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Chapter 16

Chapter 16. DNA and Its Role in Heredity. The Search for the Genetic Material. Chromosome made of Proteins DNA Frederick Griffith (1928) He studied Streptococcus pneumoniae A bacterium that causes pneumonia in mammals. R strain was harmless S strain was pathogenic

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Chapter 16

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  1. Chapter 16 DNA and Its Role in Heredity

  2. The Search for the Genetic Material • Chromosome made of • Proteins • DNA • Frederick Griffith (1928) • He studied Streptococcuspneumoniae • A bacterium that causes pneumonia in mammals. • R strain was harmless • S strain was pathogenic • Smooth, enabled the pathogen to get into the cell

  3. Living S Strain Living R Strain Heat Killed S Strain

  4. Heat Killed S Strain and Living R Strain Living S cells found in blood sample from dead mouse

  5. Heat treated S strain • Didn’t kill the DNA completely • only transformed • Heat opened up DNA • R strain took in small S strain strands • turned into live S - lethal • Griffith called this process transformation • Change in genotype and phenotype • Assimilation of external DNA

  6. For the next 14 years scientists tried to identify the transforming substance • Avery, McCarty, MacLeod (1944) • Isolated different parts of the bacteria and injected each part into a mouse • Carbohydrates • Mouse lives • Lipids • Mouse lives • Proteins • Mouse lives • Nucleic Acids • Mouse dies • Still many biologists were skeptical

  7. Hershey, Chase (1952) • Transduction • Infiltrate into cell • Replicate until the cell bursts • Lytic cycle • Worked with viruses that infect bacteria • Bacteriophages or phages • Virus • DNA (or RNA) center • Program host • Restriction enzyme • Cuts up the host cell DNA • Protein coat • Labeled the protein or DNA with radioactive isotopes • Sulfur protein • Phosphorous DNA • Tracked which entered the E. coli cell during infection

  8. Pellet = trans infected bacteria Only DNA could get into the bacteria Protein left outside

  9. What they knew about the structure of DNA • DNA was a polymer of nucleotides • Phosphate group • Deoxyribose • Nitrogenous base • Adenine, guanine, thymine, cytosine

  10. Chargaff (1947) • DNA composition changes between different organisms • Chargraff’s rule • Molecular diversity • %T = %A • %G = %C • In every organism he studied • Wilkins, Franklin (1950’s) • X-ray crystallography • DNA helical structure • Estimate the width of the helix • Could only be double stranded • Linus Pauling

  11. Watson, Crick (1950’s) • Model of Double Helix DNA • Full turn every 3.4 nm • 10 layers of base pairs per turn • Nitrogenous bases hydrophobic • Shielded by sugar back bone from aqueous medium • 2nm helix diameter indicated by the X-ray data • Pyrimidine-purine pairing • Adenine and Guanine = purine • Cytosine and Thymine = pyrimidine

  12. The base-pairing rules • A - T 2 hydrogen bonds • G - C 3 hydrogen bonds • This does not restrict the sequence of nucleotides • The linear sequence varied in countless ways • Each gene has a unique order of nitrogen bases • April 1953 - Watson and Crick published a succinct paper in Nature on the double helix model of DNA

  13. DNA Replication • Base pairing enables existing DNA strands to serve as templates for new complementary strands • Each strand can form a template when separated

  14. Semiconservative replication • Each of the daughter molecules will have one old strand and one newly made strand Conservative Semi-Conservative Dispersive

  15. Meselson, Stahl (1950’s) • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen (15N), while any new nucleotides were indicated by a lighter isotope (14N) • Replicated strands could be separated by density in a centrifuge • New strands less dense

  16. Origins of replication • Special sites where DNA replication begins • Bacteria • Single site • Both directions • Eukaryote • Hundreds or thousands per chromosome

  17. DNA polymerase • Enzyme that adds new nucleotides to the growing strand • Driven by nucleoside triphosphates • Similar to ATP • Suga component is deoxyribose instead of ribose • Loses a pyrophosphate - 2 phosphate groups • Hydrolysis supplies energy for polymerization • antiparallel

