DNA & RNA- Nucleic Acids and Protein Synthesis

1. DNA & RNA- Nucleic Acids and Protein Synthesis IB Biology Ch. 16: Campbell Ch. 5&6: Orange Book

2. Objectives • Describe the history behind the discovery of DNA and its function • Outline the structure of a nucleotide • Describe the structure of the DNA molecule • Describe the process of DNA replication including the various enzymes and that it is a semi-conservative process.

3. Introduction • Your genetic endowment is the DNA you inherited from your parents. • Nucleic acids are unique in their ability to direct their own replication. • The resemblance of offspring to their parents depends on the precise replication of DNA and its transmission from one generation to the next. • Once T.H. Morgan’s group showed that units of heredity are located on chromosomes, the two constituents of chromosomes - proteins and DNA - were the candidates for the genetic material. • Until the 1940s, the great heterogeneity and specificity of function of proteins seemed to indicate that proteins were the genetic material. • However, this was not consistent with experiments with microorganisms, like bacteria and viruses.

4. Discovery of DNA • 1868: Miescher first isolated deoxyribonucleic acid, or DNA, from cell nuclei

5. Fredrick Griffith- 1928 • First suggestion that about what genes are made of. • Worked with: 1) Two strains of Pneumococcus bacteria: Smooth strain (S) Virulent (harmful) Rough strain (R) Non-Virulent 2) Mice-were injected with these strains of bacteria and watched to see if the survived. 3) Four separate experiments were done: -injected with rough strain (Lived) -injected with smooth strain (Died) -injected with smooth strain that was heat killed (Lived) -injected with rough strain & heat killed smooth (????)

6. Mixture of heat-killed S cells and living R cells EXPERIMENT Living R cells (control) Living S cells (control) Heat-killed S cells (control) RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells

7. Griffith’s Conclusion • Somehow the heat killed smooth bacteria changed the rough cells to a virulent form. • These genetically converted strains were called “Transformations” • Something (a chemical) must have been transferred from the dead bacteria to the living cells which caused the transformation • Griffith called this chemical a “Transformation Principle”

8. Next Breakthrough came from the use of Viruses • Viruses provided some of the earliest evidence that genes are made of DNA • Molecular biology studies how DNA serves as the molecular basis of heredity • Are only composed of DNA and a protein shell. T2 Bacteriophage- a typical virus

9. Phage reproductive cycle Phage attaches to bacterial cell. Phage injects DNA. Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble. Cell lyses and releases new phages. Figure 10.1C

10. Photo of T2 Viruses Fig. 16-3 Phage head Tail sheath Tail fiber DNA 100 nm Bacterial cell

11. Hershey-Chase Experiment- 1952 • In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 • To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection • They concluded that the injected DNA of the phage provides the genetic information

12. Fig. 16-4-1 EXPERIMENT Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Radioactive DNA Batch 2: radioactive phosphorus (32P)

13. Fig. 16-4-2 EXPERIMENT Empty protein shell Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Phage DNA Radioactive DNA Batch 2: radioactive phosphorus (32P)

14. Fig. 16-4-3 EXPERIMENT Empty protein shell Radioactivity (phage protein) in liquid Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet Pellet

15. Video Clip of Hershey-Chase • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

16. Erwin Chargaff- 1950 Already known- DNA is a polymer of nucleotides- nitrogen base, pentose sugar, and a phosphate group. Chargaff noticed a ratio of the bases: 30.3% Adenine 30.3% Thymine 19.5% Guanine 19.9% Cytosine So, for DNA, the amt. of A = T, and The amt. of C= G (Chargaff’s Rules)

17. Who Discovered the Shape of DNA? • James Watson and Francis Crick (1954) are credited with finally piecing together all the information previously gathered on the molecule of DNA. They established the structure as a double helix - like a ladder that is twisted. The two sides of the ladder are held together by hydrogen bonds.

18. How did they get to their conclusions? • They built many models, always perplexed at how it fit together, until one day, when they wandered into the office of a fellow scientist, Dr. Rosalind Franklin.

