CH 10: Molecular Biology of the Gene

1. CH 10: Molecular Biology of the Gene DNA  RNA  Protein

2. Sections Covered with my titles for each section 10.1: DNA as the genetic material 10.2/3: Structure of DNA and RNA 10.4/5: DNA replication 10.6-10.14: Transcription and translation 10.15: review 10.16: Mutations

3. History of DNA • DNA as the genetic material • Griffith (1928) • Found that the genetic component of pathogenic bacterial cells was not destroyed when the cells were heated • He did not follow-up on what that component was and how/why it survived. • Griffith Experiment

4. DNA as the Genetic Material • Avery (1944) • Most believed protein to be genetic material at this time. • Avery found that pathogenic bacterial cells treated with protein digesting enzymes could still transform harmless bacterial cells. • Cells treated with a DNA digesting enzyme could not.

5. DNA as the Genetic Material • Avery (1944) • Avery concluded that DNA and not protein must be the genetic material. • Many refused to accept this conclusion. • Thought his findings only applied to bacteria and not eukaryotic cells.

6. DNA as the Genetic Material • Hershey-Chase Experiment (~1950) • Their work confirmed to the scientific community that DNA was the genetic material. • Considered an “elegant” experiment. • Very simple and demonstrates a great deal. • See page 183

7. Hershey-Chase Experiment • They took advantage in a chemical difference between DNA and protein • DNA contains the elements: C, H, O, N, P • Protein contains the elements: C, H, O, N, S

8. Hershey-Chase Experiment • Experiment utilized bacteriophages • Bacteriophages are viruses that infect bacteria. • Knew that a virus’ genetic material enters the host cell • as a result the bacterial cell makes more virus as directed by the virus’ genetic material

9. Hershey-Chase Experiment • More on viruses….. • Viruses have two components: • An outer protein coat with nucleic acid inside

10. Hershey-Chase Experiment The Experiment • Allowed one sample of viruses to infect bacteria grown on a radioactive (RA) sulfur-35 medium • Viruses made had RA Sulfur-35 in their protein coats.

11. Hershey-Chase Experiment The Experiment • Allowed another sample of viruses to infect bacteria grown on a radioactive (RA) phosphorus-32 medium • Viruses made had RA phosphorus-32 in their DNA.

12. Hershey-Chase Experiment • The two RA viral cultures were isolated and each was allowed to infect a new (non RA) bacterial culture. • Exp’t was done in a liquid medium called the supernatant. • Cultures were gently shaken in a blender to shake the virus off of the outside of the bacteria.

13. Virus infecting bacterial cell

14. Hershey-Chase Experiment • Each culture was centrifuged to separate the liquid medium (supernatant) from the infected bacteria. • The bacteria and the supernatant were checked for radioactivity. • Whatever entered the bacteria is the genetic material.

15. Hershey-Chase Experiment What they found: • Bacteria infected with the virus with a RA S-35 (protein) coat • The infected bacteria were NOT RA • The supernatant was RA • This is evidence that the protein did not enter the bacteria and thus, could not be the genetic material.

16. Hershey-Chase Experiment • For the bacteria infected by virus with RA P-32 in theirDNA • The infected bacteria were RA • The supernatant was not RA • This is evidence that the DNA entered the bacteria and thus, MUST be the genetic material. • http://www.accessexcellence.org/RC/VL/GG/hershey.php

18. Structure of DNA What was known about DNA • Chemical components are: • Deoxyribose – 5 carbon sugar • Phosphate groups • Nitrogenous bases • Adenine • Guanine • Cytosine • Thymine

19. Structure of DNA • Nitrogenous bases were of 2 types: • Purines: have a double-ring structure • Adenine (A) • Guanine (G) • Pyrimidines: have a single-ring structure • Cytosine (C) • Thymine (T) • Page 185

20. Structure of DNA • Chargaff’s findings (1949) • Studied DNA from many organisms • Found that the amount of guanine is always equal to the amount cytosine and the amount of adenine is equal to the amount of thymine. • G=C • A=T

21. Structure of DNA • X-Ray Crystallography Data on DNA • Maurice Wilkins and Rosalind Franklin • Franklin’s data suggested that DNA was a long thin molecule of 2 nm diameter • Data also indicated a repeating pattern consistent with a helix. • Wilkins shared Franklin’s data and lab notes with Watson and Crick without her permission.

