5 DNA RNA Protein Synthesis

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  • 1. DNA, RNA, and Protein Synthesis
  • 2. Importance and Structure of DNA: Deoxyribo-Nucleic Acid
    • Historical Review:
      • 1900’s – Morgan’s studies with fruit flies showed that genes were located on chromosomes and chromosomes consisted of protein and DNA
      • 1952- Hershey-Chase demonstrated that DNA (not protein) was the genetic material of a viral phage
  • 3. Figure 16.2a The Hershey-Chase experiment: phages
  • 4. Figure 16.2b The Hershey-Chase experiment
  • 5. Phages Infecting a bacterium
  • 6. Figure 16.1 Transformation of bacteria – Griffith (and later Avery, McCarty and MacLeod)
  • 7. The Structure of DNA
    • Nucleotide monomers:
      • Phosphate
      • Pentose Sugar (C5) – Deoxyribose Sugar
      • Organic Nitrogen Base :
        • Cytosine (C)
        • Adenine (A)
        • Guanine (G)
        • Thymine (T)
  • 8. Structure of DNA cont’
    • Polynucleotide chain with linkage via phosphates to next sugar, with nitrogen base away from backbone of
    • Phos-sugar-phos-sugar
    • Dehydration synthesis
  • 9. Beginning of the 1950’s several labs were studying the structure of DNA
    • Maurice Wilkins & Rosalind Franklin
      • X-ray crystallography: x-rays pass through pure DNA and diffraction of x-rays were then examined on film
    • James Watson and Francis Crick did not have the expertise of Franklin and were without proper photos until………
  • 10. Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA
  • 11. Watson and Crick
  • 12. April 1953 – Classical one page paper in Nature by Watson and Crick
    • A double helix – 2 polynucleotide strands
    • Sugar-phosphate chains of each strand are like the side ropes of a rope ladder
    • Pairs of nitrogen bases, one from each strand, form the rungs or steps
    • The ladder forms a twist every 10 bases (all from x-ray studies!)
  • 13. Figure 16.5 The double helix
  • 14. Internal Structure of DNA: Purine and pyridimine? REMEMBER X-RAY DATA
  • 15. Confirms Erwin Chargaff’s Rules
    • # of Adenine = to # of thymine
    • # of guanine equal to # of cytosine
    • This dictates the combinations of N-bases that form steps/rungs
    • Does not restrict the sequence of bases along each DNA strand
  • 16. Information storage in DNA
    • The 4 nitrogenous bases are the “alphabet” or code for all the traits the organism possesses
    • Different genes or traits vary the sequence and length of the bases
    • ATTTCGGAC vs. GGGATTCTAG vs. GATC
  • 17. Replication/Duplication of DNA
    • Due to complimentary base paring – one strand of DNA determines the sequence of the other strand
    • Therefore, each strand of double stranded DNA acts as a template
    • The double helix first unwinds – controlled by enzymes –and uses new nucleotides that are free in the nucleus to copy a complimentary strand off the original DNA strand
  • 18. Figure 16.7 A model for DNA replication: the basic concept (Layer 1)
  • 19. Figure 16.7 A model for DNA replication: the basic concept (Layer 2)
  • 20. Figure 16.7 A model for DNA replication: the basic concept (Layer 3)
  • 21. Figure 16.7 A model for DNA replication: the basic concept (Layer 4)
  • 22. Figure 16.8 Three alternative models of DNA replication
  • 23. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 1)
  • 24. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2)
  • 25. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 3)
  • 26. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)
  • 27. There are a series of enzymes that control DNA replication – enzymes which:
    • Uncoil the original double helix strand via a helicase
    • Single-strand binding protein keeps helix apart so replication can start
    • Prime an area to start replication – primase except it adds RNA nucleotides at first
    • Polymerase to join individual nucleotides (dehydration synthesis)
    • Ligases to join short segments
  • 28. DNA REPLICATION
  • 29. Figure 16.10 Origins of replication in eukaryotes
  • 30. Figure 16.11 Incorporation of a nucleotide into a DNA strand
  • 31.