19. Along with Dr. Maurice Wilkins, she had taken x-ray crystallography photos of DNA. They saw her photos and realized the great secret- that DNA was coiled like a spring. They then made their model and won the Nobel Prize in 1962. Franklin’s famous photo of DNA

20. So, What is DNA?Deoxyribonucleic Acid • blueprint of life (has the instructions for making an organism) • codes for your genes • made of repeating subunits called nucleotides • shape is the double helix (twisted ladder)

21. I. Structure of DNA- 3 parts: • Sugar- Deoxyribose • Phosphate Group • Nitrogen bases The sugar and phosphates make up the "backbone" of the DNA molecule. Nucleotide

22. DNA and RNA are polymers of Nucleotides DNA is a nucleic acid, made of long chains of nucleotides- Sugar, phosphate, nitrogen base. Phosphate group Nitrogenous base Nitrogenous base(A, G, C, or T) Sugar Phosphategroup Nucleotide Thymine (T) Sugar(deoxyribose) DNA nucleotide** Figure 10.2A Polynucleotide Sugar-phosphate backbone

23. Nitrogen bases Bases come in two types: a. Purines (adenine and guanine- A&G) b. Pyrimidines (thymine and cytosine- T&C).

24. DNA Maintains a Uniform Diameter • See pg. 310

25. Base Pairing- Chargaff’s Rules • Watson and Crick reasoned that the pairing was more specific, dictated by the base structures • They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) • The Watson-Crick model explains Chargaff’s rules: Adenine pairs to Thymine (A-T)Guanine pairs to Cytosine (G-C)**Very important- remember this!!!!

26. DNA Bonding The two sides of the helix are held together by Hydrogen bonds (weak) The sides of the DNA, the sugar and phosphate, are held together with covalent bonds.

27. Simple Diagram of DNA-Think of it like a ladder, the bases being the rungs.

28. Each strand of the double helix is oriented in the opposite direction • The ends are referred to as the 3’ and 5’ ends. 5 end 3 end P P P P P P P P 3 end 5 end Figure 10.5B

29. Summary: • Chargaff ratio of nucleotide bases (A=T; C=G) • Watson & Crick (Wilkins, Franklin) • The Double Helix • √ nucleotides: nitrogenous base (thymine, adenine, cytosine, guanine); sugar deoxyribose; phosphate group

30. Putting it all together: here are some images of what the DNA double helix looks like:

31. DNA Replication and Repair • The relationship between structure and function is manifest in the double helix • Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

32. The Basic Principle: Base Pairing to a Template Strand • Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication • In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

33. Fig. 16-9-1 A T C G T A A T C G (a) Parent molecule

34. Fig. 16-9-2 A T T A C G G C A T A T T A T A C C G G (b) Separation of strands (a) Parent molecule

35. Fig. 16-9-3 A T A T A T A T C G C G C G C G A T A T A A T T T A T A T T A A C C G C G C G G (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands (a) Parent molecule

36. Three Proposed Models of DNA Replication

37. Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand • Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new)

38. DNA Replication: A Closer Look • The copying of DNA is remarkable in its speed and accuracy • More than a dozen enzymes and other proteins participate in DNA replication

39. DNA replication depends on specific base pairing • In DNA replication, the strands separate • Enzymes use each strand as a template to assemble the new strands Nucleosomes Parental moleculeof DNA Both parental strands serveas templates Two identical daughtermolecules of DNA

40. Anti-parallel Structure of DNA

41. Antiparallel nature • 5’ end corresponds to the Phosphate end • 3’ end corresponds to the –OH sugar • Replication runs in BOTH directions • One strand runs 5’ to 3’ while the other runs 3’ to 5’ • Nucleotides are added on the 3’ end of the original strand. • The new DNA strand forms and grows in the 5’  3’ direction only

42. Building New Strands of DNA 5’ end 3’ end 5’ end

43. Building New Strands of DNA • Each nucleotide is a triphosphate: (GTP, TTP, CTP, and ATP) • Nucleotides only add to the 3’ end of the growing strand (never on the 5’ end) • Two phosphates are released (exergonic) and the energy released drives the polymerization process.

44. Getting Started • Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” • A eukaryotic chromosome may have hundreds or even thousands of origins of replication • Replication proceeds in both directions from each origin, until the entire molecule is copied

45. Fig. 16-12b Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Bubble Two daughter DNA molecules (b) Origins of replication in eukaryotes

46. Fig. 16-12 Origin of replication Parental (template) strand Daughter (new) strand Replication fork Double- stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Bubble Two daughter DNA molecules (b) Origins of replication in eukaryotes

47. Getting Started- Enzymes • At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating • Topoisomerasecorrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands • Helicasesare enzymes that untwist the double helix at the replication forks • Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template

48. Fig. 16-13 Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer 5 5 3 Helicase

49. DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end • The initial nucleotide strand is a short RNA primer

50. RNA Primers • An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template • The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand.