Molecular Biology of the Gene

1. Molecular Biology of the Gene Chapter 10

2. Is DNA the Hereditary Material? • Biologists knew that genes were located on chromosomes • 2 components of chromosomes: • Protein & DNA • Proteins were in the lead • Appeared more structurally complex & functionally specific

3. Is DNA the Hereditary Material? • Griffith (1928) • 2 Strains of Bacteria • R strain - harmless • S strain – disease-causing • Procedure of Experiment • Heat-killed S strain • Mixed with R strain • Injected mice with mixture & some of the harmless strain transformed to pathogenic

4. Is DNA the Hereditary Material?

5. Is DNA the Hereditary Material? • What materials were used & what was the set-up before the trials of the Hershey-Chase Experiment? • Materials: chem. containing radioactive isotopes, radioactivity detector, blender, centrifuge • Procedure: radioactive isotopes to label DNA & protein in T2 • Grew T2 w/ E. coli in a solution containing radioactive sulfur (protein contains sulfur, but DNA does not) - yellow • New phages were made, radioactive sulfur atoms were incorporated only into their proteins • Grew separate batch in a solution containing radioactive phosphorus (phosphorus is only in DNA) - green

6. DNA Structure

7. DNA Structure

8. RNA Structure

9. Double Helix

10. DNA Replication • “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” • Semiconservative model – each of the two daughter molecules will have one old strand and one new strand

11. DNA Replication

12. DNA Replication

13. DNA Replication

14. Replication Process • http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

15. Flow of Genetic Info

16. Flow of Genetic Info • Discovery: Archibald Garrod (1909) • Genes dictate phenotypes through enzymes • Inherited disease reflects a person’s inability to make a particular enzyme • Remember: • Each step in a metabolic pathway is catalyzed by a specific enzyme

17. Flow of Genetic Info • Tatum & Beadle (1940s) • One gene-one enzyme hypothesis: • Function of a gene is to dictate the production of a specific enzyme • Modifications: • All types of proteins (not just enzymes) • Ex: keratin & insulin • One gene-one protein • Many proteins are made from two or more polypeptide chains • Ex: hemoglobin – two kinds of polypeptides, encoded by 2 different genes • One gene-one polypeptide hypothesis

18. Flow of Genetic Info • Cells governed by a molecular chain of command • DNA  RNA  Protein • Chemical language of DNA/RNA • Polymers of nucleotide monomers • A, T(U), C, G • Written as a linear sequence of bases • Specific sequence of bases = a gene • Consists of hundreds/thousands of bases in a sequence

19. Flow of Genetic Info

20. Flow of Genetic Info • DNA/RNA nucleic acid language is translated into Protein polypeptide language • Polypeptide language • Polymers made up of monomers of amino acids • Written in a linear sequence • Sequence of RNA dictates sequence of amino acids • RNA is the messenger carrying genetic info from DNA

21. Flow of Genetic Info • DNA & RNA consist of 4 bases • In translation, these 4 must specify 20 amino acids • Triplett code: Genetic instructions for the amino acid sequence of a polypeptide chain are written in DNA & RNA as a series of 3-base words, called codons • 3-base codons in DNA are transcribed into 3-base codons in RNA, and then the RNA codons are translated into amino acids that form the polypeptide

22. Genetic Code • Set of rules giving the correspondence between codons in RNA and amino acids in proteins • 61 of 64 codons for amino acids • AUG – Methionine or start of polypeptide sequence • 3 other codons do not designate amino acids, but they stop the translation • Redundancy but no ambiguity • Nearly universal, shared by organisms from bacteria to plants and animals

23. Genetic Code

24. Genetic Code

25. Transcription • Transfer of genetic info from DNA to RNA • Occurs in nucleus (Eukaryotes) • RNA is transcribed from DNA template • RNA nucleotides follow same base-pairing rules that govern DNA replication • U pairs with A, instead of T with A • RNA polymerase – link RNA nucleotides • Promoter – nucleotide sequence that says “start transcribing” • Binding site for RNA polymerase

27. mRNA • Conveys genetic info from DNA to translation machinery • Transcribed from DNA – Translated into polypeptides • Modified before leaving nucleus • Add nucleotides • Cap (G nucleotide) & tail (50-250 A’s) • RNA splicing • Remove introns and join exons • Produce mRNA molecule that is continuous

28. mRNA

29. tRNA • Translates codons into amino acids • Amino acids can not recognize mRNA codons • Up to the tRNA to match amino acids to the appropriate codons • 2 Functions must be met: • Picking up the appropriate amino acids • Recognizing appropriate mRNA codons

30. tRNA • Structure • Single strand of RNA – 80 nucleotides • Twists & folds itself • Anticodon: single-stranded loop at 1 end that contains a special triplet of bases • Complementary to codon triplet • Opposite end of anticodon contains the amino acid • tRNA molecules a slightly different for each amino acid it specifies • Specific enzyme that joins amino acids to tRNA

