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In 1952, Martha Chase and Alfred Hershey published their "Blender" experiment, showing that phages (bacterial viruses) inject DNA (not protein) into bacteria Hershey and Chase bacteria. used radioactive isotopes to mark either DNA (phosphorous isotope 32P) or protein (sulfur isotope 35S labeling Met and Cys amino acids). The figure on the right represents two experiments conducted in parallel, one using phages containing 32P-labeled DNA (left) the other using phages that contained 35S-labeled proteins (right). After the phages had injected their genetic information into the bacteria, the phages were removed from the bacteria by shearing them off in a blender. After separation by centrifugation, b th phages and b t i were analyzed f radioactivity. B t i were t if ti both, h d bacteria l d for di ti it Bacteria only found to contain radioactive molecules, if the DNA molecules had been labeled with 32P, demonstrating that DNA was the carrier molecule of genetic information.

After publication of the Hershey-Chase Experiment, research teams at the California Institute of Technology (Caltech) the King's College in London at the Cavendish Laboratory in Cambridge competed in the "Race for DNA".

Linus Pauling at Caltech assumed a helical structure consisting of three strands with the phosphates building a central core in this structure while the bases were pointing outward structure, outward. Phosphates were thought to be joined by anhydride bonds in the center of the helix.

Top view on this "triple helix" with the central phosphate core. Two major problems arouse from this structure: It did not explain how the negatively charged phosphates could be held together. in addition, the (weak) bases are uncharged and thus hydrophobic at physiological pH. In the triple helix they would be exposed to water...

Model rebuilt from Linus Pauling's notes.

Surprisingly, Linus Pauling completely ignored Chargaff's first parity rule, based on which the DNA seemed to be a double helix (or at least a two-stranded structure) Obviously structure). there was some base pairing between a purine and a pyrimidine base required, where A paired with T and G paired with C.

Rosalind Franklin and Maurice Wilkins (King's College London) worked on the crystal (King s structure of DNA. X-ray beams can be focused onto the DNA strands and become diffracted. The diffracted X-rays give a characteristic "diffraction pattern" (a pattern of black spots) on an X-ray film. The famous original picture that Maurice Wilkins showed to J. Watson and F. Crick is shown on the left. The evaluation of the spots allowed the crystallographers the following assumptions: DNA has a helical structure and consists of more than one strand (2 or 3 were possible). In addition, one can measure distances of repeating units: the helix shows periodicity of 3.4 Angstroms (340 pm) and of 34 Angstroms. It was later shown that these observations of periodicity correspond to the stacking of bases (3.4 Angstroms) 10 bases per turn (34 Angstroms). Rosalind Franklin also gave the most important hint on the structure (communicated by Maurice Wilkins): the phosphates had to be on the outside, clearly contradicting the previous model postulated by Pauling and Corey.

After having retrieved this information from the X-ray film Watson and Crick started working X ray film, on building a model.

The original Watson/Crick DNA model is shown here However before they finally got it right here. However, right, they also had many other models (including a triple helix with a central core, just as Pauling had suggested). One of the problems was that Watson started working on the model with the wrong tautomeric forms: For example, he used the lactim (= enol) form of guanine instead of the lactam (= ketone) configuration which changes the arrangement of hydrogen bond donors and acceptors.

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Watson and Crick discovered the correct mode of base pairing of purines with pyrimidines in molecules of DNA. This is possible because the bases have very different abilities to donate and accept hydrogen bonds, as shown in the slide. H bond donors (green) and H bond acceptors (red) are indicated. Note the perfect match between A-T and G-C.

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There were different theoretical possibilities how the bases could pair. However one important aspect was that DNA always had to form the same structure irrespective of the structure, sequence of bases within this structure. Therefore whatever building blocks the DNA should be made out of, they all had to have the same dimensions and geometry. This holds true for GC and AT base pairs held together by hydrogen bonds between them. The similar geometries allow for a regular structure of the DNA macromolecule. AT and GC base pairs differ in the number of hydrogen bonds between bases (2 between A and T, 3 between C and G) and in the dipole moment due to Van der Waals interactions between th b b t the base aromatic ring structures (th l tt was not di ti i t t (the latter t discussed t d ) d today).

