Lecture 7: The structure of DNA [G] ,but revise x-ray crytsallography Flashcards
Thursday 10th October 2024
What did Phoebus Levene show in 1930?
That each building block of DNA is a nucleotide: a phosphate group linked to a deoxyribose sugar, which is linked to one of 4 nitrogenous bases.
What were Phoebus Levene’s key controbutions?
- Identified DNA’s building blocks: Deoxyribose sugar, phosphate group, and nitrogenous bases.
- Proposed the nucleotide structure: Phosphorylated nucleoside = nucleotide.
- Defined carbon naming convention: Carbons in ribose/deoxyribose named 1′ to 5′.
- Identified the sugar in DNA as deoxyribose
- Came up with the tetranucleotide molecule in 1930, which proposed DNA as a ring structure with four nucleotides, each base occurring in equal proportion (A=T=C=G). (incorrect)
What is a nucleoside?
A sugar and a base
What are the 4 DNA bases?
Adenine, guanine, cytosine, thymine
Who came up with the tetranucleotide theory?
Phoebus Levene, and this was before he had even worked out the structure of a nucleotide.
What was the tetranucleotide theory?
DNA as a ring structure with four nucleotides, each base occurring in equal proportion (A=T=C=G).
What sugar is used in DNA?
Deoxyribose
Draw out deoxyribose sugar
Draw out ribose sugar
What is the structure of purines?
They’re double-ringed
What are the 2 purines?
Adenine and Guanine
What is the structure of pyrimidines ?
They’re single-ringed
What are the 2 pyrimidines?
Cytosine, Thymine, Uracil (in RNA only)
How do glycosidic bonds form in pyrimidines?
Between the carbon 1 of the sugar and the N1.
How do glycosidic bonds form in purines?
Between the carbon 1 of the sugar and N9.
How do you get from a nucleoside to a nucletoide?
You phosphorylate the nucleoside (add a phosphate group to it)
Where do ester links form in nucleotides?
between sugar C-5’ group and the phosphate
Draw 2’- deoxyguanosine – 5’ monophosphate
Draw 2’-deoxycytidine – 5’ monophosphate
Draw 2’- deoxyadenosine - 5’ monophosphate
Draw 2’-deoxythymidine – 5’
monophosphate
Nucleotides nomenclature
What does a phosphodiester bond do?
A phosphodiester bond 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.
Is the DNA base sequence read from 5’ to 3’?
Yes
What does the 5’ end of a DNA strand have?
A phosphate group
What does the 3’ end of a DNA strand have?
A hydroxyl group
What must be considered everytime a nucleic acid is considered?
Polarity/Directionality
What did Phoebus Levene propose in 1930?
The Tetranucleotide model that said that the 4 nucleotides occurred in tetanucleotide blocks, with the bases pointing outwards.
He therefore came to the conclusion that DNA was simple and repetitive and could not be the genetic material.
This meant that Avery et al, who showed that DNA was the transforming principle (1944) were not believed and in 1952, Hershey and Chase could go no further than state that ‘…DNA has some function. Further chemical inferences should not be drawn…’
At the time, who was one of the only scientists who favoured Avery’s paper over Leuvene’s?
Erwin Chargaff
If Levene’s tetranucleotide model were correct, then the nucleotides in DNA would be present in…
equal proportion. So, % T = % A = % G = % C
What were the objectives of Chargaff’s experiment?
- To analyze the base composition of DNA in different organisms.
- To test whether the proportions of adenine (A), thymine (T), guanine (G), and cytosine (C) were equal, as predicted by Levene’s tetranucleotide model.
How did Chargaff experiment in 1950?
- He measured the base compositions in different organisms.
- He did this with: Homo Sapiens, Sweetcorn, fruit flies, and ducks.
- He also recognised that there was a certain proportion of GC’s.
- None of these fitted the accepted model. As the percentages of the bases weren’t equal. Instead, %T = %A and %G = %C, and %GC varied with the organism.
- Observed differences in GC content across species, suggesting DNA’s biological diversity. While A=T and G=C ratios were constant within a species, the relative proportions of (A+T) and (G+C) varied between species. This disproved Levene’s idea of DNA having uniform composition across all organisms.
~31% A, ~31% T, ~19% G, ~19% C.
~31% A, ~31% T, ~19% G, ~19% C.
Who was the leading structural chemist in 1950?
Linus Pauling
Who described the alpha helix in 1951?
Linus Pauling
What technique enabled Linus Pauling to work out the Alpha helix structure in 1951?
X-ray crystallography
How did x ray crytallography work ?
- Take a molecule and crystallise it to arrange its atoms in a regular, repeating pattern.
- When those crystals are formed, you hang it in front of an x-ray source and illuminate it with x-rays.
- Those x-rays hit the crystals and are deflected in different directions through a process called diffraction.
- The diffracted X-rays form a pattern of spots (reflections) on a photographic film or detector.
- You hold a piece of photographic film at the back, which gets exposed, then developed.
