BB10006: Cell & Molecular biology

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

2. 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(4th ed)byAlberts 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

3. Outline of my lectures Lecture 1. Nucleic acids – an introduction Lecture 2. Properties and functions of nucleic acids Lecture 3. DNA replication Lectures4-6. Transcription and translation Access to web lectures at http://www.bath.ac.uk/bio-sci/hejmadi/teaching%202005-06.htm

4. 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, structureand forms of DNA http://www.dnai.org/lesson/go/2166

5. 1869 Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle http://www.dnai.org/lesson/go/2166/1994

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

7. Griffith experiment

8. Griffith experiment

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

10. 1800’s Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle Avery, McCleod& McCarty- Transforming principle is DNA 1944 http://www.dnai.org/lesson/go/2166/1994

11. Avery, MacLeod, McCarty Experiment

12. Avery, MacLeod, McCarty Experiment

13. 1800’s Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle Avery, McCleod& McCarty- Transforming principle is DNA 1944 Erwin Chargaff – base ratios 1949 http://www.dnai.org/lesson/go/2166/1994

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

15. 1800’s Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle Avery, McCleod& McCarty- Transforming principle is DNA 1944 Erwin Chargaff – base ratios 1949 1952 Hershey-Chase ‘blender’ experiment http://www.dnai.org/lesson/go/2166/1994

16. 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 32P for DNA and 35S for protein

17. 1800’s Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 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 1952 http://www.dnai.org/lesson/go/2166/1994

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

19. 1800’s Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 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 1952 J Watson and F Crick – DNA structure solved 1953 http://www.dnai.org/lesson/go/2166/1994

20. 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

21. 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 project Goal: to sequence the entire human nuclear genome Humangenome = 3.3 X 10 9 base pairs Number of genes = 26 – 32,000 genes

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

23. DNA in forensicswhat can a single human hair tell you? mitochondrial DNA Hair shaft nuclear DNA Hair root

24. Types of RNA

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

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

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

28. 5’ – 3’ polynucleotide linkages 2) N-glycosidic bonds Links nitrogenous base to C1’ pentose in beta configuration • 1) Phosphodiester bonds • 5’ and 3’ links to pentose sugar

29. 5’ – 3’ polarity 5’ end 3’ end

30. 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

31. 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 DNA conformations A- DNA B-DNA Z-DNA • 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

32. Z B A

33. 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

34. Problem • http://www.dnaftb.org/dnaftb/19/concept/index.html

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

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

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

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

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

40. 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

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

42. 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