1. DNA STRUCTURE, GENETIC CODE, CHROMOSOMES MOLECULAR BIOLOGY – DNA structure, genetic code

2. GENES ARE ON CHROMOSOMES DNA is the carrier of the genetic information MOLECULAR BIOLOGY – DNA structure, genetic code

3. DISCOVERY OF THE STRUCTURE OF DNA DISCOVERY OF THE STRUCTURE OF DNA

4. DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES: 1. Provide a means for its own replication 2. Be able to encode the genetic information 3. Direct cell function 4. Accommodate changes caused by ‘mutations’ MOLECULAR BIOLOGY – DNA structure, genetic code

5. James D. Watson 15 years old accepted to University 22 years old received Ph.D. in zoology at Indiana University, Bloomington 1950 breif research stay in Denmark and attended a conference in Napoli - developing interest in DNA Lecture by Maurice Wilkins (Kings College of London: KCL) - study of DNA structure by X-ray diffraction of DNA crystals MOLECULAR BIOLOGY – DNA structure, genetic code Watson moved to Cambridge in order to learn X-ray diffraction/ crystalography

6. X-ray crystalography MOLECULAR BIOLOGY – DNA structure, genetic code ‘crystalised’ sample e.g. protein or DNA fibre X-ray source X-ray beam Diffracted X- rays Photographic film Analysis of the diffracted X-rays detected on the photographic film yields structural information about the crystalised sample

7. Cavendish Laboratory, Cambridge Watson: studing the 3D-structure of myoglobin (X-ray crystalography) Francis Crick 33 years old Ph.D. student (WWII) studing haemoglobin - physics background MOLECULAR BIOLOGY – DNA structure, genetic code Both men were interested in the problem of how genetic information was molecularly stored - favouring DNA Wanted to solve DNA structure

8. Chemical composition of DNA was known: 5-carbon sugar (2’-deoxyribose) Nitrogen containing bases Phosphate 4 bases: Adenine, Guanine, (Purine bases) Thymine (T) and Cytosine (Pyrimidine bases) DNA long polymer MOLECULAR BIOLOGY – DNA structure, genetic code Erwin Chargaff (rules) DNA of any species always had equal concentrations of A & T and G & C bases suggesting a fixed relationship in DNA The molecular structure of these componenets was however unknown

9. Linus C. Pauling Models of aminoacids cut from paper plausible 3-D models could be built from knowledge of chemical bonding and bond distances to fit experimental data. Keratin (William Astbury‘s alpha form of protein - also a beta form) alpha-helix MOLECULAR BIOLOGY – DNA structure, genetic code Very eminent and respected molecular biologist who was known to be working on uncovering the molecular structure of DNA 3D structure of proteins by X- ray crystalography

10. Rosalind E. Franklin (1920-1958) Colleague of Wilkins at KCL (albeit a fractous one) Exceptionally talented experimental chemist with extensive experience in X-ray crystalography MOLECULAR BIOLOGY – DNA structure, genetic code Discovered by careful experimentation and optimisation that DNA could exists in a dehydrated ‘A-form’ and a fully hydrated ‘B-form’

11. NOVEMBER 1951 – Rosalind Franklin gave a seminar on her DNA crystalography experiments James Watson attended the seminar: driven by the perceived competition from Pauling, Watson & Crick proposed their first model of the structure of DNA it was an embarrassing failure and was quickly discredited the model incorrectly placed the phosphate groups at the inside and bases on the outside later emerged that Watson had incorrectly recalled Franklin’s data! Franklin’s excellent background in physical chemistry and her knowledge of the different hydration forms of DNA allowed her to dispute that hydrophilic phosphate groups would be in the centre whilst the hydrophobic bases would be on the outside! MOLECULAR BIOLOGY – DNA structure, genetic code

12. Franklin was characteristically cautious about over interpretation of the data Prior to Franklin‘s identification of ‘A’ and ‘B’ forms of DNA, complete interpretation of DNA X-ray diffraction patterns was hampered by the presence of both hydration forms in the crystal - (Wilkins and Astbury) MOLECULAR BIOLOGY – DNA structure, genetic code 1952 Franklin (Ph.D. student Raymond Gosling) produced a very high resolution X-ray diffraction image from a pure crystal of B- form DNA ‘Photo 51’ IMPORTANT DEDUCTIONS/ HINTS: 1) DNA was helical and most likely a double helix consisting of 2 anti-parallel strands 2)Phosphates were on the outside of the helicies with the bases on the inside 3)The distance between bases (3.4A), the length of the period (34A i.e. 10 bases per turn of helix) and the rise of the helix (36 degrees)

13. MOLECULAR BIOLOGY – DNA structure, genetic code Early 1953 Watson: "The instant I saw the picture my mouth fell open and my pulse began to race" DOUBLE HELIX! Detailed calculations suggested two strands running in opposite directions with bases on inside Franklin about to leave KCL was instructed that the DNA work was to remain in there! She was preparing and had already submitted manuscripts. MRC grant report Data (inc. photo 51) Watson Wilkins showed Watson ‘photo 51’ without permission of Franklin Wilkins Gosling Max Perutz Crick Cavendish laboratory

14. How are the two strands held together & how do the bases interact with each other? In 1952 Crick was speculating about the potential attractive forces between the bases: Meanwhile Watson had been attempting to model base interactions by ‘playing’ with cardboard cut outs (c.f. Linus Pauling and the discovery of the protein alpha-helix) MOLECULAR BIOLOGY – DNA structure, genetic code mathematician friend John Griffith theorised which bases were most likely to be attracted to each other based quantumn mechanics Griffith suggested A-T and G-C as the most chemically attractive combinations at the time Crick was unaware of Chargaffs rules! Modelling proved unsuccessful as hydrogen bonding between base pair combinations seemed too weak and unsatisfactory!

15. ENOL FORMKETO FORMS However bases can exist in two TAUTOMERIC FORMS Jerry Donohue MOLECULAR BIOLOGY – DNA structure, genetic code Donohue advised Watson and Crick that the base tautomers in DNA are most likely to be the Keto form and not the Enol form they had been modelling

16. MOLECULAR BIOLOGY – DNA structure, genetic code Modelling using the keto form the hydrogen bonding worked! Moreover the specific base-pair combinations agreed with both Griffith’s theory and Chargaff’s rules

17. MOLECULAR BIOLOGY – DNA structure, genetic code James Watson and Francis Crick now had all the information they needed to build their model and publish the molecular structure of DNA

18. DNA STRUCTURE SOLVED! (immortality without even one experiment of their own!) MOLECULAR BIOLOGY – DNA structure, genetic code Nature April 25, 1953.

19. 5-carbon sugar Nitrogen containing base Phosphate 4 bases: adenine, guanine, thymine, cytosine D EOXYRIBO N UCLEIC A CID - DNA MOLECULAR BIOLOGY – DNA structure, genetic code Phosphodieste r backbone A-T & G-C hydrogen bonding base pairs

20. The two DNA strands have directionality as they are polarized polymers that run anti-parallel to each other MOLECULAR BIOLOGY – DNA structure, genetic code The repeating unit of the DNA polymer is the nucleotide (either; A, T, G or C), that is based around the 5 carbon sugar deoxyribose Each carbon in the deoxyribose sugar is numbered with 1’ - 5’ nomencluture 1’ 2’ 3’ 4’ 5’ DNA polymer formed by the formation of phosphodiester bonds between the 5’ phosphate group and the 3’ hyrdroxl group 5’ 3’ Therefore one end of each strand contains a 5’ phosphate group (actually triphosphate) whilst the other end contains 3’ hydroxl group 3’ OH 5’ P deoxyribose

21. 1962 Nobel Prize for medicine : Francis Crick, James Watson and Maurice Wilkins 1958 (37 years ) Rosalind E. Franklin MOLECULAR BIOLOGY – DNA structure, genetic code

22. DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES: 1. Provide a means for its own replication 2. Be able to encode the genetic information 3. Direct cell function 4. Accommodate changes caused by ‘mutations’ MOLECULAR BIOLOGY – DNA structure, genetic code

23. Watson & Crick knew that their DNA structure provided a possible copying mechanism based on specifc base-pairing How could this be achieved?

