1) Chemical analysis.

Analytical techniques of pure DNA revealed the basic constituent molecules, but did not show how they were joined together.

2) Chargaff’s work on base equivalence.

Chargaff analysed the DNA from a wide variety of organisms and found that the ratio between Purine and Pyrimidine was constant. Furthermore he showed that the ratios of Adenine to Thymine and of Guanine to Cytosine were also consistent.

Later this indicated the A-T and G-C bonding in DNA.

3) Franklin and Wilkins’ work on X ray crystallography.

DNA is a very ordered molecule and has a consistent symmetry. The technique of X ray crystallography can reveal the pattern of such regular molecules. A beam of X rays shining through the molecule is scattered by the atoms to give a distinctive X ray diffraction pattern, which may be photographed and measured.

The DNA X ray diffraction photographs showed the molecule was a double helix and also revealed the exact pitch of the helix, together with the layer distances. Each layer is of course one base pair.

The structure of DNA


DNA is a macromolecule polymer made of subunits called nucleotides. The nucleotides are arranged in two chains which are coiled into a spiral shape called a double helix.

DNA is the molecule from which the gene alleles on the chromosomes are made.

As with all nucleotides, those in DNA have three parts. These are a pentose sugar called deoxyribose, a phosphate group and a nitrogenous base.

The sugar and the phosphate are exactly the same in every nucleotide, but the base varies. There are four bases in DNA and each nucleotide contains one of them. The bases are called Adenine, Guanine, Thymine and Cytosine. (A,G,T and C for short).

The nucleotides are joined in a specific order. The order of the nucleotides means that the bases they contain are in a certain order, it is this order which forms the genetic code.

Look at these diagrams showing the sugar and the phosphate. Note that the sugar has its carbon atoms numbered according to their positions in the molecule.

The sugar and phosphate join together to make the backbone of the DNA molecule. The 3’ carbon on one sugar is joined to the 5’ carbon on the next by means of a phosphate bridge, like this.

Diagrammatically shown as:

Each time the sugar joins to a phosphate, a molecule of water is eliminated in a condensation reaction.

This sugar-phosphate-sugar bond is called a phosphodiester bond.

The process repeats so that a very long chain of nucleotides is made, a polynucleotide. Note that the bases protrude from the side of the chain.

There will be a spare 5’ sugar atom at one end of the chain and a spare 3’ atom at the other. The chain thus has a 3’ to 5’ direction reading up the page.

In DNA a second polynucleotide chain forms next to the first, but this runs in the opposite direction. The chains are therefore described as antiparallel.

The bases now find themselves opposite one another and bond with weak hydrogen bonds. When this occurs Adenine always pairs with Thymine (A-T) and Guanine with Cytosine (G-C). There is a good reason for this complementary pairing.

Adenine and Guanine both have a double ring structure and are classified chemically as Purine bases.

A purine molecule looks like this:

Thymine, uracil and Cytosine all have a single ring structure and are classified as Pyrimidines. Their molecules look like this:

When the base pairs form, a consistent spacing is obtained between the polynucleotide chains.

The whole double chained molecule is formed into a double helix spiral, caused by the bond angles between each base pair. Each complete turn of the spiral includes ten base pairs. This takes up a distance of 34 Angstrom units.

The structure of RNA


Ribonucleic acid (RNA) is also a polynucleotide. The chain of nucleotides is formed in exactly the same way as in DNA, but the molecule has some very important differences:

1) It is a single stranded molecule

2) The pyrimidine Thymine never occurs but is always replaced by Uracil, another pyrimidine.

3) It is much smaller than DNA

4) It comes in three different forms, ribosomal, transfer and messenger.

Ribosomal RNA is 80% of the total in a cell. It is involved with the formation of ribosomes and is therefore important as the site of protein synthesis in a cell.

Messenger RNA is 3-5% of the total in a cell, depending on the protein synthesis activity. It forms in the nucleus and is used to communicate the genetic code in the allele to the ribosome during protein synthesis.

Transfer RNA is a clover leaf shaped molecule and is up to 15% of the total in the cell. It is involved in carrying the amino acids through the cytoplasm to their correct places in a growing polypeptide chain.

It also follows that all new cells in an organism must gain a copy of the genes at mitosis, because they are able to continue the characteristic biochemical behaviour of that organism.

What is the mechanism for this exact copying or replication of the DNA?

Three theories existed:

1) The parent DNA molecule breaks into segments and new nucleotides fill in the gaps precisely. (fragmentation theory).

