Lecture 7 – The Structure of DNA PDF

Lecture 7 – The structure of DNA Part 1 – what stopped scientists believing that DNA is a genetic material? Chemical nature of DNA In 1930 Phoebus Levene showed that each building block of DNA is a nucleotide: phosphate grouped liked to deoxyribose sugar which in turn is linked to one of 4 nitrogeno...

Lecture 7 – The structure of DNA Part 1 – what stopped scientists believing that DNA is a genetic material? Chemical nature of DNA In 1930 Phoebus Levene showed that each building block of DNA is a nucleotide: phosphate grouped liked to deoxyribose sugar which in turn is linked to one of 4 nitrogenous bases: Adenine (A) Guanine (G) Cytosine (C) Thymine (T) But in 1909, he presented his ‘tetranucleotide theory’ before he started to work out the structure of a nucleotide. Sugar used in DNA The pentose (5 carbon) deoxyribose Ribose is used in RNA and contain hydroxyl group at 2’ Deoxyribose is used in DNA and does not contain hydroxyl group at 2’ The nitrogenous bases Purine Adenine Guanine Pyrimidine Cytosine Thymine Uracil (RNA only) Nucleoside are formed from a base and the sugar Glycosidic bonds: between sugar C-1' and N-9 (purine) or N1 (pyrimidine) Nucleotides are phosphorylated nucleotides  Ester kinks are formed between sugar C-5' group and the phosphate Nucleotides come in multiple flavours: time for nomenclature DNA is a polynucleotide, with polarity (directionality) A phosphodiester bond (creates backbone of the DNA) links the 3’C of one nucleotide to the 5’C of the next. This means that DNA strands have POLARITY: a 5’end and a 3’ end. Phoebus Levene: the tetranucleotide model – 1930 Phoebus Levene proposed that the four nucleotides occurred in tetranucleotide blocks with bases pointing outwards. DNA was therefore simple and repetitive and could not be a genetic material. This meant that Avery et al., who showed that DNA was the transforming principle (1944), were not believed … and Hershey and Chase could go no further than state in 1952 that ‘… DNA has some function. Further chemical inferences should not be drawn…’ Part 2 – The importance of a cross on X-ray images Erwin Chargaff Chargaff accepted the unusual Avery paper and concluded that genetic differences among DNAs must be reflected in chemical differences among these substances. # If Levene’s tetranucleotide model were correct, then the nucleotides in DNA would be present in equal proportion. PREDICTION: % T = % A = % G = % C He re-organised his lab to test this. Chargaff’s rules – 1950  He measured base composition in different organisms Levene’s prediction: % T = % A = % G = % C REALITY: % T = % A and % G = % C (and also % GC varies with organism) Linus Pauling Was world’s leading structural chemist in 1950 Described the alpha helix – a structural motif present in many proteins Used powerful new technique called X-ray crystallography A cross formed by the ‘reflections’ gave him the clue X-ray crystallography, a helix and the cross The crystalline target molecule diffracts X-rays and cause exposed patches (‘reflections’) on photographic films. The resulting diffraction pattern is a unique "signature" of the molecule. Diffraction: the nature of light When light passes through a small opening, comparable in size to the wavelength of the light, a wave front is propagated on the other side. A single spot appears on a screen.  Huygens showed that if the slit is wider, all points across the slit act as a point source. The result is a single slit diffraction pattern on the screen. Diffraction: 2 slits cause interference Huygens showed that if the slit is wider, all points across the slit act as a point source. The result is a single slit diffraction pattern on the screen. If there are two slits, the diffraction patterns interfere. The result is a complex double slit interference pattern on the screen. Diffraction: around solid objects Augustin Fresnel showed that diffraction also occurred around a solid object with the same width as a slit. The result is (similar to) the familiar diffraction pattern on the screen. If there are two solid objects, the diffraction patterns interfere. The result is like the familiar double slit interference pattern on the screen. Spatial separation of points alters the interference fringes Features that are close produce widely separated reflections Features that are distant produce closely separated reflections Part 3 – The race to describe the structure of DNA Photo 51 – the basic dimensions (in 1953) The height of one helical turn - In an X-ray diffraction pattern, the closer the spots, the larger the actual distance in the target. Thus, the close horizontal bars correspond to large features: the helical turns. The separation of the bases - The more distant the spots, the smaller the actual distance in the target. The distance from the middle of the pattern to the edge gives the stacking of the bases (~0.34 nm). The pitch of the helix - The helix's pitch (degree of rise) can be calculated from the angle the "X" makes with the horizontal axis. Linus Pauling – 1953 The model failed because the negative charges on the stacked phosphate groups would repel each other and destabilise this molecule. 6 key features of the Watson Crick Model Feature 1: Right-handed helix Two polynucleotide chains are wound in a right-handed double helix Feature 2: The strands are anti parallel  Nucleotide chains are anti-parallel: one reads 5’ --> 3’, and the other 3’