2. BB10006: Cell & Molecular biology Dr. MV Hejmadi Dr. JR Beeching (convenor) Prof. RJ Scott Prof. JMW Slack

3. Dr. Momna Hejmadi (bssmvh@bath.ac.uk) Structure and function of nucleic acids Books (any of these): Any bioscience textbook will do but my favourites are Biochemistry (3e) by D Voet & J Voet Molecular biology of the cell (4 th ed) by Alberts et al Essential Cell Biology by Alberts et al Key websites http://www.dnaftb.org/dnaftb/ http://www.dnai.org/lesson/go/2166/1994 http://molvis.sdsc.edu/dna/index.htm

4. Outline of my lectures Lecture 1. Nucleic acids – an introduction Lecture 2. Properties and functions of nucleic acids Lecture 3. DNA replication Lectures 4-6. Transcription and translation Access to web lectures at http://www.bath.ac.uk/bio-sci/hejmadi/teaching%202005-06.htm

5. Lecture 1 - Outline How investigators pinpointed DNA as the genetic material The elegant Watson-Crick model of DNA structure Forms of DNA (A, B, Z) References: History, structure and forms of DNA http://www.dnai.org/lesson/go/2166

6. Timeline 1869 F Miescher - nucleic acids 1928 F. Griffith - Transforming principle http://www.dnai.org/lesson/go/2166http://www.dnai.org/lesson/go/2166/1994

7. Discovery of transforming principle 1928 – Frederick Griffith – experiments with smooth (S) virulent strain Streptococcus pneumoniae and rough (R) nonvirulent strain

8. Griffith experiment

10. What is this transforming principle? Bacterial transformation demonstrates transfer of genetic material

11. Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle Avery, McCleod & McCarty- Transforming principle is DNA 1944 http://www.dnai.org/lesson/go/2166http://www.dnai.org/lesson/go/2166/1994

12. Avery, MacLeod, McCarty Experiment

14. Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1949 Avery, McCleod & McCarty- Transforming principle is DNA 1944 Erwin Chargaff – base ratios http://www.dnai.org/lesson/go/2166http://www.dnai.org/lesson/go/2166/1994

15. E. Chargaff’s ratios A = T C = G A + G = C + T% GC constant for given species

16. Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1952 Avery, McCleod & McCarty- Transforming principle is DNA 1944 Hershey-Chase ‘blender’ experiment http://www.dnai.org/lesson/go/2166http://www.dnai.org/lesson/go/2166/1994 1949 Erwin Chargaff – base ratios

17. Hershey and Chase experiments 1952 – Alfred Hershey and Martha Chase provide convincing evidence that DNA is genetic material Waring blender experiment using T2 bacteriophage and bacteria Radioactive labels 32 P for DNA and 35 S for protein

18. Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1952 Avery, McCleod & McCarty- Transforming principle is DNA 1944 Hershey-Chase ‘blender’ experiment 1952 Erwin Chargaff – base ratios 1952 R Franklin & M Wilkins–DNA diffraction pattern http://www.dnai.org/lesson/go/2166http://www.dnai.org/lesson/go/2166/1994

19. X-ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins

20. Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1952 Avery, McCleod & McCarty- Transforming principle is DNA 1944 Hershey-Chase ‘blender’ experiment 1952 Erwin Chargaff – base ratios 1952 R Franklin & M Wilkins–DNA diffraction pattern 1953 J Watson and F Crick – DNA structure solved http://www.dnai.org/lesson/go/2166http://www.dnai.org/lesson/go/2166/1994

21. The Watson-Crick Model: DNA is a double helix 1951 – James Watson learns about x-ray diffraction pattern projected by DNA Knowledge of the chemical structure of nucleotides (deoxyribose sugar, phosphate, and nitrogenous base) Erwin Chargaff’s experiments demonstrate that ratio of A and T are 1:1, and G and C are 1:1 1953 – James Watson and Francis Crick propose their double helix model of DNA structure