22. As a scientist Miss Franklin was distinguished by extreme clarity and perfection in everything she undertook. Her photographs are among the most beautiful X-ray photographs of any substance ever taken. Their excellence was the fruit of extreme care in preparation and mounting of the specimens as well as in the taking of the photographs. -- J. D. Bernal[1958 N] Rosalind Franklin

23. Franklin’s X-Ray Data

24. Structure of DNA "The instant I saw the picture my mouth fell open and my pulse began to race.... the black cross of reflections which dominated the picture could arise only from a helical structure... mere inspection of the X-ray picture gave several of the vital helical parameters." Watson

25. Structure of DNA • In 1953 Watson, Crick, and Wilkins put the pieces together and proposed their famous double helix structure for DNA. • Watson, Crick, and Wilkins were awarded a Nobel Prize for deciphering the structure of DNA

26. Watson and Crick

27. Structure of DNA • DNA is a double-stranded helix • Each strand is a long chain of covalently bonded nucleotides • Phosphates can bond to carbon 5 or carbon 3 of deoxyribose • Phoshpates link the sugars to form the backbone of the chain • Bases bond to carbon 1 of deoxyribose • Page 187

29. Structure of DNA • Each strand has a 5’ and a 3’ end • Two DNA strands run in opposite directions • One runs 5’  3’ and the other 3’ 5’

30. Structure of DNA • The two strands are joined by hydrogen bonds between the bases • Two H bonds form between A and T. • Three H bonds form between G and C.

31. C G A T

32. Structure DNA

33. DNA Replication • DNA replication – DNA synthesis • Occurs in the nucleus during ___ of the cell cycle • Goal is to make an exact copy of the cell’s DNA • Put another way -- goal is to duplicate the chromosomes. Replication

34. Semi-Conservative Model • Each newly made piece of DNA is ½ old DNA and ½ new DNA (page 188) Simple animation of replication

35. DNA Replication-enzymes needed • Helicases • Open the H bonds between the strands • Stabilizing proteins • Hold the two strands apart

36. DNA Replication: enzymes needed • DNA polymerase III • Adds nucleotides to the 3’ end of DNA • Say…synthesizes DNA in the 5’  3’ direction • It cannot initiate (start) a new DNA strand • DNA polymerase I • Removes primer sequences and fills in the gaps with DNA • Other DNA polymerases • Proofread the DNA and correct mutations

37. DNA Replication-enzymes needed • “Primer” enzyme – not shown in text • Starts synthesis in the 5’  3’ direction • Makes a primer sequence to which DNA polymerase III can add DNA • DNA ligase • Joins newly made DNA segments after the primer sequences have been removed and replaced by DNA polymerase I.

38. DNA Replication • Helicases and stabilizing proteins open and unwind small sections of DNA and hold the strands apart. • Occurs at specific locations on the DNA – called origins of replication • Primer enzymes synthesize primer strands in the 5’  3’ direction on each DNA strand.

39. DNA Replication • DNA polymerase III adds DNA to each primer sequence in the 5’  3’ direction.

40. DNA Replication • Proteins open more of the DNA (replication fork opens more). • DNA synthesis continues in the 5’ 3’ direction on one strand. (leading strand) • Another primer is laid down on the other strand and then DNA synthesis continues. (lagging strand)

41. Primer sequences

43. DNA Replication • Process continues until all of the DNA has been replicated. • Primer sequences are cut out, the gaps filled in with DNA • DNA ligase joins the new DNA sequences. http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swfAnimation