I. REVIEW OF MICROBIAL GENETICS

B. DEOXYRIBONUCLEIC ACID (DNA)

Fundamental statements for this learning object:

1. Deoxyribonucleic acid (DNA) is a long, double-stranded, helical molecule composed of building blocks called deoxyribonucleotides.
2. A deoxyribonucleotide is composed of 3 parts: a molecule of the 5-carbon sugar deoxyribose, a nitrogenous base, and a phosphate group.
3. There are four nitrogenous bases found in DNA: adenine, guanine, cytosine, or thymine. Adenine and guanine are known as purine bases while cytosine and thymine are known as pyrimidine bases.
4. Deoxyribose is a ringed 5-carbon sugar. The 5 carbons are numbered sequentially clockwise around the sugar. The first 4 carbons actually form the ring of the sugar with the 5' carbon coming off of the 4' carbon in the ring. The nitrogenous base of the nucleotide is attached to the 1' carbon of the sugar and the phosphate group is bound to the 5' carbon.
5. During DNA synthesis, the enzyme DNA polymerase can only attach the phosphate group of a new deoxyribonucleotide to the 3' carbon of a nucleotide already in the chain.
6. During DNA replication, each parent strand acts as a template for the synthesis of the other strand by way of complementary base pairing.
7. Complementary base pairing refers to DNA nucleotides with the base adenine only forming hydrogen bonds with nucleotides having the base thymine (A-T). Likewise, nucleotides with the base guanine can hydrogen bond only with nucleotides having the base cytosine (G-C).
8. While the two strands of DNA are complementary, they are oriented in opposite directions to each other. One strand is said to run 5' to 3'; the opposite DNA strand runs antiparallel, or 3' to 5'.
9. In prokaryotic cells there is no nuclear membrane surrounding the DNA. Prokaryotic cells lack mitosis and meiosis.
10. To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. Then an enzyme called DNA gyrase supercoils each domain around itself forming a compacted, supercoiled mass of DNA. A topoisomerase called DNA gyrase catalyzes the negative supercoiling of the circular DNA found in bacteria. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.
11. The DNA in eukaryotic cells is packaged in basic units called a nucleosomes, a beadlike structure consisting of DNA wrapped around eight histone molecules. The DNA is organized as multiple chromosomes located within a nucleus surrounded by a nuclear membrane. The nucleus divides by mitosis and gametes are produced by meiosis in eukaryotes reproducing sexually.
12. The structural nature of the deoxyribonucleoprtein contributes to whether or not DNA is transcribed into RNA. For example, chemical changes to the chromatin can enable portions of it to condense or relax. When a region is condensed, genes cannot be transcribed. In addition, chemical can attach to or be removed from the histone proteins around which the DNA wraps. The attachment or removal of these chemical groups to the histone determines whether nearby gene expression is amplified or repressed.

 

LEARNING OBJECTIVES FOR THIS SECTION

Deoxyribonucleic Acid (DNA)

DNA is a long, double-stranded, helical molecule composed of building blocks called deoxyribonucleotides (def). A deoxyribonucleotide is composed of 3 parts: a molecule of the 5-carbon sugar deoxyribose, a nitrogenous base (def), and a phosphate group (see Fig. 1 and Fig. 2).

a. Deoxyribose. Deoxyribose (def) is a ringed 5-carbon sugar (see Fig. 3). The 5 carbons are numbered sequentially clockwise around the sugar. The first 4 carbons actually form the ring of the sugar with the 5' carbon coming off of the 4' carbon in the ring. The nitrogenous base of the nucleotide is attached to the 1' carbon of the sugar and the phosphate group is bound to the 5' carbon. During DNA synthesis, the phosphate group of a new deoxyribonucleotide is covalently attached by the enzyme DNA polymerase (def) to the 3' carbon of a nucleotide already in the chain.

b. A nitrogenous base (def). There are four nitrogenous bases found in DNA: adenine, guanine, cytosine, or thymine. Adenine and guanine are known as purine bases (def) while cytosine and thymine are known as pyrimidine bases (def) (see Fig. 4).

c. A phosphate group (see Fig. 5).

To synthesize the two chains of deoxyribonucleotides during DNA replication, the DNA polymerase enzymes involved are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl (OH) group of the 3' carbon of a nucleotide already in the chain. The covalent bond that joins the nucleotides is called a phosphodiester bond (def). Each DNA strand has what is called a 5' end and a 3' end. This means that one end of each DNA strand, called the 5' end (def) , will always have a phosphate group attached to the 5' carbon of its terminal deoxyribonucleotide (see Fig. 2). The other end of that strand, called the 3' end (def) , will always have a hydroxyl (OH) on the 3' carbon of its terminal deoxyribonulceotide.