  18. The strands of the helix are antiparallel • DNA polymerases can only add nucleotides to the free 3’ end of a growing DNA strand • A new DNA strand can only elongate in the 5’→3’ direction • Leading strand • 3’→5’ parental strand • Can be used as a template for a continuous complementary strand • Lagging Strand • 5’→3’ parental strand • Copied away from the fork in short segments • Okasaki fragments • DNA ligase • Joins together fragments • sealing repairs • sealing recombination fragments

  19. Priming • DNA polymerase cannot initiate synthesis • Can only add nucleotides to an existing chain • Primase • RNA polymerase • Adds a short segment of RNA • DNA polymerase can now add nucleotides to the primer • The primer is later replaced with DNA nucleotides • Leading strand • Requires only one primer • Lagging strand • Requires each fragment be primed

  20. Helicase • Untwists and separates DNA strands • Single strand binding proteins • Helps separate and prevent ssDNA from reforming • Topoisomerase • Single strand breaks to allow for DNA unwinding • Prevent supercoiling • Strand breakage during recombination

  21. Replication factories • Mechanisms create a machine • Stationary – fixed by the nuclear matrix • Spits out daughter strands

  22. Enzymes Proofread and Repair • DNA polymerase proofreads each new nucleotide against the template as soon as it is added • If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis • Mutations that occur after DNA synthesis is completed can often be repaired • Nucleotide excision repair • Nuclease cuts out a segment of a damaged strand • The gap is filled in by DNA polymerase and ligase

  23. Exonuclease • Enzyme that removes nucleotides from the end of a polynucleotide • Has direction • Used to edit DNA, remove RNA primers • Related to Endonuclease

  24. Chromosome Ends

  25. Telomeres • Ends of eukaryotic chromosomal DNA molecules • Special nucleotide sequences • TTAGGG • Repeated between 100 and 1,000 times • Protect genes from being eroded • Telomerase • Uses a short molecule of RNA as a template to extend the 3’ end of the telomere • Present in germ-line cells

  26. Chapter 17 From DNA to Protein: Gene Expression

  27. DNA RNA • ATCG • Nucleic acid • Double helix • Generally stays in nucleus • Storing and transferring genetic information • Stable under alkaline conditions • 5 carbon sugars • Deoxyribose • Only an -H on its second carbon • AUCG • Nucleic acid • Single strand • Outside the nucleus • Codes for amino acids • Messenger btw DNA/ribosomes • Unstable under alkaline conditions • 5 carbon sugars • Ribose • -OH group attached to second carbon

  28. Gene to protein • Archibald Garrod • Genes dictate phenotypes thru enzymes • Diseases reflect inability to produce an enzyme • Beadle and Tatum • Mutant bread mold in mimimal medium + 1 animo acid • One gene – one enzyme • Amended to one gene – one polypeptide

  29. An Overview • Genes provide the instructions for making specific proteins • The bridge between DNA and protein synthesis is RNA • To get from DNA to protein there are two major stages • Transcription • DNA provides a template to make RNA • Translation • Information contained in RNA nucleotides determines amino acid sequence

  30. Transcription and translation Pro Eu • Very quick transcription to translation • Basically simultaneously • All occur in Cytoplasm • mRNA directly transcribed from DNA • Much slower translation and transcription • Different space and time • Transcription in nucleus • Translation in cytoplasm • Pre - mRNA • RNA processing into mature mRNA http://pediaa.com/difference-between-prokaryotic-and-eukaryotic-transcription/

  31. Transcription • Messenger RNA is transcribed from the template strand of a gene • Prolene • CGG • Found in every living thing

  32. In DNA or RNA, the four nucleotides act like the letters of the alphabet • Triplet code • Three consecutive bases specify an amino acid • Unit called codon • 61 of 64 triplets code for amino acids • AUG • Methionine • Also, start of translation • 3 stop codes • UAA, UAG, UGA

  33. RNA polymerase • Separates the DNA strands • Bonds the RNA nucleotides • Can add nucleotides only to the 3’ end of the growing polymer • Genes are read 3’→5’ • Creating a 5’→3’ RNA molecule

