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The Chemical Nature of DNA Md. Habibur Rahaman (HbR) Lecturer Dept. of Biology & Chemistry North South University Characteristics of Genetic Material The coding instructions of all living organisms are written in the same genetic language—that of nucleic acids. The idea that genes are made of nucleic acids was not widely accepted until after 1950. Until the structure of DNA was fully elucidated, it wasn’t clear how DNA could store and transmit genetic information. Even before nucleic acids were identified as the genetic material, biologists recognized that, whatever the nature of genetic material, it must possess four important characteristics. First Genetic material must contain complex biological information in stable form: •The genetic material must be capable of storing large amounts of information—instructions for all the traits and functions of an organism. •This information must have the capacity to vary, because different species and even individual members of a species differ in their genetic makeup. •At the same time, the genetic material must be stable, because most alterations to the genetic instructions (mutations) are likely to be detrimental. Second Genetic material must replicate faithfully: •Genetic material must have the capacity to be copied accurately. •Every organism begins life as a single cell, which must undergo billions of cell divisions to produce a complex, multi-cellular creature like yourself. •At each cell division, the genetic instructions must be transmitted to descendent cells with great accuracy. •When organisms reproduce and pass genes to their progeny, the coding instructions must be copied with fidelity. Third Genetic material must encode phenotype: •The genetic material (the genotype) must have the capacity to “code for” (determine) traits (the phenotype). • The product of a gene is often a protein; so there must be a mechanism for genetic instructions to be translated into the amino acid sequence of a protein. Fourth Genetic material must be capable of variation: •This requirement is somewhat contradictory to the first requirement, which demanded stability of the genetic material. • There is, in fact, no a priori reason why genetic material should have built-in provisions for change; one could certainly design a hypothetical genetic system in which information would be rigidly conserved from generation to another. •The dominant theme in the history of life is, however, organic evolution, and this demands that genetic material be capable of change, if only infrequently. The Nature of Genetic Material Historical Background • Miescher isolated nuclei from pus (white blood cells) in 1869 – – • • Found a novel phosphorus-bearing substance and named nuclein Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins End of 19th century – DNA and RNA separated from proteins Levene, Jacobs, et al. characterized the basic composition Transforming Principle • Key experiments done by Frederick Griffith in 1928 • Observed in Streptococcus pneumoniae • Heat-killed virulent colonies could transform avirulent colonies of bacteria Griffith’s Experiment DNA: The Transforming Material In 1944 a group used a transformation test similar to Griffith’s procedure taking care to define the chemical nature of the transforming substance – Techniques used excluded both protein and RNA as the chemical agent of transformation – Other treatments verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent DNA: The Transforming Material Avery–MacLeod–McCarty experiment DNA Confirmation • In 1940s geneticists doubted use of DNA as it appeared to be monotonous repeats of 4 bases • By 1953 Watson & Crick published the double-helical model of DNA structure and Chargaff had shown that the 4 bases were not present in equal proportions • Hershey and Chase demonstrated confirmed that DNA is the genetic material Hershey-Chase Experiments The Hershey–Chase (“blender”) experiment Summary • Genes are made of nucleic acid, usually DNA • DNA has the hereditary and transforming activity Nucleotides • Nucleotides are the unit structure of nucleic acids. • Nucleotides composed of 3 components: – Nitrogenous base (A, C, G, T or U) – Pentose sugar – Phosphate Nitrogenous bases • There are 2 types: – Purines: • Two ring structure • Adenine (A) and Guanine (G) – Pyrimidines: • Single ring structure • Cytosine (C) and Thymine (T) or Uracil (U). Nucleotide bases Types of Nucleic acids There are 2 types of nucleic acids: Deoxy-ribonucleic acid (DNA) • • Pentose Sugar is deoxyribose (no OH at 2’ position) Bases are Purines (A, G) and Pyrimidine (C, T). Ribonucleic acid (RNA) • • Pentose Sugar is Ribose. Bases are Purines (A, G) and Pyrimidines (C, U). Linear Polymerization of Nucleotides • Nucleic acids are formed of nucleotide polymers. • Nucleotides polymerize together by phospho-diester bonds via condensation reaction. • The phospho-diester bond is formed between: – Hydroxyl (OH) group of the sugar of one nucleotide. – Phosphate group of other nucleotide Polymerization of Nucleotides • The formed polynucleotide chain is formed of: – Negative (-ve) charged SugarPhosphate backbone. • Free 5’ phosphate on