Welcome to Introduction to Bioinformatics Introduction to Molecular Biology DNA to protein

1. Welcome toIntroduction to Bioinformatics Introduction to Molecular Biology DNA to protein

2. Molecular biology forms part of a long intellectual tradition • c. 350 BCE - Aristotle begins the Western tradition of natural philosophy • 1664 - Robert Hook coins the term “cell” in his treatise Micrographia • 1676 - Anton van Leeuwenhoek discovers bacteria, later spermatozoa • 1735 - Carl Linnaeus’ Systema Naturae lays the foundations for taxonomy • 1859 - Charles Darwin’s Origin of the Species • 1860s - Louis Pasteur disproves abiogenesis and develops the “Germ theory” • 1866 - Gregor Mendel describes the “inheritance of traits” in peas. • 1868 - Ernst Haeckel postulates that the nucleus responsible for heredity • 1869 -Friedrich Miescher isolates a crude nucleic acid preparation • 1880s - Cytologists work out the details of mitosis, meiosis and fertilization

3. Molecular biology forms part of a long intellectual tradition • 1903 - Walter Sutton proposes a chromosomal theory of heredity • 1908 - Thomas Hunt Morgan discovers that genes can mutate • 1909 – Archibald Garrod proposes the “gene-enzyme” hypothesis • 1915 – Morgan and colleagues publish linkage maps of D. melanogaster • 1927 – H.J. Muller and L.J. Stadler show that radiation can induce mutation • 1928 - Fredrick Griffith demonstrates genetic transformation • 1931 - Barbara McClintock proves genetic recombination – “crossing over” • 1944 - Avery, MacLeod & McCarty show that DNA carries genetic information • 1940s - The “modern synthesis” - E. Mayr, T. Dobzhansky, & J. Huxley • 1949 – Erwin Chargaff formulates “Chargaff’s rules” of DNA composition • 1952 - Hershey and Chase demonstrate that DNA is the genetic material • 1953 - James Watson and Francis Crick describe the double helix of DNA • 1957 – Vernon Ingram - genes determine the sequence of amino acids • 1958 - Matt Meselson and Frank Stahl prove semiconservative replication

4. Molecular biology forms part of a long intellectual tradition 1958 - Arthur Kornberg discovers DNA polymerase 1960 – Sam Weiss and Jerard Hurwitz independently discover RNA polymerase 1960s - Genetic code cracked by Crick, Marshall Nirenberg, Har Gobind Khorana, etc. 1961 - Charles Yanofsky & Sydney Brenner show colinearity of DNA & protein 1965 - Holley and Zamir determine the structure of a tRNA 20xx – Your contribution!!

5. Transcription Translation Replication The “Central Dogma” of Molecular Biology Term coined by Francis Crick in 1956 to describe the flow of information in the cell DNA RNA Protein

6. Information flow is compartmentalized DNA Transcription Pre-mRNA Processing mRNA Export mRNA mRNA Decay Translation protein Protein Decay

7. What is the nature of the Gene? Frederick Griffiths demonstrates “Transformation” of a heritable character in the bacteria Streptococcus pneumoniae

8. What is the nature of the Gene? Oswald Avery, Colin MacLeod & Maclyn McCarty first show that DNA is the “genetic principle” Enzymes used to degrade proteins

9. The Hershey-Chase experiment confirms that DNA is the stuff of heredity

10. Erwin Chargaff and his rules • Ratio of nucleotides depends on species • A=T and G=C no matter the organism

11. What is the structure of DNA? 1952 Rosalind Franklin and Maurice Wilkins produce X-ray diffraction images of DNA crystals that suggested that DNA must have some helical arrangement

12. What is the structure of DNA? 1953 Francis Crick and James Watson put together all of the clues and correctly deduce that DNA is a Double Helix

13. DNA base pairing occurs through hydrogen bonds A:T pairs: 2 bonds G:C pairs: 2 bonds

14. The double helix strongly suggested that DNA replication might proceed by a “semiconservative” process

15. The Meselson-Stahl experiment argues for strand separation during DNA replication

16. Genes control the amino acid sequence of proteins • 1957 – Vernon Ingram shows that sickle cell haemoglobin varies from wild type by the substitution of one amino acid

17. Genes control the amino acid sequence of proteins Alteration of amino acid sequence is also observed in all other hereditary anaemias!

18. DNA cannot directly specify the sequence of amino acids in proteins • Protein synthesis in eukaryotic cells known to take place in the cytoplasm • There must therefore be a SECOND information containing molecule that gets its specificity from DNA, but then moves to the cytoplasm • Attention immediately focuses on RNA – was easy to imagine that it could be produced from a DNA template • Torborn Caspersson and Jean Brachet demonstrated that RNA was mostly in the cytoplasm Jean Brachet (1909-1998)

19. Discovery of mRNA • T2 is a bacteriophage that infects E. coli • Completely shuts down normal cellular transcription. Only viral protein is made • T2 RNA always has the same composition as T2 DNA • T2 carries none of its own RNA • 32P is incorporated into RNA made after T2 infection • Only about 3-5% of cellular RNA • is messenger RNA!