As will be seen in the next section, each parent strand, during DNA replication, acts as a template for the synthesis of the other strand by way of complementary base pairing. Complementary base pairing (def) refers to DNA nucleotides with the base adenine only forming hydrogen bonds with nucleotides having the base thymine (A-T). Likewise, nucleotides with the base guanine can hydrogen bond only with nucleotides having the base cytosine (G-C). (In the case of RNA nucleotides, as will be seen later, adenine nucleotides form hydrogen bonds with nucleotides having the base uracil since thymine is not found in RNA.) As a result of this bonding, the DNA assumes its helical shape. Therefore, the two strands of DNA are said to be complementary. Wherever one strand has an adenine-containing nucleotide, the opposite strand will always have a thymine nucleotide; wherever there is a guanine-containing nucleotide, the opposite strand will always have a cytosine nucleotide (see Fig. 2).

While the two strands of DNA are complementary, they are oriented in opposite directions to each other. One strand is said to run 5' to 3'; the opposite DNA strand runs antiparallel, or 3' to 5' (see Fig. 2).

We will now briefly compare the genome of prokaryotic cells with that of eukaryotic cells.

1. The Prokaryotic (Bacterial) Genome

The area within a bacterium where the chromosome can be seen with an electron microscope (see Fig. 6) is called a nucleoid (def).

The chromosome of most prokaryotes is typically one long, single molecule of double stranded, helical, supercoiled DNA (def) which forms a physical and genetic circle (def). The chromosome is generally around 1000 µm long and frequently contains around 4000 genes (see Fig. 8). Escherichia coli, which is 2-3 µm in length has a chromosome approximately 1400 µm long. To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. A DNA topoisomerase (def) enzyme called DNA gyrase then supercoils the chromosome into a tight bundle forming a compacted, supercoiled mass of DNA approximately 0.2 µm in diameter.

Bacterial enzymes called DNA topoisomerases (def) are essential in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA (def) (see Fig. 9). They are also essential in transcription of DNA into RNA, in DNA repair, and in genetic recombination in bacteria.

The prokaryotic nucleoid has no nuclear membrane surrounding the DNA and the nuclear body does not divide by mitosis (def). The cytoplasmic membrane plays a role in DNA separation during bacterial replication. Since bacteria are haploid (def) (have only one chromosome), there is also no meiosis (def).

 

2. The Eukaryotic Genome

Prokaryotic and eukaryotic cells differ a great detail in both the amount and the organization of their molecules of DNA. Eukaryotic cells contain much more DNA than do bacteria, and this DNA is organized as multiple chromosomes located within a nucleus.

The nucleus (def) in eukaryotic cells is surrounded by a nuclear membrane (see Fig. 7) and contains linear chromosomes (def) composed of negatively charged DNA associated with positively charged basic proteins called histones (def) to form structures known as nucleosomes (def). The nucleosomes are part of what is called chromatin (def), the DNA and proteins that make up the chromosomes. The nucleus divides my mitosis (def) and haploid (def) sex cells are produced from diploid cells (def) by meiosis (def).

The DNA in eukaryotic cells is packaged in a highly organized way. It consists of a basic unit called a nucleosome (def), a beadlike structure 11 nm in diameter that consists of 146 base pairs of DNA wrapped around eight histone molecules. The nucleosomes are linked to one another by a segment of DNA approximately 60 base pairs long called linker DNA (see Fig. 10). Another histone associated with the linker DNA then packages adjacent nucleotides together to form a nucleosome thread 30nm in diameter. Finally, these packaged nucleosome threads form large coiled loops that are held together by nonhistone scaffolding proteins. These coiled loops on the scaffolding proteins interact to form the condensed chromatin seen in chromosomes during mitosis (see Fig. 11).

In recent years its been found that the structural nature of the deoxyribonucleoprotein contributes to whether or not DNA is transcribed into RNA. For example, chemical changes to the chromatin can enable portions of it to condense or relax. When a region is condensed, genes cannot be transcribed. In addition, chemical can attach to or be removed from the histone proteins around which the DNA wraps. The attachment or removal of these chemical groups to the histone determines whether nearby gene expression is amplified or repressed.

The epigenome (def) refers to a variety of chemical compounds that modify the genome typically by adding a methyl (CH₃) group to the nucleotide base adenine at specific locations along the DNA molecule. This methylation can, in turn, either repress or activate transcription of specific genes. By basically turning genes on or off, the epigenome enables the genome to interact with and respond to the cell's environment. The epigenome can be inherited just like the genome.

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