Lecture 2 (2015)Apunte Inglés
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Lecture 2: DNA from shape to function
History of genetics:
Mendel defined Genes as a conceptual idea.
Since 1944, when it’s discovered that DNA are genes, scientists start to think about how do genes work instead of thinking about what are genes.
• The cell: During the investigation about mitosis, scientist discovered that the nucleus of the cell contains chromatin, which is made of DNA and proteins. They thought that proteins had the information to codify genes because they have a more complex structure but it was proved that DNA contained that information.
To prove this, they made an experiment: o Pneumococcus typeàinfection of mice § This experiment was made by Avery and proved that DNA is the molecule that contains genes.
• Structure of DNA: o The nucleotide: is the basic component of DNA. It is made of: § Nitrogen base Molecular Biology • • Small basesàpyrimidines: thymine and cytosine • Large basesàpurines: adenine and guanine § Pentose (deoxyribose) § Phosphate group Components of the DNA: The nitrogen bases are linked to the C1 of the pentose. The most important positions of the pentose are the ones we find in 5’ and 3’.
o Ester links: phosphate groups are used to link one nucleotide to another. These links are an ester link and connect the phosphate group to the pentose. This connection creates a polarity 5’à3’ which makes the DNA asymmetric.
o Hydrogen bonds: nitrogen bases can form pairs through hydrogen bonds between the radicals of the bases. This kind of bonds affects to those atoms with a slight negative charge and those atoms with a slight positive charge.
§ The more hydrogen bonds you have between nitrogen bases the strongest will be the union between the two molecules.
o Distance: the distance between bonds linking the bases to the pentose rings is almost the same independent of the pair (A-T or G-C).
o Angle: the angle of bonds linking the bases to the pentose rings is almost the same independent of the pair (A-T or G-C).
o Sequence: the key property of DNA is its specific sequence. No matter what the sequence is, the angle and distance are going to be the same and this makes the DNA a very stable molecule.
Codes with small number of components (as low-level languages) are more precise than those with large number of components. The low-level language of DNA allows the creation of more precise information.
• DNA double helix: The three-dimensional structure of DNA arises from the chemical and structural features of its two polynucleotide chains. The hydrogen bonds between the bases of the two different polynucleotides hold the two chains together.
Molecular Biology A double helix can be right-handed or left-handed. To know the direction of the helix you can follow it with your own finger.
While one polynucleoide runs 5’à3’ the other one goes the other way aroundàantiparallel conformation • Stability of DNA: The fact that the two pairs share angles and distances permits DNA to be a very homogeneous and stable molecule.
o There is a base paring with hydrogen bonds o Base stacking: one base pair is placed on top of the next base pair. The distance between the bases allows the atoms of the bases to interact with Van del Waals forces, which stabilize the double helix.
o Hydrophobic effects: The inner region of DNA is hydrophobic because the polar groups that belong to the bases cancel each other by making hydrogen bonds. This hydrogen bonds are found inside the double helix. With the hydrogen bonds atoms can’t interact with water because atoms interact with each other.
o Interaction with cations: we have free negative charges that create repulsion between consecutive nucleotides and to compensate this we need magnesium (Mg2+) because this cation interacts with negative charges of the double helix. Prevent the phosphates to break the structure with negative charges.
All these characteristics allow the double helix to be very stable. Stability means reaching very low internal energy. To break the DNA helix you need to heat up DNA up to 100ºC • Interaction between bases: Red: slight negative charge Blue: slight positive charge Molecular Biology The interaction between bases that are not complementary is less stable because of the number of hydrogen bonds. Chemically the bases are going to interact to be as stable as possible so they will pair to create as much hydrogen bonds as possible.
As more hydrogen bonds are between two bases, more stable is the structure.
In any combination that we try the number of the hydrogen bonds is lower than 5. The combination that we find in DNA is the more stable one. All chemical systems try to be in a state of minimal internal energy, so that’s why the bases are put together creating the maximum number of hydrogen bonds.
Only if the polynucleotide is antiparallel the bases can pair with the maximum number of hydrogen bonds. In addition, a parallel structure would not keep similar distance and angles between bonds linking the bases to the pentose rings.
DNA is a helix because of a “mistake”. It couldn’t be anything else but a helix because of the inevitable turn produced by linking a base pair to the next one.