one end (5’ end) • Free 3’ hydroxyl on other end (3’ end) – Nitrogenous bases are not in the backbone • Attached to the backbone • Free to pair with nitrogenous bases of other polynucleotide chain Polymerization of Nucleotides • Nucleic acids are polymers of nucleotides. • The nucleotides formed of purine or pyrimedine bases linked to phosphorylated sugars (nucleotide back bone). • The bases are linked to the pentose sugar to form Nucleoside. • The nucleotides contain one phosphate group linked to the 5’ carbon of the nucleoside. Nucleotide = Nucleoside + Phosphate group Polymerization of Nucleotides • The polymerization of nucleotides to form nucleic acids occur by condensation reaction by making phospho-diester bond between 5’-phosphate group of one nucleotide and 3’-hydroxyl group of another nucleotide. • Polynucleotide chains are always synthesized in the 5’ to 3’ direction, with a free nucleotide being added to the 3’ OH group of a growing chain. Complementary base pairing • It is the most important structural feature of nucleic acids • It connects bases of one polynucleotide chain (nucleotide polymer) with complementary bases of other chain • Complementary bases are bonded together via: – Double hydrogen bond between A and T (DNA), A and U (RNA) (A═T or A═U) – Triple H-bond between G and C in both DNA or RNA (G≡C) Base pairing Significance of complementary base pairing • The importance of such complementary base pairing is that each strand of DNA can act as template to direct the synthesis of other strand similar to its complementary one. • Thus nucleic acids are uniquely capable of directing their own self replication. • The diameter of the helix could only be kept constant at about 2 nm or 20 Å if one purine and one pyrimidine base made up each stair/rung Summary • DNA and RNA are chain-like molecules composed of subunits called nucleotides • Nucleotides contain a base linked to the 1’-position of a sugar and a phosphate group at 5’-position • The two polynucleotide strands run in opposite directions—they are antiparallel DNA Structure The Double Helix Rosalind Franklin’s x-ray data suggested that DNA had a helical shape The data also indicated a regular, repeating structure Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned to the interior DNA Helix • Structure compared to a twisted ladder – Curving sides of the ladder represent the sugarphosphate backbone – Ladder rungs are the base pairs – There are about 10 base pairs per turn • Arrows indicate that the two strands are antiparallel Forms of DNA 1- B-form helix: It is the most common form of DNA in cells. • Right-handed helix • Turn every 3.4 nm. • Each turn contain 10 base pairs (the distance between each 2 successive bases is 0.34 nm) Minor groove Contain 2 grooves; • • Major groove (wide): provide easy access to bases Minor groove (narrow): provide poor access. Minor groove Major groove 2- A-form DNA: – Less common form of DNA , more common in RNA • Right handed helix • Each turn contain 11 bp/turn • Contain 2 different grooves: – Major groove: very deep and narrow – Minor groove: very shallow and wide (binding site for RNA) Minor groove 3- Z-form DNA: Radical change of B-form Left handed helix, very extended It is GC rich DNA regions. The sugar base backbone form Zig-Zag shape The B to Z transition of DNA molecule may play a role in gene regulation. Minor groove Major groove Major and Minor Grooves (Side view) The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together. The major and minor grooves are opposite each other, and each runs continuously along the entire length of the DNA molecule. Major and Minor Grooves (other view) obtuse angle acute angle Significance of Major and Minor Grooves • Most sequence specific DNA-binding proteins (regulatory proteins) bind DNA via major groove • Major grooves primarily help in transcription (serve as recognition sites for transcription initiation factors, promote DNA strand separation) • Minor grooves are thought to accommodate smaller molecules, intercalators (e.g., anti-cancer drugs, nonprotein ligands) to stop DNA replication (non-sequence specific binding, so has a global effect as expected) RNA as Genetic Material Fraenkal-Conrat and Singer’s experiment Physical Chemistry of Nucleic Acids DNA and RNA molecules can appear in several different structural variants – Changes in relative humidity will cause variation in DNA molecular structure – The twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA was identified in 1979 Variation in DNA between Organisms • Ratios of G to C and A to T are fixed in any specific organism • The total percentage of G + C varies over a range to 22 to 73% • Such differences are reflected by differences in physical properties • Higher GC content correlates with increased thermo-tolerance DNA Melting • • • • • With heating, non-covalent forces holding DNA strands together weaken and break When the forces break, the two strands come apart in denaturation or melting Temperature at which DNA strands are ½ denatured is the melting temperature or Tm GC content of DNA has a significant effect on Tm with higher GC content meaning higher Tm Melted DNA absorbs more UV light than double-helical DNA