22. Human genome project Public consortium Headed by F Collins Started in mid 80’s Working draft completed in 2001 Final sequence 2003 Nature: Feb 2001 Celera Genomics Headed by C Venter Started in mid 90’s Working draft completed in 2001 Science: Feb 2001 Human genome = 3.3 X 10 9 base pairs Number of genes = 26 – 32,000 genes Goal: to sequence the entire human nuclear genome

23. The human genome Nuclear genome (3.2 Gbp) 24 types of chromosomes Y- 51Mb and chr1 -279Mbp Mitochondrial genome

24. DNA in forensics what can a single human hair tell you? nuclear DNA Hair root mitochondrial DNA Hair shaft

25. Types of RNA

26. Nucleotides DNARNA Originally elucidated by Phoebus Levine and Alexander Todd in early 1950’s 2’-deoxy-D-ribose 2’-D-ribose Made of 3 components 1) 5 carbon sugar (pentose) 2) nitrogenous base 3) phosphate group 1) SUGARS

27. 2) NITROGENOUS BASES planar, aromatic, heterocyclic derivatives of purines/pyrimidines adenine uracil thymine cytosine guanine pyrimidines purines Note: Base carbons denoted as 1 etc Sugar carbons denoted as 1’ etc

28. Nucleotide monomer nucleotide = phosphate ester monomer of pentose dinucleotide - Dimer Oligonucleotide – short polymer (<10) Polynucleotide – long polymer (>10) Nucleoside = monomer of sugar + base

29. 1) Phosphodiester bonds 5’ and 3’ links to pentose sugar 2) N-glycosidic bonds Links nitrogenous base to C1’ pentose in beta configuration 5’ – 3’ polynucleotide linkages

30. 3’ end 5’ end 5’ – 3’ polarity

31. Essential features of B-DNA Right twisting Double stranded helix Anti-parallel Bases on the inside (Perpendicular to axis) Uniform diameter (~20A) Major and minor groove Complementary base pairing

32. DNA conformations Right-handed helix intermediate planes of the base pairs nearly perpendicular to the helix axis tiny central axis wide + deep major groove narrow + deep minor groove Right-handed helix Widest planes of the base pairs inclined to the helix axis 6A hole along helix axis narrow + deep major groove Wide + shallow minor groove Left-handed helix Narrowest planes of the base pairs nearly perpendicular to the helix axis no internal spaces no major groove narrow + deep minor groove B-DNA A- DNA Z-DNA

33. BA Z

34. Structurally, purines (A and G) pair best with pyrimidines (T and C) Thus, A pairs with T and G pairs with C, also explaining Chargaff’s ratios

35. Problem http://www.dnaftb.org/dnaftb/19/conce pt/index.html

36. Maybe because RNA, not DNA, is prone to base-catalysed hydrolysis Why has DNA evolved as the genetic material but not RNA?

37. linear human chromosomes Double stranded DNA Genetic material may be DNA Single stranded DNA circular linear circular Prokaryotes Mitochondria Chloroplasts Some viruses (pox viruses) Parvovirus adeno-associated viruses

38. reoviruses Double stranded RNA Genetic material may be RNA Single stranded RNA Retroviruses like HIV

39. RNA / DNA hybrids e.g. during retroviral replication

40. What is the base found in RNA but not DNA? ? A) Cytosine B) Uracil C) Thymine D) Adenine E) Guanine

41. What covalent bonds link nucleic acid monomers? A) Carbon-Carbon double bonds B) Oxygen-Nitrogen Bonds C) Carbon-Nitrogen bonds D) Hydrogen bonds E) Phosphodiester bonds

42. What sugar is used in in a DNA monomer? A) 3'-deoxyribose B) 5'-deoxyribose C) 2'-deoxyribose D) Glucose

43. Each deoxyribonucleotide is composed of A) 2'-deoxyribose sugar, Nitrogenous base, 5'- hydroxyl B) 3'-deoxyribose sugar, Nitrogenous base, 5'- hydroxyl C) 3'-deoxyribose sugar, Nitrogenous base, 5'- Phosphate D) Ribose sugar, Nitrogenous base, 5'-hydroxyl E) 2'-deoxyribose sugar, Nitrogenous base, 5'- phosphate