In physics and chemistry there’s not an absolute 0. This little “mistake” (the angle between base pairs) prevents DNA from being a perfect straight structure and creates the helix.
• Separation of the two DNA stands Molecular Biology The helical structure of the DNA causes lots of problems to the cell because the cell has to separate the two strands in order to express genes and also during replication.
Every fifty thousand pairs of nucleotides, our DNA is fixed in the nucleus and it can’t turn around to separate the strands. To separate the stands at this point, it is necessary a force that must pull on the gap and compress the turns at the end. This is called supercoiling. Supercoiling is a way to prevent the superhelical tension introduced into the DNA by helix opening.
o Positive supercoiling: Is created when the two strands of the DNA are separated. This supercoiling occurs following the direction of the DNA, so it will be right-handed. The DNA is separated around the region where strands are being separated.
During transcription, the cell has to compensate this positive supercoiling only in specific locations but, in order to complete the process of replication, the cell must separate the whole length of strands by separating all the polynucleotides unions. To do this, there are enzymes called topoisomerases that avoid the accumulation of supercoiling.
§ Topoisomerase I: it doesn’t require energy from ATP. Its function is breaking one of the phosphodiester links in the polynucleotide strand. These enzymes remain linked into the chain (DNA backbone phosphate) covalently. This allows the strands to rotate freely relative to each other.
Once the energy contained on the strand’s tension is released, this topoisomerase creates a 3’à5’ link to close the strands. The action of the topoisomerase I causes the release of energy contained on the strand’s tension. The relaxation of the molecule and release of this energy helps DNA to loose the supercoiling structure. It relaxes DNA decreasing both positive and negative supercoiling.
§ Topoisomerase II: it consumes ATP. It forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double strand break in the helix. This Molecular Biology enzyme creates a dimer by the junction of one left-side monomer and one right-side monomer. When the dimer is created, three gates are created (C-gate, N-gate and DNAgate). Firstly, it breaks one double helix segment reversibly to create a DNA gate. Then the other DNA segment enters to the N-gate. The molecule embraces the DNA and it doesn’t let it go until the C-gate is opened. This occurs spontaneously when the DNA is supercoiled.
Uses energy to add some negative supercoiling to the DNA thus facilitating other enzymes to open and transcribe the DNA.
o Negative supercoiling: Our DNA has a little degree of negative supercoiling, which is not totally relaxed. Our DNA in addition to the double helix turns, has extra left-handed turns. This supercoiling occurs in the other direction of the DNA, so it’s left-handed.
This kind of supercoiling facilitates local separation of DNA strands.
• Histones and nucleosome: DNA is packed along with proteins forming a complex called chromatin. These proteins are mostly histones and they bind the DNA to form eukaryotic chromosomes.
There are up to five types of histones with their different subtypes.
Histones are responsible for the first and most basic level of chromosome packing, the nucleosome.
Molecular Biology The nucleosome can be seen in the microscope as a line of 10nm. If we see the superhelical structure, we will see the 30 nm fiber. This fiber is structured in loops onto a protein scaffold (bastida, andamio) to constitute the metaphase chromosome.
Each individual nucleosome core consists of a complex of eight histone proteins and double stranded DNA that is 147 nucleotide pairs long. Each nucleosome core particle is separated from the next by a region of linker DNA so, on average, nucleosomes repeat at intervals of about 200 base pairs.
Every 10 bases pairs, the double helix turns 360º, so around the nucleosome we will have 20 full terms of the double helix.
Nucleosomes wrap DNA to the left (just like negative supercoiling) because DNA needs negative supercoiling to facilitate transcription and replication. If nucleosomes wrapped DNA to the right, during this processes they will have to make an enormous waste of energy that is not productive.
Histones are globular proteins and in their C-terminal have their amino acid backbone (formed basically by Lysine with full positive charge). This amino acid backbone of the histones and the phosphodiester backbone of DNA interact creating hydrogen bonds, hydrophobic interactions and salt linkages that hold the DNA and the protein together in the nucleosome.
The ability of histones to make nucleosomes can be regulated. Lysine can be modified (changing their charge to a negative one) by different reactions (like phosphorylation or acetylation) and repeal DNA.