- You see spots on the photographic film called reflections.
- Because you know the distance from the target to the film, and because you know the wavelength of the x-rays, you can use maths to find out the structure of the molecule and what exactly caused the diffraction.
- Using the tops of both the blobs, a cross could be drawn.
- Worked out that whenever you see an x-ray image of a cross and worked backwards, you’d end up with a helix.
Pauling noticed a characteristic cross pattern in the X-ray diffraction images of proteins, indicating a helical arrangement.
Pauling noticed a characteristic cross pattern in the X-ray diffraction images of proteins, indicating a helical arrangement.
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.
What happens when you add a photographic film and then expose and develop this?
A single spot appears on a screen
What happens when you widen the slit?
All points across the slit act as a point source.
The result is a single slit diffraction pattern on the screen, with a central beam of light that is surrounded by 2 other beams of light.
When was the wave theory of light published?
In 1678
What happens if there are 2 slits?
The diffraction pattern interferes, and the result is a complex double slit interference pattern on the screen.
What are the size and shape of the pattern dependant on?
- The width of the slits
- How many slits there are
- How far apart the slits are
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The more slits there are…
the sharper the pattern becomes
How can you calculate how far apart the slits are?
By knowing the wavelength of the incident light.
What did Augustin Fresnel show in 1815?
That diffraction also occurred around a solid object with the same width as the slit, and that the result was similar to the familiar diffraction pattern on the screen.
Is it true that feautures that are close produce widely separated reflections?
Yes
Is it true that features that are distant produce closely separated reflections?
Yes
What do vertical gratings produce?
Horizontal spots
What do horizontal gratings produce?
Vertical spots
What do grids produce?
A cross
What do angled grids produce?
An angled cross
Why do reflections from a helix form a cross?
Because the aligned helices effectively form an angled diffraction grid, which produces a cross.
How do we know that the DNA is helical?
Because photo 51 shows a cross image.
Could DNA be crystallised at the time?
No, instead it could be stretched into long fibres that could be mounted in front of the X-ray source. This is called X-ray fibre diffraction.
Who stretched DNA and air dryed it, and then illuminated it with X-rays?
Maurice Wilkins
Who stretched out DNA and left it hydrated before illuminating it?
Raymond Gosling, Rosalind Franklin’s DNA student
Describe Maurice Wilkin’s X-ray image of DNA that he produced in 1950
- He produced an image using the process of X-ray diffraction of dried DNA (the A form) .
- It was high resolution, but without a clear model in mind, it was difficult to interpret.
Describe Gosling and franklin’s photo 51
- In 1952, after 100 h of exposure, Gosling and Franklin produced ‘Photo 51’ using hydrated DNA – the ‘B’ form.
- The cross was unmistakable and so it was concluded that DNA must be helical .
Who was involved in the race to find out the structure of DNA?
- Linus Pauling
- Rosalind Franklin
- James Watson, Francis Crick, Maurice WIlkins
What structure of DNA did Linus Pauling propose?
He proposed there being 3 strands of DNA, wrapped around each other, with the bases pointing outwards.
Placed phosphates in the core, which repelled due to negative charges.
Why did many people believe Pauling’s proposed DNA structure?
Because he was the foremost structural chemist
What DNA model did James Watson, Francis Crick, Maurice WIlkins propose?
a double helix 2-strand model, with the bases pointing inwards. This was quickly debunked by Watson and Crick.
What was each turn of the helix of DNA measured to be?
3.4 nm
What was the distance between the bases measured to be?
0.34 nm (10 bases per turn of the helix)
What was the pitch of the helix measured to be?
2nm
How did Watson and Crick come to the conclusion that the DNA was a double helix?
- The data from photo 51 were consistent with a double helix, with the bases paired.
- Remembered Chargaff’s base pairing. (one of purine and pyrimidine was perfect)
- Realised that the strands must be antiparallel, as this was the only way that the bond angles would work.
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What are the 6 key features of the Watson-Crick model?
1) The two polynucleotide chains are wound in a right-handed (clockwise) double-helix.
2) The strands are anti-parallel (from consideration of bond angles)
3) Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis.
4) Complementary base pairs from opposite strands are bound together by weak hydrogen bonds.
5) The B form of DNA is now regarded as having 10.5 base pairs per turn, the helix turn is now regarded as 3.6 nM.
6) The sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves.
Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because the backbones do not get in the way.
How many hydrogen bonds do Adenine and Thymine form?
2
How many hydrogen bonds do Guanine and Cytosine form?
3
What are the structural variants of DNA
A DNA
B DNA
Z DNA
A DNA
only in low hydration conditions.
right-handed helix
non-physiological
B DNA
the most structurally stable form under physiological conditions.
hydrated
right-handed helix
Z DNA
taken up physiologically by stretches of alternating pyrimidines and purines.
left-handed helix
Who got the nobel prize in 1962 for discovering the structure of the DNA?
Watson, Crick, and Wilkins.
Franklin’s contributions were only recognized after she had passed away.