  34. Specific sequences of nucleotides along the DNA mark where gene transcription begins and ends • The transcription unit • Part of DNA transcribed into RNA • The promotor • Where the RNA polymerase attaches • Will not work if attached inproperly • “Upstream” of the transcription unit • The terminator • Signals the end of transcription • Transcription can be separated into three stages: • Initiation • Elongation • Termination

  35. Initiation • Promoter • Transcription start point • Nucleotide where RNA synthesis begins • TATA box • DNA sequence upstream of start point • Transcription factors • Collection of proteins • Recognizes TATA • Only certain ones RNA poly binds to • Transcription initiation complex = TF and RNA poly

  36. Elongation • RNA poly • Untwists • Exposes 10 – 20 bases • Adds nucleotides to the 3’ end of the growing strand • Behind the point of RNA synthesis • DNA rewinds • RNA peels away • 60 nucleotides per sec • A single gene can be transcribed multiple times • Mass production of proteins

  37. Termination • Pro • Stops at the termination signal • Releases both DNA and RNA • Eu • Continues ~ 100 nucleotides beyond termination signal • AAUAAA in pre-mRNA • Stop codon only finishes translation • Termination signal after stop codon finishes transcription • Pre-mRNA cut free 10-35 nu past TS

  38. RNA Processing • Prokaryotic cells • Many proteins synthesized at a time • Similar to each other • RNA after transcription is mRNA

  39. Eukaryotic cells • Gene only codes for 1 protein • RNA formed after transcriptionis pre-mRNA • Needs to be modified • Ends modified to prevent degradation from hydrolytic enzymes • 5’ cap • Modified guanine (guanine triphosphate) • Guides attachment of the polypeptide • Poly-A tail • 50 – 250 adenine • Assists in export from nucleus • Nucleoplasm to cytoplasm

  40. RNA splicing • Covalently altered • Long noncoding stretches of nucleotides • Introns • Noncoding segments between coding segments • Exons • Exit nucleus • Includes leader/trailer segments and translated segments • RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence Leader segment help recognize start codon Trailer segment

  41. Pre-mRNA HnRNA • Before introns are cut out • Spliceosome • SnRNPs • Recognize nucleotide segments on the ends of introns • Cuts points on the RNA, releases the intron and joins the flanking portions • Catalytic process • Ribozymes • Self splicing in ribosomal RNA • Catalysts • Alternative RNA splicing/more possibilities for crossing over

  42. Translation • Transfer RNA (tRNA) • Transfers amino acids from the cytoplasm to the ribosome • Anticodon to polypeptide • Hydrogen bonds to the mRNA • 45 types • Anticodon can recognize more than 1 codon • Wobble • Relaxed 3rd base • U can bond with A or G • Inosine, anticodon, can bond with U, C, or A

  43. Aminoacyl-tRNA synthetase • Enzyme • Join amino acid to correct tRNA • 20 different types • One for each amino acid • Specific combination at active site • Powered by hydrolysis of ATP • Creates activated animo acid Fig. 17.14

  44. Ribosomes • Large and small subunit • Formed in the nucleolus • Composed of proteins and 60% rRNA • A binding site for mRNA • Three binding sites for tRNA molecules. • The P site • Holds the tRNA carrying the growing polypeptide chain • The A site • Carries the tRNA with the next amino acid • Holds amino acid to carboxyl • Catalyzes peptide bond • The E site • tRNAs leave the ribosome

  45. Translation can be divided into three stages: • Initiation • Elongation • Termination • All three phase require protein “factors” • Initiation and elongation require energy • GTP

  46. Initiation • mRNA • A tRNA with the first amino acid • The two ribosomal subunits • Initiation factors • MET • Always first Fig. 17.17

  47. Elongation • Series of 3 steps • Codon recognition • Peptide bond formation • Translocation Peptidyl transferase

  48. Termination • Stop codon reaches the A site • Release factor • Binds to the stop codon • Adds a water molecule • Hydrolyzes the bond between the polypeptide and its tRNA in the P site Fig. 17.19

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