Summary • GC content of a natural DNA can vary from less than 25% to almost 75% • GC content has a strong effect on physical properties that increase linearly with GC content – Melting temperature, the temperature at which the two strands are half-dissociated or denatured – Density – Low ionic strength, high pH and organic solvents also promote DNA denaturation DNA Renaturation • After two DNA strands separate, under proper conditions the strands can come back together • Process is called annealing or renaturation • Three most important factors: – Temperature – best at about 25C below Tm – DNA Concentration – within limits higher concentration better likelihood that 2 complementary will find each other – Renaturation Time – as increase time, more annealing will occur Polynucleotide Chain Hybridization Hybridization is a process of putting together a combination of two different nucleic acids – Strands could be 1 DNA and 1 RNA – Also could be 2 DNA with complementary or nearly complementary sequences DNA Sizes DNA size is expressed in 3 different ways: – Number of base pairs – Molecular weight – 660 g/mol/base pair – Length – 33.2 Å per helical turn of 10.4 base pairs Measure DNA size either using electron microscopy or gel electrophoresis DNAs of Various Sizes and Shapes • Phage DNA is typically circular • Some DNA will be linear • Supercoiled DNA coils or wraps around itself like a twisted rubber band Summary • Natural DNAs come in sizes ranging from several kilobases to thousands of megabases • The size of a small DNA can be estimated by electron microscopy • This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled DNA Size VS Genetic Capacity How does one know how many genes are in a particular piece of DNA? – Can’t determine from DNA size alone – Factors include: • How DNA is devoted to genes? • What is the space between genes? – Can estimate the upper limit of number genes a piece of DNA can hold DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions • Gene encodes protein • Protein is abut 40,000 Da – How many amino acids does this represent? • • • • Average mass of an amino acid is about 110 Da Average protein – 40,000 / 110 = 364 amino acids Each amino acid = 1 codon= 3 DNA base pairs 364 amino acids requires 1092 base pairs I Da = 1 g/mol DNA size and Genetic Capacity How large is an average piece of DNA? – E. coli chromosome • 4.6 x 106 bp • ~4200 proteins – Phage l (infects E. coli) • 4.85 x 104 bp • ~44 proteins – Phage x174 (one of smallest) • 5375 bp • ~5 proteins C-Value Paradox • C-value is the DNA content per haploid cell • We would expect that, the more complex the organism, the more DNA is needed to “run it” (larger C-value) • Therefore, we would expect a linear relationship between genome size and organism complexity • Bacteria have smaller genomes than eukaryotes, and viruses have smaller genomes than bacteria • In larger organisms, relationship breaks down • Yet the frog has 7 times more DNA per cell than humans C-Value Paradox • The observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradox • Most likely explanation for the paradox is that organisms have DNA apparently in excess of what is needed; repetitive sequences, “junk DNA” Summary • There is a rough correlation between DNA content and number of genes in a cell or virus • This correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widely • C-value paradox is probably explained not by extra genes, but by extra noncoding DNA in some organisms The Tertiary Structure of DNA Packing DNA into Small Spaces E. coli, a single molecule of DNA with approximately 4.64 million base pairs Stretched out straight 1,000 times of the cell Human cells contain 6 billion base pairs of DNA, 1.8 meters stretched end to end The human body collectively contain perhaps 25 billion kilometers of DNA (the distance from the earth to the sun is only 58 million kilometers). This much DNA weights only about 200 grams, or less than half a pound, underscoring how incredibly thin it is. The Tertiary Structure of DNA Packing DNA into Small Spaces DNA can be considered at three hierarchical levels: Primary structure: the nucleotide sequence Secondary structure: the double-stranded helix Tertiary structure: refers to higher-order folding that allows DNA to be packed into the confined space of a cell Histones Package DNA in Eukaryotes Histones Package DNA in Eukaryotes beads on a strig Histones Package DNA in Eukaryotes https://www.youtube.com/watch?v=gbSIBhFwQ4s Palindromic sequence and Significance 5’–ATCGAT–3’ 3’–TAGCTA–5’ ROTATOR ATOYOTA DNA methylation and Significance • In bacteria, adenine and cytosine are commonly methylated, whereas, in eukaryotes, cytosine is the most commonly methylated base. • Bacterial DNA is frequently methylated to distinguish it from foreign, un-methylated DNA that may be introduced by viruses; bacteria use proteins called restriction enzymes to cut up any un-methylated viral DNA (Host-controlled restriction and modification system). • In eukaryotic cells, Sequences that are methylated typically show low levels of transcription while sequences lacking methylation are actively being transcribed. 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