DNA Replication
History of DNA:
1. Frederick Griffith: Experiments concerning the transformation of S and R strain of a bacterium. First evidence of DNA.
2. Oswald Avery: Proved that DNA was the agent of transfer.
3. Erwin Chargaff: determined that the bases A-T and C-G are found in definite ratios.
4. Linus Pauling, Maurice Wilkins, and Roseland Franklin: worked on the structure of DNA.
5. Watson and Crick: determined the shape and structure of DNA.
DNA Replication:
1. Complex.
2. Extremely rapid.
3. Very accurate, one in a billion are incorrectly paired.
4. Requires the cooperation of over a dozen different enzymes.
Replication begins at special sites along the DNA molecule called Origins of replication. It is at these sites that the helix opens and moves away from. This movement produces the replication fork. Here the new strands of DNA are added.
In order for the DNA strands to separate,3 types of enzymes are needed. Helicases, single-strand binding proteins and topoisomerase.
Priming: Once the replication fork is established a short segment of RNA,formed by an enzyme called primase, initiates the process of replication. With the primer in place, DNA polymerase catalyzes the synthesis of a new strand of DNA. The new nucleotides align themselves with the old DNA. These strands grow from the 5'--3' direction. The energy for this process comes from the 2 P molecules attached to the nucleotide as it is moved into place. There are 2 strands of DNA being formed at the same time. The Leading strand , which is completed in one piece, and moves toward the replication fork; and the lagging strand, which is produced in pieces called Okazaki fragments. These fragments are formed in the 5'--3' direction and linked together by the enzyme DNA ligase.
After the process has been completed the DNA molecules undergo proof reading. DNA polymerase is the enzyme that checks for these errors. If a repair is needed DNA polymerase and ligase will identify, cut, remove, and correct the mistake. This is called Excision repair.
Protein Synthesis

Scientists that worked on Protein Synthesis:

1. Archibald Garrod: First proposed the relationship between genes and proteins.

2. George Beadle and Edward Tatum: working with Neurospora , formulated the one-gene-one-protein hypothesis. This was later changed to one-gene-one-polypeptide. Since some proteins are produced from more than one gene.

The Problem: Since there are 20 different amino acids; and these amino acids are used to make proteins, how does the information get from the DNA to the final protein product? In a DNA molecule the only variable part is the 4 different bases. How can 4 bases, or combinations there of , code for the 20 different amino acids?

1. If 1 base coded for 1 amino acid = 41 (4) amino acids coded for.

2. If 2 bases coded for 1 amino acid = 42 (16) amino acids coded for.

3. If 3 bases coded for 1 amino acid = 43 ( 64) amino acids coded for.

Notice the large gap between 2 and 3. It was concluded that each coding part of DNA (codon) consisted of 3 bases. Since 64 is larger than the 20 amino acids, it stands to reason that some amino acids are coded for by different codons. Why is this important?

Transcription: DNA is too large to leave the nucleus. If the program it contains must leave the nucleus, an intermediate must be produced. This intermediate is called m-RNA. m-RNA is formed in the following way, RNA polymerase binds to, a region of the DNA called, the promoter. Here is found the start codon TAC which codes for the amino acid methionine. Once this has been established the m-RNA sequence is produced growing from the 5' - 3' region. This occurs at the rate of 60 nucleotides / sec. The terminator sequence will keep the process from going on indefinitely. The eukaryotic m-RNA must be modified before going to the ribosome, while the prokaryotic does not.

Translation: t-RNA : a specialized form of RNA used to carry a specific amino acid to the ribosome and place it in the proper position. t-RNA contains 80 nucleotides in the form of a clover leaf. The t-RNA must attach the correct amino acid to itself. The process is as follows:

1. The specific amino acid reacts with a specific enzyme and ATP to release 2 P and combine the AMP and the amino acid.

2. The t-RNA will replace the AMP and bond with the amino acid.

Ribosomes: Before translation ribosomes consist of 2 separate sub-units. As translation proceeds the ribosome parts come together to allow protein synthesis to begin.

There are 3 steps to protein synthesis:

1. Initiation: m-RNA binds to the small subunit of the ribosome. There is 1 binding site for the m-RNA, and 2 binding sites for the t-RNA. The P site holds for the t-RNA carrying the growing polypeptide and the A site for the t-RNA carrying the next amino acid. AUG ( start codon ) is in place and the appropriate t-RNA delivers the amino acid Methionine. The large sub unit then joins and creates a functional ribosome.

2. Elongation: The chain moves from the 5' - 3' direction caused by the use of GTP and several protein-enzyme complexes. As the polypeptide chain is produced the t-RNA move from site A to site P, and then are released into the cytoplasm. The polypeptide chain continues to grow, and is held to the ribosome by the t-RNA at the P site.

3. Termination: The termination codons: UAA, UAG, and UGA stop the process by adding water to the end of the chain instead of an amino acid. In order for the protein to become functional, it must be folded into its appropriate shape,and/ or have some amino acids removed, and/or have some of the polypeptides modified by adding sugars or phosphate groups to them.

The Wobble Phenomenon: There are only 40 different types of t-RNA and 64 codons. This means that some of the t-RNA can pair up with several different codons. This can occur because the third base of a t-RNA molecule can form a hydrogen bond with more than one kind of base. U in the third position can bond with A or G in the corresponding position.

Much of the DNA does not code for the protein. Some eukaryotic DNA has long non coding portions called Introns. The coded portions of the DNA are called exons. Before the m-RNA leaves the nucleus the introns are cut out and the exons are spliced together and can now carry on its function.

Types of Mutations:

1. Base-pair substitutions: is the replacement of one nucleotide and its partner from the other DNA strand with another pair of nucleotides. Most of the time they are not serious.

2. Insertion or deletion: adding or subtraction a pair of nucleotides into a DNA strand. These are more disastrous and can lead to a frameshift mutation.

3. Conditional mutations: temperature sensitive mutations that react at high temperatures and occasionally at cold temperatures.