20. The case for RNA Missing methyl group in uracil relative to thymine Hydroxyl group Chemically very similar to DNA

21. Easy to imagine RNA being produced from a DNA template

22. There must be a molecular machine that makes RNA from a DNA template • Jerard Hurwitz and Sam Weiss independently discover an enzyme that will only make RNA in the presence of DNA. • The enzyme uses ATP, GTP, CTP, and UTP as precursors POOR GUYS! 1959 Nobel prize was already awarded to Severo Ochoa for what turned out later to be the WRONG ENZYME!

23. RNA Polymerase is a molecular machine that carries out transcription

24. RNA is synthesised in the nucleus but travels to the cytoplasm Cells pulse-labelled with 3H coupled cytidine T = 15 minutes T = ~90 minutes D.M. Prescott

25. Ribosomes are the site of protein synthesis ribosomes studding the endoplastic reticulum Shown using radio labelled amino acids in conjunction with ultracentrifugation to isolate Different cell fractions. Where does the radioactivity end up at various times?

26. Ribosomesand associated rRNAs are the factories for protein synthesis

27. Crick’s adaptor hypothesis • Can folded RNA act directly as the template for protein synthesis? • Seems unlikely: • the nucleosides chemically want to react with water soluble groups • but many amino acids are polar • no clear way to discriminate chemically similar amino acids Crick proposes that an adaptor molecule must fit between RNA and the incoming amino acids, but its nature is unknown Incoming amino acid Adaptor molecule RNA

28. Crick’s adaptors (tRNAs) are themselves RNA molecules • Self-folding by complementary base pairs gives a structure with several functional domains • Account for ~10% of cellular RNA • abundance • Typically includes several modified, non-standard bases.

29. Zamecnick and Hoagland discover aminoacyl synthetases Enzymes that added an adaptor (that we now know to be tRNA) to amino acids prior to their incorporation in proteins It turns out these tRNAs are Crick’s proposed adaptors Mahlon Hoagland Paul Zamecnik

30. Translation proceeds through a tRNA intermediate

31. Nature of the genetic code • Obvious early on most likely a triplet code in order to code 20 amino acids: • 4 x 4 nucleotides can specify 42 = 16 amino acids • 4 x 4 x 4 nucleotides can specify 43 = 64 amino acids • Code must be redundant • Not overlapping – Sydney Brenner’s thought experiment • Marshall Nirenberg and Heinrich Matthaei showed that a homopolymer (UUUUUU…. etc. ) produced a poly-phenylalanine protein

32. Khorana's synthetic RNA approach to cracking the genetic code • Example RNA with two repeating units • RNAs with two repeating units: • (UCUCUCU → UCU CUC UCU) produced a polypetide consisting of alternating Serine and CUC codes for Leucine • RNAs with three repeating units: • (UACUACUA → UAC UAC UAC, or ACU ACU ACU, or CUA CUA CUA) produced three different strings of amino acids • RNAs with four repeating units including UAG, UAA, or UGA, produced only dipiptides and tripeptides thus revealing that UAG, UAA and UGA are stop codons.

33. The genetic code is (almost) universal

34. Amino acids fall into five functional categories

35. Study Question 11Degeneracy and frequency of amino acids Most commonLeu Gly Ser Least commonTrp Met His

36. Study Question 12Single mutation from AGA Silent: | Hydrophilic/Hydrophilic: |

37. Study Question 12Single mutation from AGA Silent: | Conservative: | Hydrophilic/Hydrophilic: | | Hydrophilic/Hydrophobic: |

38. Study Question 12Single mutation from AGA Silent: | | Conservative: | Hydrophilic/Hydrophilic: | | | Hydrophilic/Hydrophobic: | Other: |

39. Proteins have four levels of structure

40. The primary structure of proteins is determined by peptide bonds between amino acids Formation of this bond is associated with a small +ve DG Protein synthesis depends on coupled reactions!

41. Secondary structure - the alpha helix H bonds Alpha helical conformation is stabilised by hydrogen bonding

42. Secondary structure – beta sheets Parallel configuration Antiparallel configuration

43. Secondary structures combine to determine tertiary structure The enzyme acetylcholinesterase bound to acetylcholine

44. Proteins combine to form quaternary structures Collagen Haemoglobin

45. Allosteric interactions +ve -ve Interactions (usually with a small molecule) can alter the shape and activity of an enzyme

46. Enzymes lower the activation energies associated with biochemical reactions DG Typical energy of activation is 20-30 kcal/mol

47. Eukaryotic mRNA must often must be spliced in order to produce a mature transcript Exons often correspond to functional protein domains and alternative splicing can give rise to variant proteins