodon hydrogen bonds codon Peptide bond 3-tRNA G A A aa3 ElongationElongation
  38. 38. Peptide Bond Formation: - The formation of the peptide bond is catalyzed by by the rRNA in the 50S large subunit - After formation of the peptide bond the ribosome is ‘translocated’ or moved down the RNA - The new tRNA which now has the nascent peptide is now moved to the P site - The tRNA that was previously attached to the peptide is then moved to the E site, then released
  39. 39. mRNAmRNA A U G C U A C U U C G 1-tRNA 2-tRNA U A C G aa1 aa2 A U A peptide bond 3-tRNA G A A aa3 Ribosomes move over one codon (leaves)
  40. 40. Translocation and peptidyl transferase PT
  41. 41. mRNAmRNA A U G C U A C U U C G 2-tRNA G aa1 aa2 A U A peptide bonds 3-tRNA G A A aa3 4-tRNA G C U aa4 A C U
  42. 42. mRNAmRNA A U G C U A C U U C G 2-tRNA G aa1 aa2 A U A Peptide bonds 3-tRNA G A A aa3 4-tRNA G C U aa4 A C U (leaves) Ribosomes move over one codon
  43. 43. mRNAmRNA G C U A C U U C G aa1 aa2 A Peptide bonds 3-tRNA G A A aa3 4-tRNA G C U aa4 A C U U G A 5-tRNA aa5
  44. 44. mRNAmRNA G C U A C U U C G aa1 aa2 A Peptide bonds 3-tRNA G A A aa3 4-tRNA G C U aa4 A C U U G A 5-tRNA aa5 Ribosomes move over one codon
  45. 45. VVP Fig. 26-28
  46. 46. Ribosomal binding sites in the elongation cycle.
  47. 47. mRNAmRNA A C A U G U aa1 aa2 U primaryprimary structurestructure of a proteinof a protein aa3 200-tRNA aa4 U A G aa5 C U aa200 aa199 terminatorterminator or stopor stop codoncodon TerminationTermination
  48. 48. End ProductEnd Product • The end products of protein synthesis is a primary structure of a proteinprimary structure of a protein. • A sequence of amino acidamino acid bonded together by peptide bondspeptide bonds. aa1 aa2 aa3 aa4 aa5 aa200 aa199
  49. 49. PolyribosomePolyribosome • Groups of ribosomes reading same mRNAmRNA simultaneously producing many proteinsproteins (polypeptides).(polypeptides). incoming large subunit incoming small subunit polypeptidepolypeptide mRNAmRNA 1 2 3 4 5 6 7
  50. 50. Question:Question: • The anticodonThe anticodon UACUAC belongs to abelongs to a tRNAtRNA that recognizes and binds to athat recognizes and binds to a particularparticular amino acidamino acid.. • What would be theWhat would be the DNA base codeDNA base code for thisfor this amino acid?amino acid? • What is Reverse Central Dogma?What is Reverse Central Dogma? • AnswerAnswer • tRNA - UAC (anticodon)tRNA - UAC (anticodon) • mRNAmRNA - AUG (codon)- AUG (codon) • DNADNA - TAC- TAC • mRNA →cDNA →Protein (TAMINISM)mRNA →cDNA →Protein (TAMINISM)
  51. 51. The Action of Antimicrobial Drugs Figure 20.4
  52. 52. Summary of Prokaryotic vs. Eukaryotic Translation - General processes are very similar - Eukaryotic ribosomes are larger. 4.2 MDa vs 2.7 MDa - In Eukaryotes, special MetinitiatortRNA is NOT formylated - IMPORTANT: No eukaryotic. Shine-Delgarno sequence. Initiation occurs at the first start codon after 5’-cap - Additional initiation and Elongation Factors required in Eukaryotes
  53. 53. Thank You Dr.D.Surendra Nadh Benerji. M.Sc, M.Phil, Ph.D.(Biotechnology), E Mail: dsnbenerji@gmail.com Mobile: +91 9247407189 Analytical R&D

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