24. DNA replication theories MOLECULAR BIOLOGY – DNA structure, genetic code How do we experimentally test these theories? - proposed by Watson and Crick

25. DNA replication: Meselson-Stahl experiment normal 14 N MOLECULAR BIOLOGY – DNA structure, genetic code DNA extracted from E-coli grown for many generations on a heavy 15 N isotope of nitrogen will specifically sediment in a salt gradient heavy isotope 15 N cell generation by following the sedimentation characteristics of DNA extracted from E-coli transferred back to normal 14 N containing media one can infer the mechanism of DNA replication after each cell division possible replication mechanisms The DNA must replicate in a semi-conservative fashion as predicted by Watson & Crick (expanded upon on later lectures)

26. MOLECULAR BIOLOGY – DNA structure, genetic code http://www.sumanasinc.com/webcontent/animations/content/meselson.html Meselson-Stahl experiment video/ tutorial

27. DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES: 1. Provide a means for its own replication 2. Be able to encode the genetic information 3. Direct cell function 4. Accommodate changes caused by ‘mutations’ MOLECULAR BIOLOGY – DNA structure, genetic code

28. The Central Dogma of Molecular Biology - Francis Crick 1958 The genetic flow of information in a cell starts with DNA ‘instructions’ and passes through RNA ‘intermediates’ that dictate the synthesis of ‘functional’ protein INFORMATION Details in later lectures BUT WHAT IS THE CODE BEHIND THIS TRANSFER OF GENETIC INFORMATION ?

29. Marshall Nirenberg & Heinrich Matthaei Cracking the Genetic Code MOLECULAR BIOLOGY – DNA structure, genetic code Proteins consist of 20 different amino acids whereas DNA/ RNA have only 4 different nucleotides (Uracil, replacing T in RNA): If a sequence of 2 nucleotides encoded a single amino acid the code could only accommodate 16 amino acids (i.e. 4 2 ) however a triplet nucleotide could code for potentially up to 64 amino acids (4 3 ) Lysed E-coli cell lysate (protein synthesis apparatus intact) Synthetic poly-uracil RNA + 1 radiolabelled amino acid + 19 unlabelled amino acids Observe if the radioactively labelled amino acid would be incorporated into protein? Only when using labelled phenylalanine did the poly-uracil RNA lead to the production of radioactive protein The genetic code for the incorporation of phenylalanine into proteins had been cracked

30. MOLECULAR BIOLOGY – DNA structure, genetic code The Genetic Code Similar experiments identified the other three letter ‘codons’ found in messenger RNAs (mRNAs) responsible for the incorporation of the remaining amino acids into protein - Nobel Prize of 1968 N.B. that the genetic code is largely redundant with most amino acids having more than one codon Methionine and tryptophan only have one codon Three codons do not lead to incorporation of any amino acids - play role in terminating protein synthesis

31. MOLECULAR BIOLOGY – DNA structure, genetic code Cracking the genetic code video/ tutorial http://bcs.whfreeman.com/thelifewire/content/chp12/1202002.html