2) The complete parent DNA molecule acts as a template for the new daughter molecule, which is assembled from new nucleotides. The parent molecule is unchanged. (Conservative hypothesis).

3) The parent DNA molecule separates into its two component strands, each of which acts as a template for the formation of a new complementary strand. The two daughter molecules therefore contain half the parent DNA and half new DNA. (Semi-conservative hypothesis).

The semi conservative Hypothesis


The semi conservative hypothesis was shown to be the true mechanism by the work of Meselsohn and Stahl (1958).

In their experiment they grey the bacterium E. coli in the presence of radioactive 15N until a culture was obtained in which all the DNA was labelled with 15N.

A subculture of this labelled bacterium was than transferred for growth in the presence of normal 14N. The generation time of E. coli is known, so it was possible to take samples of this growing subculture after exactly one, two, three generations and so on.

Each sample had its DNA extracted and the isolated DNA was then centrifuged in a caesium chloride solution (high viscosity) at 40,000G for 20 hours, causing the DNA to sediment out.

The heavier the DNA, the further it moved down the centrifuge tubes. 15N DNA is heavier than 14N DNA. Mixed 14N and 15N DNA is intermediate in mass between the two.

The original 15N DNA moved to the lowest position in the tube.

After one generation all the DNA moved to an intermediate position, indicating the presence of only mixed 14N and 15N DNA. This was because the DNA in this generation contained one strand of the parent molecule and one new 14N strand.

Had the conservative hypothesis been true, two DNA masses would have been visible, one heavy and the other light.

In the second generation half the DNA was intermediate and half was light, for the same reason.

Process of DNA replication


The actual process is simple. To begin with one strand in the DNA duplex is nicked by the enzyme DNA topoisomerase, allowing part of the molecule to unravel to form a replication fork (the DNA is replicated a bit at a time and the whole molecule is never completely uncoiled).

Next, the enzyme DNA helicase splits the two strands by breaking the hydrogen bonds. This exposes the bases.

DNA polymerase enzyme then moves along the exposed bases sequences, creating a new complementary strand as it goes. DNA polymerase reads the exposed code from the 3’ to the 5’ end and therefore assembles the new strand from the 5’ to the 3’.

Several molecules of DNA polymerase act simultaneously, each assembling a separate section of the new strand of DNA. Each DNA polymerase is preceded by an RNA polymerase enzyme, which constructs an RNA primer to guide the action of the DNA polymerase.

These new DNA segments are then joined together by the enzyme DNA ligase. The two new daughter molecules then coil up again to reform the double helix structure. This process occurs during the S phase of the cell cycle.

If the harmless bacteria were allowed to grow in the presence of the dead virulent cells, they became virulent themselves and killed the mice. Some part of the dead virulent strain had transformed the previously harmless strain. This phenomenon is transformation.

Avery later (1940’s) showed that it was almost certainly the DNA from the dead virulent bacteria that caused this effect.

Radioactive assay techniques


Hershey and Chase grew cultures of T2 bacteriophage viruses in the presence of radioisotopes. A virus only contains protein and nucleic acid.

One culture was given 32P so that all the virus DNA became radioactive (proteins do not contain P).

The second culture was given 35S so that all the virus protein became radioactive (DNA does not contain S).

It is known that when bacteriophages attack bacteria, only their DNA enters the host, while the protein is always left outside the parasitised cell.

Separate bacterial cultures were infected, one by each labelled virus. A blender was then used to dislodge the empty virus protein coats from the infected bacterial cells. A centrifuge was then used to separate the heavier bacteria from the light virus coats.

Bacteria infected with 32P viruses became radioactive, but bacteria infected with 35S viruses did not.

Only the 32P ever entered the bacteria. Thus it was shown that the DNA is the molecule used by viruses to pass their genetic information

Other evidence


Other evidence includes:

1) The mass of DNA in somatic cells is constant, while gametes have half this mass.

2) DNA is associated with the chromosomes (but so is protein)

3) Mutagens that affect DNA molecules cause increased phenotypic mutation.

4) The wavelengths of UV radiation which cause the most mutation are also those which pure DNA absorbs the strongest.

5) All cells in one organism have the same type of DNA, as shown by Chargaff’s work, but the DNA varies between different species.

7) In non dividing tissue such as nervous tissue, the DNA is not broken down, but other cellular molecules are constantly being recycled and replaced.

8) DNA is copied exactly during each cell cycle and is therefore consistent and self-perpetuating (Meselson and Stahl).