    • The carbons of the deoxyribose sugar are numbered
    • #3 carbon attached to an -OH group
    • #5 carbon holds the phosphate molecule of that nucleotide
    • #3 ready to bond with another nucleotide to form a polynucleotide link (5’ to3’)
    • Notice complimentary strand in opposite direction (5’ to 3’)
    • DNA always grows 5’ to 3’ never 3’ to 5’
    Antiparallel Arrangement of Double Strands
  • 32. Definitions
    • Origins of Replication – where replication of the DNA molecule begins
      • Bacteria – circular DNA – 1 origin of replication (RF)
      • Eukaryotes – multiple origins of replication (ORFS)
        • ORF = Replication Fork
  • 33. More Definitions
    • DNA Polymerases – enzymes that catalyze DNA replication
    • Leading Strand – Synthesized continuously towards the replication fork by the DNA polymerase in one long fashion
    • Lagging Strand – Synthesized by short fragments away from the replication fork by the DNA polymerase
  • 34. Definitions Cont’
    • Ligase – combines (joins) short fragments
    • Primer – starts replication of DNA (in this case it’s RNA)
    • Primase – an enzyme that joins the RNA nucleotides to make the primer
    • Helicase – an enzyme that untwists the double helix at the replication fork
    • Nuclease – a DNA cutting enzyme
  • 35. DNA REPLICATION -VIDEO
  • 36. Figure 16.13 Synthesis of leading and lagging strands during DNA replication
  • 37. Figure 16.14 Priming DNA synthesis with RNA
  • 38. Figure 16.15 The main proteins of DNA replication and their functions
  • 39. Figure 16.16 A summary of DNA replication
  • 40. Figure 16.17 Nucleotide excision repair of DNA damage
  • 41. A PROBLEM!
    • The end of the leading strand was initiated with an RNA primer
      • Normally removed by other DNA polymerase
    • Removal of gaps by DNA Polymerase doesn’t work on lagging strand end
      • RNA primer removed with no replacement
      • A GAP!
      • SHORTER AND SHORTER FRAGMENTS?
  • 42. Prokaryotes have circular DNA – no problem at ends (there aren’t ANY!
    • Eukaryotes – have special terminal sequences of 6 nucleotides that repeat from 100-1000 times with no genes included
      • Telomers
    • Protect more internal gene materials from being eroded
    • Germ cells / sex cells have a special enzyme (telomerase) that actually restore shortened Telomers
    • Somatic cells – telomer continues to shorten and may play a role in aged cell death
    • Cancer cells
      • A telomerase prevents very short lengths
  • 43. Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes
  • 44. Ribonucleic Acid (RNA)
    • Structure of RNA
      • Nucleotide monomer
        • Phosphate
        • Pentose sugar = ribose (extra oxygen)
        • Nitrogenous base (A/G/C/U)
        • Single stranded
        • 3 types (mRNA, tRNA, rRNA)
  • 45. Synthesis of RNA - transcription
    • DNA acts as a template, but only one strand of DNA utilized at a given time
    • This exposed strand is controlled by specific enzymes that pair the DNA nucleotides with free RNA nucleotides which are also present in the nucleus
    • These RNA nucleotides form a single stranded RNA nucleic acid
    • DNA = ATTGGCT
    • RNA = UAACCGA
    • Short segments of DNA are transcribed at a time with start and stop messages
  • 46. Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 1)
  • 47. Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 2)
  • 48. Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 3)
  • 49. Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 4)
  • 50. Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 5)
  • 51. Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 1)
  • 52. Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 2)
  • 53. Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 3)
  • 54. Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 4)
  • 55. Figure 17.6 The stages of transcription: elongation
  • 56. Three types of RNA
    • mRNA : messenger RNA
      • Transcribed from a specific segment of DNA which represents a specific gene or genetic unit
    • tRNA : transfer RNA
      • Transcribed from different segments of DNA and their function is to find a specific amino acid in the cytoplasm and bring it to the mRNA
    • rRNA : ribosomal RNA
      • Transcribed at the nucleolus - with proteins function as the site of protein synthesis
  • 57. Three types of RNA
  • 58. Protein Synthesis = Translation
    • Ribosomes = sites of protein synthesis
      • 30% - 40% protein
      • 60% - 70% RNA (rRNA)
      • Assembled in nucleus and exported via nuclear pores
      • Antibiotics can paralyze bacterial ribosomes, but not eukaryotic ribosomes (not targeting them)
      • 2 ribosomal subunits –a large and a small
      • Small subunit has been used as a means of classifying different bacteria and different invertebrates (16S)
        • Eukaryotes – 18S
      • There are three sites on the ribosome that are involved in protein synthesis
  • 59. Ribosomes bring mRNA together with amino acid bearing tRNA’s
    • Three ribosomal sites
      • P Site (peptidyl-tRNA) holds the tRNA carrying the growing peptide chain after several amino acids have been added
      • A site (aminoacyl-tRNA) holds the next single amino acid to be added to the chain
      • E site (exit site) site where discharged tRNA minus amino acids leave ribosome
  • 60. Figure 17.15 Translation – the basic concept
  • 61. Preparation of Eukaryotic mRNA
    • RNA splicing- a cut and paste job to remove nucleotides from transcribed mRNA
      • 8000 nucleotides transcribed but the average gene contains 1200+ nucleotides
      • Long non-coding segments (introns) interspersed between coding segments (exons) expressed via amino acids
  • 62.  
