1 Nucleotide Chemistry and DNA Structure PDF

Lecture 1: Nucleotide Chemistry Prepared by: Biochemistry Department , BMC  Nucleic acids  Nucleic acids are required for the storage and expression of genetic information. Includes DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid). ...

Lecture 1: Nucleotide Chemistry Prepared by: Biochemistry Department , BMC  Nucleic acids  Nucleic acids are required for the storage and expression of genetic information. Includes DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid).  DNA carries all the genetic information of the individual.  Site:  Eukaryotic cells: Mostly found in the nucleus in chromosomes and small amount is found in the mitochondria. 1. Nuclear DNA: carries the genetic information that encode functional proteins or functional RNA. 2. Mitochondrial DNA: contains  Genes encode proteins of the electron transport chain.  Genes encoding transfer RNA (tRNA) and the small & large subunits of ribosomal RNA (rRNA).  Prokaryotic cells: which have a single chromosome but may also contain non-chromosomal DNA (plasmids).  The chloroplasts of plants.  DNA Structure: DNA is formed of two strands. The 2 strands wind around each other forming a double helix (Watson and crick model). Each strand is a polymer of deoxynucleotides linked together covalently by 3’-5’ phosphodiester bonds.  Nucleotides structure The nucleotide has three characteristic components  Nitrogenous base  Pentose sugar  Phosphate Nitrogenous Bases  They are derivatives of Purines and pyrimidines  DNA and RNA contain the same purine bases and the pyrimidine base, Cytosine.  Thymine found only in DNA while Uracil found only in RNA (A) (G) (C) (U) (T) Pentoses of Nucleotides D-ribose (in RNA) 2-deoxy-D-ribose (in DNA) The difference is presence of OH group in 2nd carbon atom in ribose vs the presence of H atom in 2nd carbon atom in deoxyribose This difference affects secondary structure and stability Nucleoside and Nucleotide Linking of the base to the sugar (at the 1st carbon of the sugar) is called nucleoside Linking of nucleoside to phosphate group (at the 5th carbon of the sugar) is called nucleotide  DNA Primary structure Nucleotides are linked in each DNA strand through 3′ 5′ Phosphodiester bond between the OH group at 3rd carbon of sugar and phosphates at 5th carbon of next sugar Each DNA strand (chain) has 2 Polar ends: 1. The 5’ end has a free phosphate group. 2. The 3’ end has a free hydroxyl group.  DNA Secondary structure It is called Watson and Crick double helix model (or B-form) The 2 DNA strands wind around each other in antiparallel manner forming double helix in which one strand is oriented in the 5′ 3′ direction and the other in the 3′ 5′ direction. The double helix is stabilized by hydrogen bonding between the bases of the 2 strands and by the hydrophobic interactions between the stacked bases (spiral staircase). The hydrophilic (polar) deoxyribose phosphate backbone of each chain is on the outside of the DNA molecule, whereas the hydrophobic (nonpolar bases) are stacked inside perpendicular to the axis of the helix. Each turn contains 10 base pairs ( 3.4 nm length, 1.9 nm width). The spatial relationship between the two strands in the helix creates a major (wide) groove and a minor (narrow) groove. These grooves provide access for the binding of regulatory proteins.  Base Complementarily Base pairing rule: Thymine always pair with adenine while cytosine always pair with guanine. So, one polynucleotide chain of the double helix is always complementary to the other. The amount of adenine equal thymine and the amount of guanine equal the cytosine. The total amount of purines equal the total amounts of pyrimidines Thymine and adenine bases are connected by 2 hydrogen bonds, while cytosine and guanine are connected by 3 hydrogen bonds. This makes G-C base pairs more stable than A-T base pairs  DNA Tertiary structure Double stranded linear: Eukaryotic nuclear chromosome). Double stranded circular: Mitochondrial, plasmid, viral and chloroplast). Single stranded circular: (small viruses).  Structural forms of DNA Parameters A -DNA B-DNA Z-DNA Direction of helical Right Right Left rotation Residues (base pairs) 11 10 12 per turn of helix Rise per base pair (nm) 0.29 0.34 0.37 Distance per complete 3.2 3.4 4.5 turn (nm) Base tilt relative to 20 6 7 helix axis (in degrees) Major groove narrow and deep wide and deep Flat wide and Minor groove shallow narrow and shallow narrow and deep Structural forms of DNA  DNA Denaturation (Melting) Definition: Separation of the 2 DNA strands by disruption of hydrogen bonds between them. Denaturation factors: 1. Change in pH: which also cause ionization of bases 2. Heating (thermal denaturation). Melting temperature (Tm):  Temperature at which 50% of the DNA molecule exist as single strands.  The Tm differs according to base composition of the DNA molecule, with high Tm value in DNA rich in C-G base pairs because the regions that have predominantly A-T base pairs will be less stable than those rich in G-C base pairs.  DNA denaturation can be monitored by measuring its absorbance at 260 nm. Single stranded DNA has a higher relative absorbance at this wavelength. Complementary DNA strands separated by denaturation can reform double helix when cooling occur (renaturation).  Genetic Terminology  A typical (diploid) human somatic cell contains 46 chromosomes (23 pairs).  Chromosomes are made up of two identical chromatids attached at the centromere.  Chromatids are made up of condensed chromatin.  Chromatin consists of DNA and protein (Histones and Non-Histone).  Centromere is rich in A-T bp and is about 130 bp long. It is connected to specific proteins to form a complex known as kinetochore which is connected to mitotic spindle. This complex is essential structure for chromosomal segregation during mitosis.  DNA Organization (Packing) The total DNA is about 2m in length. It is difficult to imagine how the DNA can be packaged into the nucleus of the cell to be efficiently replicated, and its genetic information expressed. To do so requires the interaction of DNA with many proteins, each of which performs a specific function in the ordered packaging of these long molecules of DNA.  Histones The chromatin in eukaryotic cells consists of Nuclear DNA bound to Histones proteins & smaller amounts of non histone proteins (Enzymes involved in DNA replication and transcription) & Small amount of RNA (snRNA). The Histones are basic proteins (having positive charges as a result of their high content of lysine and arginine. So, they form ionic bonds with the negatively charged DNA (phosphate group). They have 5 types: H1, H2A, H2B, H3, H4. Functions of histones: 1. They help in the condensation of DNA into more compact chromosomes. 2. Protection of the DNA from digestion by exonucleases. 3. Modified histones: Has an important role in changing the structure of chromatin and chromatin function e.g., acetylation of H3 and H4 is associated with activation or inactivation of gene expression (epigenetics).  Nucleosome It consists of : 1- DNA core:  Formed of supercoiled DNA (150 bp) surrounding histone octamer (2 molecules of each of H2A, H2B, H3, H4). 2- Linking region:  It joins one nucleosome core to the next.  Formed of 50 bp of DNA and 1 molecule of histone (H1) which protects the linker DNA from digestion by exonucleases  Chromatin Chromatin is built from repeating nucleosomes units. If the chromatin viewed with E/M, it resembles a string of beads (with the beads representing nucleosome cores and the string representing the DNA linker). Central Dogma of Biology  References 1. Lippincott’s Illustrated Reviews: Biochemistry. 8th Edition 2. Harper's Illustrated Biochemistry, 32nd Edition 3. Marks' Basic Medical Biochemistry: A Clinical approach