We will cover DNA first : deoxyribonucleic acid
DNA is a polymer of deoxyribonucleotides
All living cells (both prokaryotic and eukaryotic) contain double stranded DNA as their genetic material.
Chemical Structure of DNA
Individual monomers - deoxyribonucleotides (nucleotides)
Components:
1) deoxyribose (5 carbon sugar, pentose sugar)
see text for structure of deoxyribose
2) phosphate group (1 phosphorus and 4 oxygens)
see text for structure of phosphate group
At pH 7.0 the phosphate group has a net negative charge. The phosphate group is attached to deoxyribose at the 5' carbon position and the 3' carbon position.
The alternation of deoxyribose-phophate-deoxyribose, etc. is referred to as the phosphate sugar backbone of DNA. The type of chemical bond that holds the backbone together is called a phosphodiester bond. It is a very strong covalent bond (oxygen from phosphate group forms an ester linkage with either the 3' or 5' carbon).
3) Nitrogenous Base - attached to deoxyribose at the first carbon. Four types are found in DNA:
adenine, guanine, thymine, cytosine
Two structural families:
purines - double ring structure (adenine and guanine)
pyrimidines - single ring (thymine and cytosine)
see text for structure of purines and pyrimidines
Base + Sugar = nucleoside
Base + Sugar + Phosphate Group = nucleotide
So a DNA molecule is a long polymer of subunits called deoxyribonucleotides. The backbone of the polymer is composed of phosphate groups alternating with a sugar (deoxyribose) to form a covalently linked chain. Just like with proteins N-----> C the chain has directionality 5'P--------->3'OH Attached to the sugar ring is one of 4 nitrogenous bases. The bases project out from the sugar-phosphate backbone toward the center of the structure. (Sort of like the R groups of the proteins projecting out).
*The order of nucleotide bases along the chain, provides the information that specifies the composition of a cell's protein molecules.
Chargoff - Examined base ratios from bacteria, animals, and plants. Whenever he determined the ratio of the two purines (adenine / guanine) the A:G ratio was found to vary dramatically.
0.4 for bacteria 1.72 for yeast
Interestingly, the ratio of the two pyrimidines (thymine to cytosine) also varied
However, from such data, a common theme emerged: the ratio of purines to pyrimidines was always 1:1.
Chargoff's Rule A+G = T+C
Out of these observations the idea of base pairing between purines and pyrimidines emerged.
You may often hear reference to G:C content
G+C
G+C+A+T relative amount of G+C to total number of bases
Some evidence was also coming in concerning the physical structure of DNA. X-ray diffraction revealed that DNA was a very long, thin structure - stacking of bases periodicity.
Watson - Crick
Compiled available data to propose a model for the structure of DNA. They themselves contributed no data to the model, they simply interpreted other people's findings into an hypothesis.
Nature 1953 171:737-738 The structure of deoxyribonucleic acid.
So...DNA consists of two strands held together by hydrogen bonding between adjacent base pairs. The two strands are then twisted to the right to form a helix.
*10 base pairs per turn
*each base pair is 0.34 nm thick
*10 base pairs would be 0.34 nm X 10 = 3.4 nm length
If you are given the length of a segment of DNA you can calculate the number of base pairs. If a DNA molecule is 1 um
1u = 1000nm
1000 nm/0.34 nm/bp = 2941 base pairs
B -DNA has major and minor grooves because of the helix.
Very importantly, it is these grooves which allow DNA binding proteins access to the bases. Sequence specific proteins, must make contact with base pairs. Major groove is most accessible.
DNA is anti-parallel
One strand runs from 5' ------>3' while the opposite strand runs 3'----->5'.
Both DNA replication and transcription occur from the 5' end toward the 3' end. In other words new nucleotides are always added onto the 3' hydroxyl group.
Two DNA strands are complementary due to specific base-pairing.
adenine is always hydrogen bonded to thymine (2 hydrogen bonds)
cytosine is always hydrogen bonded to guanine (3 hyrdrogen bonds)
The structure of DNA we have described today is designated B-DNA.
Other forms are known to exist. i.e. Z-DNA, left handed DNA in a zig zag pattern.
Probably exerts a regulatory activity when in Z conformation.
The two DNA strands are held together by base pairing (hydrogen bonding) between complementary bases. Cumulative effect of all of the hydrogen bonds is pretty strong, but only a little stronger than energy of thermal motion at RT.
At elevated temperatures, the 3D structure of both proteins and nucleic acids (or any strucuture held together by H bonding) is disrupted.
denaturation - disordered 3D structure, disrupted due to broken H bonds
native - ordered 3D state, presumably as in biological systems.
Native DNA is double-stranded DNA
Upon heating double-stranded DNA------------------> single stranded DNA
Absorbance - ability of a substance to absorb light
Visible light 400-700 nm
DNA absorbs maximally at 260 nm (in the UV range)
Proteins absorb maximally at 280 nm
The ability of DNA to absorb light (260nm, UV) increases as denaturation progresses. Absorbance is primarily a property of the nitrogenous bases. When they are hydrogen bonded and in close proximity they cannot absorb light as well as when they are exposed. Absorbance goes up at 260nm even higher when the DNA is completely hydrolysed.
If you measure the absorbance of a sample under increasing denaturing conditions (i.e. heat) you can generate a melting curve.
For example, lets assume that for a particular sample of DNA:
Absorbance of double stranded DNA =1.00
Absorbance of single stranded DNA=1.37
Tm=melting temperature
or the temperature at which the rise in A260 is half complete
Notice from the curve:
Absorbance remains constant up to temperatures well above those encountered by living cells in nature.
When absorbance rises, the rise is steep over 6-8C.
Curve plateaus as the 2 strands separate.
If you hold DNA length constant and the concentration of DNA in the sample constant, the Tm is a pretty good indication of base content.
RULE: Tm increases as [G+C] increases. The higher the GC content of the DNA the higher the Tm.
GC base pairing involves 3 hydrogen bonds (vs two in AT pairing) Therefore it takes more heat to disrupt the pairing.
pH changes can also be used to denature DNA
Hydrogen bonding is very sensitive to H+ concentration
At pH greater than 11.3 (alkaline conditions) all hydrogen bonds are eliminated and DNA is completely denatured (in single stranded form).
Acid conditions also denature DNA but... there is damage to phosphodiester linkages. Phosphodiester bonds are resistant to alkaline pH so use of alkaline pH is method of choice for deliberately denaturing DNA in the laboratory.
Interestingly - repulsive forces also are at play in the DNA double helix Recall the negatively charged phosphate groups. Charges are neutralized by bound + ions such as Na+ and Mg++. *In dH2O, repulsions are so great, strand sepration or denaturation occurs.
Breathing - Double stranded state is stable, but double stranded regions frequently open to become single-stranded bubbles.
In AT rich areas, breathing is much more common.
Denaturation is reversible
Renaturation or reannealing
Very valuable technique which can be used to:
1) show genetic relatedness between organisms
2) can detect particular sequences (i.e. particular genes) if you have a DNA or RNA probe.
3) Can determine the frequency with which certain sequences appear [highly repetitive sequences will reanneal very rapidly].