32. AUG UAA double stranded DNA TRANSLATION protein coding sequence or open reading frame Functional Protein MAPSSRGG….. MOLECULAR BIOLOGY – DNA structure, genetic code DNA sequence driven Genetic Code of the Central Dogma ATG GCT CCT TCT TCC AGA GGT GGC...... TAA TAC CGA GGA AGA AGG TCT CCA CCG...... ATT 5’ 3’ single stranded mRNA AUG GCU CCU UCU UCC AGA GGU GGC...... UAA TRANSCRIPTION THE SEQUENCE OF SPECIFC NUCLEOTIDES IN DNA DICTATES THE SEQUENCE OF AMINO ACIDS IN THE FUNCTIONAL PROTEINS e.g. enzymes

33. DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES: 1. Provide a means for its own replication 2. Be able to encode the genetic information 3. Direct cell function 4. Accommodate changes caused by ‘mutations’ MOLECULAR BIOLOGY – DNA structure, genetic code

34. AUG UAA TRANSCRIPTION single stranded mRNA double stranded DNA Functional Protein TRANSLATION protein coding sequence or open reading frame MAPSSRGG….. MOLECULAR BIOLOGY – DNA structure, genetic code Mutations are variations in the DNA sequence e.g. single base pair substitution ATG GCT CCT TCT TCC AGA GGT GGC...... TAA TAC CGA GGA AGA AGG TCT CCA CCG...... ATT 5’ 3’ AUG GCU CCU UCU UCC AGA GGU GGC...... UAA SUCH MUTATIONS CAN INFLUENCE THE FUNCTIONALITY OF THE PROTEIN e.g. changing which amino acid is incorporated ATG GCT CCT TCA TCC AGA GGT GGC...... TAA TAC CGA GGA AGT AGG TCT CCA CCG...... ATT 5’ 3’ AUG GCU CCU UCA UCC AGA GGU GGC...... UAA MAPSSSGG….. Functional Protein

35. MOLECULAR BIOLOGY – DNA structure, genetic code Most DNA is not coding for proteins ! Only 1.5% of the human DNA genome directly encodes amino acids for incorporation into proteins

36. 600x Human DNA: 3 200 000 000 letters 200x 500-pages books A single cells stretched out DNA = 1.8m MOLECULAR BIOLOGY – DNA structure, genetic code How is DNA organised in the cell?

37. Bacterial DNA ~ 1 mm long ~ 1  m 1000 x more than …thanks to mobiles no more twisted telephone cords! Most bacterial DNA exists in a covalently closed circular form SUPERCOILING MOLECULAR BIOLOGY – DNA structure, genetic code

38. protein scaffold A typical bacterial chromosome consists of about 50 giant supercoiled loops of DNA TOPOISOMERASES – enzymes that insert or remove supercoils Type I … break only one strand -> relaxing or twisting of the helix Type II … break both strands and pass another part of the double helix through the gap MOLECULAR BIOLOGY – DNA structure, genetic code

39. Eukaryotic DNA is complexed with HISTONE proteins that together form more and more ordered structures of CHROMATIN resulting in chromosomes H2A, H2B, H3, H4 200 bp 80 bp 40 bp Nucleosome

40. MOLECULAR BIOLOGY – DNA structure, genetic code Eukaryotic chromatin hierarcheal structure Net result is that a eukaryotic (human) cell’s DNA is packaged into a mitotic chromosome 10,000 fold shorter than it extended length! ‘beads on a string’ 30nm ‘solenoid fibre’ scaffold associated fibres Condensed chromosome

41. SECOND SUMMARY – CRUCIAL KNOWLEDGE MOLECULAR BIOLOGY – DNA structure, genetic code

42. T A C C G T T A G T T C A C G A T T A T G G C A A T C A A G T G C...... T A A START STOP CODING SEQUENCE A U G G C A A U C A A G U G C U A A RNA densely packed chromosome part of the chromosome where gene X is located A U G G C A A U C A A G U G C U A A RNA TRANSCRIPTION DNA  RNA Met Ala IleLysAla PROPERLY FOLDED PROTEIN executes its function in cell double-strand DNA MOLECULAR BIOLOGY – DNA structure, genetic code The Central Dogma TRANSLATION Ribosomes, tRNAs (expanded later)