DNA and Protein Synthesis

Introduction


DNA is the molecule which controls the synthesis of proteins. Proteins are used for growth and repair and also as enzymes, in which form they catalyse all other cellular activities.

Thus DNA is able to exert a controlling influence over the whole cell and ultimately, the whole organism. The segments of DNA which hold the key to this control are the genes.

Beadle and Tatum performed experimental work using the haploid mould Neurospora. Being haploid, this organism expresses all genes present, regardless of dominance.

Beadle and Tatum demonstrated that irradiated moulds developed mutations which affected their ability to synthesise amino acids. One mutation affected only one amino acid synthesis (a critical enzyme was absent) and was also passed to subsequent generations.

Their work led to the principle of one gene = one enzyme.

Later this principle was modified to one gene = one polypeptide.

It has since has become one cistron = one polypeptide.

A cistron is the shortest length of DNA that can code for a whole polypeptide.

Protein Synthesis


Protein synthesis relies on the effective communication of the coded information held in the genes to the sites of protein manufacture, the ribosomes in the cytoplasm.

Since DNA is part of larger structures (chromosomes) which are unable to move from the nucleus, intermediate messenger molecules are needed. These are messenger RNA molecules.

To begin with the DNA duplex unzips to expose the base sequence on the coding strand. RNA nucleotides then move in and align themselves according to the rules of base pairing (A-U and G-C) with U replacing T in the RNA molecule.

Transcription

The RNA is assembled using the enzyme RNA polymerase. This process is called transcription.

The DNA template strand is read from the 3’ to the 5’ end and the mRNA is made from the 5’ to the 3’ end. During transcription only the coding parts of the DNA are copied (the exons). Non coding parts or introns are ignored.

The completed mRNA molecule detaches from the DNA template and exits the nucleus via the nuclear pores, moving into the cytoplasm.

Translation

The mRNA is now ready for translation, which is organised by the ribosomes, which now attach themselves to the mRNA.

If more than one ribosome attaches then a polysome (polyribosome) is formed, with the appearance of beads on a string. Each ribosome controls the formation of one polypeptide. The mRNA is read as triplets of bases called codons.

The ribosome attaches to the mRNA by its small subunit. Magnesium ions are involved in the attachment process.

The larger subunit of the ribosome can accommodate two codons of the mRNA. One is held in the P (peptidyl) site and the other in the A (aminoacyl) site. Each codon triplet then attracts a complementary triplet or anticodon.

Each anticodon forms part of one transfer RNA (tRNA) molecule. Each tRNA carries one specific amino acid in the cytoplasm. The anticodon and codon bind together temporarily by means of hydrogen bonds.

This causes two amino acids to be held next to one another long enough for the formation of a peptide bond between them.

The first amino acid in any polypeptide is usually methionine. The codon for this is AUG, which has come to be known as the initiation codon as a result. The formation of the peptide bond is catalysed by the enzyme peptidyl transferase, which is an integral part of the large ribosome subunit.

Translocation

Once the peptide bond has formed, the first tRNA detaches and travels into the cytoplasm to pick up another amino acid.

The ribosome shifts along the mRNA by exactly one codon, so that the second codon now occupies the P site and the third the A site.

This movement is a process called translocation.

A third tRNA now moves to the correct position and a second peptide bond forms. This process is then repeated until the polypeptide is complete.

The ribosome moves along the mRNA from the 5’ towards the 3’ end. The completion of this process of translation is signalled by nonsense or stop codons. These do not correspond with any tRNA, but signal the ribosome to detach from the mRNA.

The polypeptide is then ready for modification into a specific protein. A stop codon may be UAA, UAG or UGA.

Watson and Crick originally proposed the triplet theory on the grounds that this is the minimum number which could give at least one unique combination for each amino acid. There are in fact 64 possible codons, so each amino acid has more than one to code for it. The genetic code is thus described as being degenerate.

Evidence for the genetic code being in the form of triplets included the work of Crick (1962). He showed that if single bases were removed from the DNA of T4 bacteriophages, then frame shifts were caused in the translation to polypeptides.

The code was broken by the work of Nirenberg. He synthesised mRNA with known base sequences and observed the resulting amino acid sequences. For example, mRNA made only of uracil (polyuradylic acid or poly-U) gives polypeptides made only of phenylalanine. Thus the codon UUU corresponds with the amino acid phenylalanine.

The genetic code is also universal (the same in all organisms) and non overlapping (triplets are adjacent).