  • 63. Figure 17.17 The initiation of translation
  • 64. Figure 17.18 The elongation cycle of translation
  • 65. Protein Synthesis (cont’) Initiation – elongation - termination
    • Starting at one end of the mRNA, the small ribosomal subunit associates with the mRNA and accepts the first tRNA with its activated amino acid attached = Initiation
    • tRNA associate with a triplet codon exposed on the mRNA – these are 3 nitrogenous bases that bond with 3 complementary bases exposed (anticodon) on the tRNA opposite the attached amino acid
    • Wobble
      • Aren’t 61 tRNAs, are 54tRNAs
  • 66. Figure 17.4 The dictionary of the genetic code
  • 67. Figure 17.3 The triplet code
  • 68. tRNA complexes with its amino acid in the cytoplasm using ATP – activated tRNA
    • The activated tRNA-amino acid complex moves towards the ribosomal area and finds a triplet codon exposed that is complementary to the anticodon of the tRNA
    • The first activated tRNA-amino acid, after its anticodon is bound to the mRNA codon, associates with the large ribosomal subunit which now joins the smaller subunit and the mRNA and the tRNA (TAKE A BREATH!)
  • 69. The first tRNA and its amino acid now occupy the P site of the large ribosomal subunit
    • Review – at this point the 2 part ribosome is assembled, the mRNA has started to be read, and one tRNA plus amino acid is occupying the P site
    • That means the adjacent A site is free to accept a second activated tRNA and its amino acid, but only if the anticodon of this tRNA matches the next three base pairs exposed (codon)
  • 70. Protein Synthesis (continued)
    • At this point, there are 2 tRNA-amino acid complexes adjacent to each other – Elongation involved one amino acid being added in a three step process:
      • Codon recognition – the mRNA codon in the A site matches with the anticodon of the tRNA –amino acid complex
      • Peptide bond formation between the new amino acid in the A site and the amino acid (later peptide) in the P site
      • Translocation
  • 71. Translocation – the ribosome moves the tRNA into the A site, and its attached peptide to the P site, as the previous tRNA from the P site moves to the E (Exit) site and leaves the ribosome
    • Review: once this process is under way, an activated tRNA with its amino acid finds an exposed codon in the A site, attaches via H-bonds, then forms a peptide bond with the polypeptide associated with the tRNA sitting in the adjacent P site. For a moment, the longer polypeptide chain is only attached to the tRNA in the A site. Now the entire ribosome shifts so that the………
  • 72. Yet More Protein Synthesis
    • The empty tRNA from the P site moves in to the E site and leaves the ribosome
    • As the tRNA with the polypeptide chain moves from the A site to the now empty P site ….exposing a new codon.
      • GUESS WHAT HAPPENS NEXT?!
  • 73. A question?
    • Every time a new codon is exposed in the A site, a specific tRNA-AA complex moves into the site. What originally determined this mRNA Codon?
  • 74. The Answer!
    • The original DNA that was transcribed
    • This elongation of 1 AA takes about 0.1 s
    • Termination – the above continues (dozens to hundreds or more AA added) until the STOP CODON is reached (codon at the end of the mRNA)
    • This codon does not have a matching tRNA anticodon so the tRNA-AA attaches in the A site and the tRNA moves to the E site and releases the polypeptide chain
  • 75. FINALLY - SUMMARY
    • The take home message:
      • At the ribosome, the genetic language of DNA is translated into a different language – Via RNA – into the functioning language of PROTEINS!!!!
  • 76. Figure 17.17 The initiation of translation
  • 77. Figure 17.18 The elongation cycle of translation
  • 78. Figure 17.19 The termination of translation
  • 79. Figure 17.20 Polyribosomes
  • 80. Table 17.1 Types of RNA in a Eukaryotic Cell
  • 81. Figure 17.23 The molecular basis of sickle-cell disease: a point mutation
  • 82. Figure 17.24 Categories and consequences of point mutations: Base-pair insertion or deletion
  • 83. Figure 17.24 Categories and consequences of point mutations: Base-pair substitution
  • 84. Figure 17.25 A summary of transcription and translation in a eukaryotic cell
  • 85. Figure 18.19 Regulation of a metabolic pathway
  • 86. Control of Protein Synthesis Regulation of Gene Expression
    • Every cell has the same numbers and types of chromosomes
    • Development and normal gene function requires precise gene expression in an on and off manner
    • Operon – cluster of gene segments on DNA and its controlling segments
      • Repressible
      • Inducible
  • 87. Regions of the Operon (DNA)
    • Promoter region : promotes transcription by binding with RNA polymerase
    • Operator region : binds a regulatory protein or chemical
      • Overlaps with the RNA polymerase binding site
    • Structural genes : code for a particular peptide or several peptides
      • Start or stop codes
  • 88. Figure 18.20a The trp operon: REPRESSIBLE
  • 89. Figure 18.21a The lac operon: INDUCIBLE
  • 90. Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells