Lecture 1-Molecular_biology BASICSn.pptx

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1. Yarsin LWASA Jr. Fundamentals of Molecular Biology

2. Reference Textbooks Fundamental Molecular Biology Lizabeth A. Allison, 2007 Edn . Molecular Biology of the Cell Bruce Alberts , 2008 Edn . Molecular Diagnostics for the Clinical Laboratorian William Coleman 2010 Edn .

3. Topical outline Fundamental Molecular Biology Historical perspective Definition and Scope of Molecular Biology Central Dogma of Biology; from structure and functional significance of essential molecules to description of processes therein Signal Transduction Genes, Genomes, Introduction to Bioinformatics Bacterial Genetics Molecular Diagnostics; Definition and Scope Basic techniques in Molecular Biology Molecular Diagnostics Practicum

4. 1. Fundamental Molecular Biology I -Biological molecules -Nucleic acids structure & function -Amino acids & Proteins

5. Objectives To describe the vital Molecules that constitute cells and cellular processes Re-cap: Eukaryotic and Prokaryotic cell structure What defines all life forms ? To introduce, define & outline the scope of “Molecular Biology” To describe the “ Central Dogma of Biology ” and the cellular processes therein

6. Fundamental Molecular B iology Outline: Origin of Molecular Biology The Biological Molecules (Biomolecules) Definition and Scope of ‘Molecular Biology’ From Gene to Protein : The Central Dogma of Molecular Biology Nucleic Acids: structure and functional significance DNA Replication, Mutation and repair Transcription Translation The Genetic code, Amino acids, Proteins Genes, Genomes, Introduction to Bioinformatics

7. Origin of Molecular Biology Mendel (1860): Heredity is the transmission of characteristics (gene products) from parent to offspring by means of genes (characters)

8. Origin of Molecular Biology: Classical Genetics Mendel’s experiments Mendel’s laws: Genes (characters) segregate 2. Genes (characters) recombine independently ( Independent assortment ) Mendel (1860): Heredity is the transmission of characteristics (gene products) from parent to offspring by means of genes (characters)

9. Origin of Molecular Biology: Biochemistry Enzymes and catalysis

10. 1928 – 1944: insight into the nature of hereditary material; transforming factor is DNA Griffith, 1928: Genes are DNA Transformation: uptake of naked/free DNA by bacteria First gene transfer mechanism to be discovered Encapsulated Streptococcus pneumoniae : Smooth ( S ), virulent, kills mouse Non-encapsulated: rough ( R ), harmless to mouse Mouse died when injected with mixture of heat-killed S + and live R , but not alone Live S with a capsule was isolated from dead mice A factor from dead S converted live R to S Avery et al, 1944: t he transforming factor i s DNA! Hence Genes (characters) are DNA!

11. Avery and Griffith : Genes are DNA

12. 1953: the double helix & birth of Molecular Biology

13. Molecular Biology birthed; but what is the pathway of info from DNA to characteristics (phenotype) -Proteins The central dogma of molecular biology : DNA (genes or characters) to mRNA to Proteins (phenotype, characteristics)

14. The Biological Molecules

15. Science > Biology > Medicine Science: The study of “Living” and “Nonliving” things Biology: The study of “Living things” (plants, animals, microorganisms) Life sciences: Biology, Microbiology, Medicine, Immunology, Biochemistry, Cell biology, etc. Medicine is an applied science within biology Other sciences: Chemistry, Physics, Mathematics; (Nonliving things)

16. Biology & the domains of Life The tree of life: what unites all living things?

17. The tree of life based on whole genome data What is common among living things?

18. All Living t hings have common characteristics What are living things? Anything exhibiting all these 7 characteristics; Reproduction Growth Movement Response to stimuli Excretion Cellular structure Use of energy NB: the characteristics (phenotypes) are encoded by genes (characters)

19. Biological molecules (Biomolecules) Enable life in living things, i.e. life (on earth) depends on these molecules; Nucleic acids (DNA & RNA) Proteins Carbohydrates: energy Lipids/Fatty acids: energy, hormones etc. Water and trace elements: essential

20. Then, what is Molecular Biology? T he study Biological Molecules? All life depends on Biological Molecules: Nucleic acids (DNA & RNA) Proteins Carbohydrates: energy Lipids/Fatty acids: energy, hormones etc. Water and trace elements: essential

21. Molecular Biology: definition, scope While life depends on Biological Molecules; Nucleic acids (DNA & RNA) Proteins CHOs: energy Lipids/Fatty acids: energy, hormones etc. Water and trace elements: essential Biological information (i.e. from which characteristics are encoded) is stored and preserved as DNA , and is transmitted into proteins via RNA; hence, Molecular Biology only deals with nucleic acids, proteins and the cellular processes underlying them Molecular Biology: a science that studies how biological information is stored , transmitted and preserved in living things (cells)

22. The Central Dogma of Biology Universal in eukaryotes & prokaryotes Violated by retroviruses (HIV) and other RNA viruses -The flow of biological information from genes (DNA) to proteins via mRNA: DNA → RNA → protein ( function/phenotype/characteristic) -DNA makes RNA makes Protein (Wikipedia) Structure Structure Structure Molecular Biology

23. Central Dogma in every aspect of life 23 Replication Function/Phenotype

24. The Central Dogma of Biology Watch!

25. The central dogma of biology

26. The Central Dogma of Biology To appreciate Molecular Biology & related disciplines (e.g. Molecular Diagnostics), you must fully understand the dogma of biology. Focus on; Nucleic acids structure Processes in the dogma: DNA replication, Transcription, Translation. Relate (1) and (2) to Cell structure, eukaryotic and prokaryotic Structure Structure Structure Molecular Biology

27. Re-cup: ultra-cell structure, Prokaryotes vs. eukaryotes 27 Eukaryotes in addition have a cytoskeleton

28. Amazing interconnectedness of eukaryotic membranous structures 28

29. The rough endoplasmic reticulum has ribosomes on its surface and it transports proteins made by the ribosome through the cisternae. Rough endoplasmic reticulum Copyright 1998 Terry Brown. All rights reserved. 29

30. The Central Dogma of Biology To appreciate Molecular Biology & related disciplines (e.g. Molecular Diagnostics), you must fully understand the dogma of biology. Focus on; Nucleic acids structure Processes in the dogma: DNA replication, Transcription, Translation. Relate (1) and (2) to Cell structure, eukaryotic and prokaryotic Structure Structure Structure Molecular Biology

31. Structure of Nucleic Acids , and F unctional S ignificance

32. Nucleic A cids Nucleic acids DNA and RNA Polymers of Nucleotides NB: other biomolecules that are polymers of simple units; Proteins, CHOs, Lipids

33. Nucleic acids structure Primary structure –the components of Nucleic acids The 5-Carbon sugars Nitrogenous bases The Phosphate functional group Nucleotides and Nucleosides The significance of 5’ and 3’ Nomenclature of N ucleotides Secondary structure: the double helix Tertiary structure: Supercoiling

34. Primary structure The 5-Carbon sugars Nitrogenous Bases The Phosphate Functional group Nucleotides and Nucleosides

35. 5-C Sugars in N ucleic Acids The 5-C atoms numbered 1’ to 5’ ; primes are used to distinguish with numbering in nitrogenous bases Both sugars have an O2; 5’-C is outside the ring Sugars differ in Presence or Absence of an O2 in 2’-C

36. The Nitrogenous bases Bases: N2-containing molecules having the chemical properties of a base (a substance that accepts an H+ ion or proton in solution)

37. Before the double helix, Erwin Chargaff ; [A] = [T]; [G] = [C] [ A ] + [ G ] = [ T ] + [C] i.e. [Purines] = [ P yrimidines ] % G + C (base composition of DNA) differs among species but constant in all cells of an organism within a species G + C content varies from 22% - 73%

38. The Phosphate functional group It gives DNA and RNA the property of an acid (a substance that releases a H+ in solution), h ence the name nucleic acids

39. Nucleotides and Nucleosides Nucleotides are the monomeric constituents of DNA and RNA. They are composed of; 5-C Sugar Nitrogenous base Phosphate group Nucleosides are composed of; A sugar Nitrogenous base

40. Nucleosides vs. Nucleotides

41. Nucleotides, cont’d Nucleotides are joined (polymerized) by condensation reactions to form chains of DNA and RNA The –OH on the 3’-C of a sugar of one nucleotide forms an ester bond to the phosphate at 5’-C of another nucleotide; The 5’ – 3’ P hosphodiester bond

42. The Phosphodiester bond

43. Nomenclature of N ucleotides -The triphosphate form is the precursor building block for DNA and RNA chain -Knowing complex names of nucleotides is important for understanding scientific literature a nd for ordering the correct compound when planning an experiment

44. Significance of 5’ and 3’ The two ends of DNA/RNA are designated by symbols 5’ and 3’ 5’ refers to C in the sugar to which a phosphate (PO4) functional group is attached 3’ refers to C in the sugar to which a –OH functional group is attached Thus, symmetry of the end of a DNA strand implies that each strand has a polarity determined by which end bears the 5’-PO4 and which end bears the 3’-OH group

45. Significance of 5’ and 3’ 5’ – 3’ directionality of nucleic acids is an extremely important property. Understanding this polarity is critical for understanding; DNA replication Transcription Reading DNA/RNA sequences Carrying out an experiment in a lab Genomics / Bioinformatics By convention DNA sequences are written with the 5’-end to the left and 3’-end to the right; 5’-TGGCCCGGGTCGACGGTGACACCGTGTTC-3’

46. Secondary structure of DNA: the Double H elix DNA is a double helix; an icon for modern biology Characterized with H-bonding between bases of two strands H-bonding is also referred to as “ Watson-Crick ” or “ Complementary base pairing ” A with T, two H-bonds; G with C, three H-bonds Watson-Crick base pairing explains Chargaff’s rules 1’-C atoms in the two strands are exactly the same distance apart (1.08 nm)

47. Watson-Crick base-pairing in DNA

48. Structure of Watson-Crick double helix Alternating deoxyribose sugars and PO4 groups form the backbone of DNA Bases are attached to sugars, located between the backbones of the DNA strands, lying perpendicular to the long axis of the strands As backbones of the two strands wind around each other, they form a double helix Polarity in each strand is 5’ to 3’ of the double helix; one end has a 5’-PO4, the other end 3’-OH H-bonding occurs only if polarity of the two strands runs in opposite direction; hence the two strands of the double helix are antiparallel Double helix has major and minor grooves

49. The double helix, cont’d

50. http:// www.bbc.com/news/science-environment-29760212

51. Significance of complementary bp Ensures storage, preservation and transmission of genetic information ; one strand is coding (sense, non-template strand) , the other is noncoding (antisense, template strand) Ensures fidelity during DNA replication; each of the two daughter DNA molecules has one old strand derived from the parent and one newly made strand; c omplementary bp results in the two daughter DNA molecules being identical The arrangement of bases determine: Functionality; i.e., genes vs. non-coding DNA Organism or species identity; Evolution Each strand is the predictable counterpart of the other Once you know the sequence of one strand, you can easily figure out the sequence of the other strand; Molecular Biology is the easiest discipline?

52. DNA can undergo reversible strand separation Reversible strand separation allows DNA replication, Transcription, Translation with high fidelity Same features make it possible to manipulate DNA in vitro Unwinding and separation of DNA strands is referred to as “Denaturation” The temperature at which half the bases in a DNA sample have denatured is called Melting Temperature (Tm)

53. Denaturing, R enaturing , Hybridization of DNA

54. DNA tertiary structure: Supercoiling DNA supercoiling is a twisted 3-D structure which is more favorable energetically Supercoiled DNA leads to localized denaturation in which the complementary strands come apart in a short reaction This is important for cellular processes like replication and transcription DNA topoisomerases relax supercoiled DNA

55. Topoisomerases relax supercoiled DNA

56. Drugs targeting DNA topoisomerases Some anti-cancer drugs inhibit topoisomerases Antibacterial agents: DNA gyrase inhibitors Ciprofloxacin and derivatives; Novobiocin , etc.