I. Comparison of Prokaryotes vs Eukaryotes genomes
    Organism                          genome size               # chromosomes            shape
1. Virus (s stranded)              5 x 10³ bp                             1                       circular
2. E. coli                                5 x 10⁶ bp                             1                       circular
3. Yeast                               1.5 x 10⁹ bp                           16                       linear
4. Human                               3 x 10⁹ bp                            23                       linear

Some thought: the genome in E. coli is 5 x 10⁶ bp = 1.7 x 10⁶ nm = 1.7 x 10³mm.  However, the size of E. coli is only about 5 mm!!! How can they pack so much genetic information in such a small area?

II. Basic structure of DNA
1. Antiparallel
2. Complementarity with regard to bases
3. Chargaff's ratio: always 1 (A=T, C=G).
4. Phosphodiester linkage (ribose to ribose)
5. Base gives:    specificity
                          genetic code
                          H-bonds (required for double helical structure)
 6. H-bonds:       melting/denaturation (G-C content)

Nucleotide components:
1. Sugar: DNA contains the sugar deoxyribose while RNA contains the sugar ribose
2. Bases: the bases are flat, aromatic and nitrogenous. They can absorb UV light and are capable
    of hydrogen bonding.
    Purine bases: (double ring) adenine and guanine (DNA or RNA)
    Pyrimidine bases: (single ring) thymine, cytosine (DNA or RNA) and uracyl (RNA)
3. Phosphate: up to three phosphate groups are linked as an ester to the sugar. The phosphates
    link nucleotides in the structure of nucleic acids. They confer a negative charge.
4. N-glycosidic linkage: refers to the type of bond between the sugar and base

Oligonucleotides
1. Oligonucleotide growth occurs in the 5'to 3' direction via an attack by 3' hydroxyl groups
    upon the 5' alpha phosphate of nucleotide triphosphates.
2. The resulting linkage is a phosphodiester

III. GENETIC ELEMENTS (are structures that contain genetic information)
(This is just extra information and the only thing that you need to know many kinds of genetic elements are found)

A. Chromosomes carry the information required for life under all conditions. In bacteria we have a
    single chromosome.

B. Non-chromosomal elements are: mitochondria and chloroplastDNA,plasmids.
    1 . Mitochondria and chloroplasts are organelles believed to have arisen by endosymbiosis.
         They contain DNA, but cannot exist independently.
    2. Plasmids are usually circular and composed of double-stranded DNA. They have their own
        orgin ofreplication(ori) and do not exist extracellularly. They may confer a selective
        advantage (e.g. antibiotic resistance).

DNA Replication:

In 1957, Matthew Meselson and Franklin Stahl did an experiment to determine which of the following models best represented DNA replication:

1. Did the two strands unwind and each act as a template for new strands? This is
    semiconservative replication, because each new strand is half comprised of molecules from
    the old strand.
2. Did the strands not unwind, but somehow generate a new double stranded DNA copy of
    entirely new molecules? This is conservative replication.

Biochemical Mechanism of DNA Replication
It is very important to know that DNA replication is not a passive and spontaneous process. Many enzymes are required to unwind the double helix and to synthesize a new strand of DNA. We will approach the study of the molecular mechanism of DNA replication from the point of view of the machinery that is required to accomplish it. The unwound helix, with each strand being synthesized into a new double helix, is called the replication fork.

The Enzymes of DNA Replication
1. DNA template

2. Topoisomerase is responsible for initiation of the unwinding of the DNA. The tension holding
    the helix in its coiled and supercoiled structure can be broken by nicking a single strand of
    DNA.

3. Helicase accomplishes unwinding of the original double strand, once supercoiling has been
    eliminated by the topoisomerase.

4. DNA polymerase (III) proceeds along a single-stranded molecule of DNA, recruiting free
        dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond with their appropriate
        complementary dNTP on the single strand (A with T and G with C), and to form a covalent
        phosphodiester bond with the previous nucleotide of the same strand.
    DNA polymerases cannot start synthesizing de novo on a bare single strand. It needs a primer
        with a 3'OH group onto which it can attach a dNTP.
    DNA polymerase also has proofreading activities, so that it can make sure that it inserted the
        right base, and nuclease (excision of nucleotides) activities so that it can cut away any
        mistakes it might have made.