31. tRNA

32. rRNA • Make up the large and small subunits of a ribosome • Ribosome is the site where translation occurs • tRNA & mRNA bind here • Prokaryotic & Eukaryotic Differences • Medically significant • Antibiotic drugs can inactivate prokaryotic ribosomes while leaving eukaryotic ribosomes unaffected

33. rRNA

34. Steps of Translation • Initiation, Elongation, & Termination • Initiation • mRNA, tRNA w/ 1st amino acid, 2 subunits of ribosome • Role: establish exactly where translation begins – get correct sequence of amino acids • 2 Steps: • mRNA molecule binds to small subunit, and tRNA (UAC) binds to start codon (AUG) – carries methionine • Large subunit binds to small; initiator tRNA fits into large subunit – P site – holds growing peptide; A site ready for next tRNA

35. Steps of Translation • Elongation • Codon recognition: anticodon pairs w/ codon at site A • Peptide bond formation: polypeptide separates from tRNA on P site & attaches by a peptide bond to the amino acid carried by the tRNA on the A site • Translocation: P site tRNA leaves, the ribosome translocates the tRNA in the A site to the P site • Codon & anticodon remain bonded • Brings next codon & anticodon to the A site

36. Steps of Translation • Termination • When stop codon reaches the A site (UAA, UAG, UGA) • Don’t code for an amino acid • Stop translation • Polypeptide is released from last tRNA & exits ribosome • Ribosome subunits separate

37. Steps of Translation

38. Mutations • Any change in the nucleotide sequence of DNA • Large regions of a chromosome • Single nucleotide pair • Gene Mutations • Base substitutions • Replacement of one nucleotide with another • No change, insignificant or significant • Base insertions or deletions • Alters the reading frame (triplet grouping) • Nucleotides downstream will be regrouped • Nonfunctional polypeptide

39. Mutations • Chromosome Mutations • Deletion – fragment lost • Cause serious physical and mental problems • Cri du chat syndrome • Duplication – fragment of one chromosome joins to a sister chromatid • Cause serious physical and mental problems • Inversion – fragment reattaches in reverse direction • Less likely to produce harmful effects • Translocation – attachment of a chromosomal fragment to a nonhomologous chromosome

40. Mutations • Chromosomal, cont • Somatic Cell mutations may lead to cancer • CML • Chromosomal translocation • Cells in bone marrow • Part of chromosome 22 switches place with the tip of chromosome 9 • Not usually inherited

41. Mutations • Mutagenesis: production of mutations • Spontaneous: • Errors from replication or recombination • Mutagen: • Physical or chemical agent that causes it • High energy radiation: X-rays and UV light • Chemicals that are similar to normal DNA, but pair incorrectly

42. Viruses • Plant Viruses • Plant must be damaged for a virus to enter • Wind, chilling, injury or insects • Insects transmit plant viruses • Farmers & gardeners • Infected plants pass virus to offspring • No cure, but: • Reduce # of infected plants • Genetically modified – resistant to viruses

43. Viruses • Emerging Viruses • HIV/AIDS, Ebola, West Nile, SARS • 3 Processes Contribute to the Emergence • Mutations • Contact b/w species • Spread from isolated populations • Mutations: • RNA viruses – high rates of mutation • Influenza virus – change each year

44. Viruses • Emerging • One species to another • 3/4 of new human diseases originated in animals • Hantavirus – mice • SARS • Flu epidemic of 1918-1919: 40 million people • Isolated populations • AIDS • Travel, blood transfusions, sexual promiscuity, intravenous drugs

45. Transfer of Bacterial DNA • Structure of DNA • 1 circular, chromosome • 100x longer than the cell, but many folds allow it to fit inside • Binary Fission • Asexual process of bacterial repro • Colonial bacteria are genetically identical

46. Transfer of Bacterial DNA • 3 Methods of DNA Transfer in Bacteria • Transformation • Uptake of foreign DNA from the surrounding environment • Griffith’s experiment • Transduction • Transfer of bacterial genes by a phage • Fragment of phage’s previous host cell accidentally packaged w/ the phage • Phage attacks new cell and injects this fragment

47. Transfer of Bacterial DNA • 3 Methods of DNA Transfer in Bacteria • Conjugation • Mating b/w bacterial cells • ‘Male” cell has a sex pili attached to ‘female’ cell • Cytoplasmic bridge forms b/w • Donor cell replicates its DNA as it transfers it to the other cell • Once in the new cell, crossing over and part of this DNA gets integrated by crossing over • Rest is broken down

48. F Factor & Plasmids • F factor – fertility factor; allows cells to carry out conjugation • Carries gene for making the sex pili • What are the 2 ways in which this F factor is used? • What is an R plasmid?