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Base pairs Base-pairs are perpendicular to the long axis and to the sugars The sugars and phosphates sugars. form the backbone of the DNA helix which is right-handed. One full helical turn comprises 10 layers of bases. Under physiological conditions, two layers of base pairs are 3.4 (340 pm, = helical rise) apart from each other. The spatial arrangement between the sugars and bases, electrostatic repulsion between phosphates as well as stacking forces between bases contribute to the helical twist in DNA. The DNA molecule then has a diameter of approximately 20 .

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Base pairing also means that each sequence on one strand has only one "complementary" sequence defining the opposing DNA strand Here you see how the two complementary strand. strands make up a right handed helix.

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This is the first page of the article that Watson and Crick wrote to the journal Nature On the journal, Nature. left you see them next to their famous DNA model with a hand-drawn model of the double helix.

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Double stranded Double-stranded DNA helices have a major (larger) and a minor (smaller) groove at their surface.

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On the right, you see a top view of the DNA double helix showing the beautiful 10-fold symmetry of the molecule The top base pair (TA how can one tell?) is highlighted molecule. highlighted.

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DNA molecules have a minor and a major groove because the bonds that connect the bases to their sugars (fat blue lines in the picture) are not at an angle of 180. Thus, the sugars are closer together on one side of the base pair. When layer is put upon layer, one obtains two helical grooves, one much bigger than the other.

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Here you see the major and minor grooves for both GC and AT base pairs. The same atomic color code has been used for the double helix on the right This may help relate the right. base pair structures to the double helix.

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Interestingly, Interestingly both strands of a double-stranded DNA molecule carry the same information double stranded information. They can be regarded as the positive and negative of the same image. This means that each single strand can be used to recreate an identical double stranded molecule. Watson and Crick immediately realized that this could be the way in which genetic information is transmitted from one mother cell to two daughter cells.

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DNA can be denatured by heat at a temperature that depends on its base composition composition. The opposite process (renaturation) is called annealing. Annealing takes place when temperature is lowered. The complementary strands associate and the many local interactions are reestablished.

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Denatured DNA (meaning single strand DNA) shows a higher absorbance as compared to native (double-stranded) DNA This can be explained by the fact that in the DNA double DNA. helix the stacking of bases prevents absorption. If DNA is denatured, the bases become unstacked and can thus absorb more light. The absorption maximum at 260 nm remains unaltered. In the laboratory, DNA is thus usually analyzed spectrophotometrically at 260 nm. For concentration determination (Lambert-Beer-Law) one just has to know whether the sample contains single or double stranded DNA.

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Tm is the temperature at which half of the DNA molecules are denatured (separated into single strands). DNA rich in AT has a lower denaturation temperature than CG rich DNA. This is because of the different number of hydrogen bonds formed between the bases (2 for AT, 3 for CG). During the denaturation process no covalent bonds are broken, only very many small-scale interactions are broken. The number of the interactions is large and many of them need to be broken before the strands separate. This leads to the sigmoidal (cooperative) shape of the melting curve.

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DNA hybridization takes place when two complementary DNA strands that originally come from two different DNA molecules anneal together. This is used in many techniques in genetic engineering that we will talk about in a later class.

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RNA is usually single stranded in the cell However in principle RNA is capable of forming cell. However, complex secondary structures by similar base pairing as DNA. In these RNA base pairs, Adenine pairs with a Uracil base instead of Thymine.

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RNA is less stable than DNA particularly because of the additional alcohol group on C2 of DNA, C2 the sugar. The OH group is reactive and can lead to self-hydrolysis of the RNA molecule (see the figure).

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RNA can adopt complex secondary structures (most common is a so called stem-loop or hairpin structure) based on local base-pair interactions Note the UA base pairs instead of interactions. AT base pairs. In theory, there are multiple secondary structures one could generate from a single RNA molecule. However, many RNA molecules have a specific secondary structure. This structure may be recognized by RNA-binding proteins.

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Transfer RNAs (tRNAs) acquire a complex secondary structure They participate in structure. transferring (name!) single amino acids onto a growing polypeptide chain (will be discussed in more detail in another lecture). All tRNAs are very similar in their overall structure but differ in two respects: A single tRNA is specific for a single amino acid. In this figure a Tryptophan-specific tRNA from yeast is shown (note that the Trp is attached to 3' end of the tRNA). In a specific region, the "anticodon stem", a sequence consisting of three nucleotides is later responsible for reading the genetic code (will be discussed later).

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