5. Primase: This enzyme attaches a small RNA primer to the single-stranded DNA to act as a
    substitute 3'OH for DNA polymerase to begin synthesizing from. This RNA primer is
    eventually removed and the gap is filled in by DNA polymerase (I).

6. Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent
    3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is
    removed and filled in.

7. Single-stranded binding proteins are important to maintain the stability of the replication fork.
    Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains
    single stranded and keep it from being degraded.

The Replication Fork

Why can DNA polymerase only act from 5' to 3'? The reason is the relative stability of each end of DNA. A triphosphate is required to provide energy for the bond between a newly attached nucleotide and the growing DNA strand. However, this triphosphate is very unstable and can easily break into a monophosphate and an inorganic pyrophosphate, which floats away into cell. At the 5' end of the DNA, this triphosphate can easily break, so if a strand has been sitting in the cell for a while, it would not be able to attach new nucleotides to the 5' end once the phosphate had broken off. On the other hand, the 3' end only has a hydroxyl group, so as long as new nucleotide triphosphate are always brought by DNA polymerase, synthesis of a new strand can continue no matter how long the 3' end has remained free.

This presents a problem, since one strand of the double helix is 5' to 3', and the other one is 3' to 5'. How can DNA polymerase synthesize new copies of the 5' to 3' strand, if it can only travel in one direction? This strand is called the lagging strand, and DNA polymerase makes a second copy of this strand in spurts, called Okazaki fragments, as shown in the diagram. The other strand can proceed with synthesis directly, from 5' to 3', as the helix unwinds. This is the leading strand.

TRANSCRIPTION:
Transcription involves the construction of an RNA copy of the genetic information in a DNA template

I. NUCLEIC ACIDS (DNA and RNA)
1. DNA (deoxyribonucleic acid) contains the sugar deoxyribose. RNA (ribonucleic acid)
    contains the sugar ribose. The difference is DNA contain a hydrogen RNA contain a hydroxyl
    group in the2' position.
2. DNA is chemically stable. Due to the presence of the 2' hydroxyl group, RNA is less stable
    than DNA.
3. DNA is double stranded. RNA (except for some viruses) is single stranded.
4. DNA contains the bases adenine (A), guanine (G), cytosine (C), and thymine (T). RNA
    contains the bases adenine (A), guanine (G), cytosine (C), and uracyl (U).

II. Basic Prokaryotic and Eukaryotic Processess
    Prokaryotes: Polycistronic - single mRNA contains more than one coding region
                           Coupled transcription/translation - both functions can occur simultaneously
    Eukaryotes:  Monocystronic - single mRNA contains only one coding region
                      Starts at 5' cap + scans --------> Splicing (noncoding regions are removed
                                                                                from RNA before translation
    Both: Polysomes - several ribosomes translating the same mRNA simultaneously.

        Why polycistronic mRNAs and coupled transcription/translation occur in eukaryotes?

III. Transcription in Prokaryotes and Eukaryotes.

• Transcription involves the construction of an RNA copy of the genetic information in a DNA template
• Eukaryotic transcription is generally more complex than prokaryotic transcription.
• mRNA synthesis and processing in eukaryotes

1. Capping - a "cap" is added to the 5' end of eukaryotic RNA transcripts. A methyl
    group is added to the 2' hydroxyl group of the 5' terminal base of the transcript and
    attach a 7-methyl-guanosine "cap" to the 5' triphosphate end via a 5'--->5' linkage.
3. Addition of a poly-A tail (hundred adenosine) residues to the 3' end of the transcript.
            a. Suppose poly-A is not added - without a poly-A tail, intron splicing will not
                occur, and the mRNA will not be transported out of the nucleus.
5. Removal of introns - introns are cut out of mRNA and the remaining exons are spliced
    together.
6. Export to the cytoplasm - processed transcripts travel out of the nucleus and enter the
    cytoplasm  where they will be translated.

2. In eukaryotes, genes are not clustered in operons. In addition, eukaryotic genes often contain
    non-coding introns ("intervening sequences") interspersed among the coding regions (exons).
    During RNA processing, introns are removed from RNA transcripts and the exons are spliced
    together. Mature mRNA, after being transcribed and processed in the nucleus, is transported
    into the cytoplasm where translation occurs. Because transcription and translation occur in
    different "compartments" in eukaryotes, "coupling" of these two processes is not possible.