Nucleic Acids (DNA and RNA) PDF

Nucleic Acids (DNA and RNA) A nucleic acid is a macromolecule composed of chains of monomeric nucleotide. these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid...

Nucleic Acids (DNA and RNA) A nucleic acid is a macromolecule composed of chains of monomeric nucleotide. these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are universal in living things, as they are found in all cells and viruses.  Types of nucleic acids Ribonucleic acid Ribonucleic acid, or RNA, is a nucleic acid polymer – consisting of nucleotide monomers, which plays several important roles in the processes of translating genetic information from.deoxyribonucleic acid (DNA) into proteins RNA is usually single-stranded, but any given – strand may fold back upon itself to form.secondary structure as in tRNA and rRNA Deoxyribonucleic acid Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms. The main role of DNA molecules is the long-term storage of information and DNA is often compared to a set of blueprints, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. DNA is usually double-stranded, though some viruses have single-stranded DNA as their genome. Retroviruses have single-stranded RNA as their genome.  The sugars and phosphates in nucleic acids are connected to each other in an alternating chain, linked by shared oxygens, forming a phosphodiester bond. In conventional nomenclature, the carbons to which the phosphate groups attach are the 3' end and the 5' end carbons of the sugar. This gives nucleic acids polarity. The bases extend from a glycosidic linkage to the 1"carbon of the pentose sugar ring. Bases are joined through N-1 of pyrimidines and N-9 of purins to 1' carbon of ribose through N-β glycosyl bond. Chemical differences between DNA & RNA Both RNA and DNA are composed of repeated units. The repeating units of RNA are ribonucleotide monophosphates and of DNA are.2'-deoxyribonucleotide monophosphates Both RNA and DNA form long, unbranched polynucleotide chains in which different purine or pyrimidine bases are joined by N-glycosidic bonds.to a repeating sugar-phosphate backbone The chains have a polarity. The sequence of a.'nucleic acid is customarily read from 5' to 3 ,The base sequence carries the information  Consequences of RNA/DNA chemistry The DNA backbone is more stable, especially to alkaline conditions. The 2' OH on the RNA forms 2'3'phosphodiester intermediates under basic conditions which breaks down to a mix of 2' and 3' nucleoside monophosphates. Therefore,.the RNA polynucleotide is unstable The 2' deoxyribose allows the sugar to assume a lower energy conformation in the backbone. This helps to increase.the stability of DNA polynucleotides Cytidine deamination to Uridine can be detected in DNA but not RNA because deamination of Cytidine in DNA leads to Uridine not Thymidine. Uridine bases in DNA are removed by a specific set of DNA repair enzymes and replaced with.cytidine bases  Nucleic acid components Each nucleotide consists of three components: a nitrogenous heterocyclic basee, which is either a purine (a heterocyclic compound, aromatic organic consisting of a pyrimidine ring fused to an imidazole)or a pyrimidine; a pentose sugar; and a.phosphate group  DNA Structure The building blocks of nucleic acids are nucleotides, each composed of: – a 5-carbon sugar called deoxyribose – a phosphate group (PO4) – a nitrogenous base adenine, thymine, cytosine, guanine, uracil 10 Nucleobases are heterocyclic aromatic organic compounds RNA containing nitrogen atoms. Nucleobases are the parts of and DNA involved in base pairing. Cytosine, guanine, adenine, thymine are found predominantly in DNA, while in RNA uracil replaces thymine. These are abbreviated as C, G,.A, T, U, respectively  Nucleosides Nucleosides are glycosylamines made by attaching a nucleobase (often referred to simply as bases) to a ribose or deoxyribose (sugar) ring. In short, a nucleoside is a base linked to sugar. The names derive from the nucleobase names. The nucleosides commonly occurring in DNA and RNA include cytidine, uridine, adenosine, guanosine and thymidine. When a phosphate is added to a nucleoside (by phosphorylated by a specific kinase enzyme), a nucleotide is produced. Nucleoside analogues, such as acyclovir, are used as antiviral agents.  ?What is the blueprint of life It is the genetic blueprint, or recipe, for making all living things. Almost every cell in your body contains DNA and all the information needed to make you what you are, from the way you look to which hand you write with. DNA is shaped like a long ladder that's twisted into a spiral.  …AMAZING DNA FACTS DNA from a single human cell extends in a single thread for almost 2 meters long!!! It contains information equal to some 600,000 printed pages of 500 words each!!! (a library of about 1,000 books) DNA discovery timeline  First isolation of Nucleic acids J. Friedrich Miescher (13 August 1844 – 26 August 1895) was a Swiss physician and biologist. He was the first researcher to isolate and identify nucleic acid. Miescher isolated various phosphate-rich chemicals, which he called nuclein (now nucleic acids (now nucleic acids), from the nuclei of white blood cells in 1869 in Felix Hoppe-Seylerin Felix Hoppe-Seyler's laboratory at the University of Tübingenin Felix  How does DNA relate to proteins? 1908: Garrod inborn errors of metabolism (hereditary disease) Alkaptonuria (AKU): accumulation of homogentisic acid 1:200,000 A defective enzyme results from a mutant gene HOW????  Genes and Proteins Inborn Errors of Metabolism shown by Garrod to cause hereditary disease. Study of Biochemical Pathways lead to understanding that mutant genes result in defective proteins (enzymes).  Biochemical Genetics Archibald Garrod (1902) - an English doctor Described “alkaptanurea” disease Symptom: urine turns black when exposed to air Found it was due to oxidation of homogentisic acid in urine homogentisic acid = an intermediate in Phe degradation homogentisic further Phe Tyr acid metabolites Accumulation of homogentisic acid  Biochemical Genetics Archibald Garrod : important contributions Described “alkaptanurea” disease Deduced that it is due to a defective metabolic enzyme Disease is a hereditary condition (ran in his patients’ families) Led to concept of “inborn errors of metabolism” A novel phenotype may reflects a discrete biochemical difference  George W. Beadle Did work in the 1930’s & 40’s on Drosophila eyes and on Neurospora (bread mold) “one gene - one enzyme” hypothesis (1941) awarded Nobel prize in 1958 (with research colleagues J. Lederberg and E. Tatum)  George W. Beadle & Edward Tatum Bread Mold: Neurospora crassa can grow on minimal media sucrose Inorganic salts biotin Beadle selected for nutritional mutants (auxotrophs: a mutant organism (typically a bacterium or fungus) that requires a particular additional nutrient that the normal strain does not.) irradiated fungal spores, grew these up on complete media, and transferred part of the stock to minimal media He looked for mutants that can grow on complete media but NOT on minimal media These mutants are lacking an enzyme for the synthesis of an essential nutrients  Mutations Mutation = change in the base sequence of DNA Any mutation that causes the insertion of an incorrect amino acid in a protein can impair its function Base substitutions alter the genetic code which specifies amino acid placement in proteins  Types of DNA Damage Summarised G A T C ds DNA Break Mismatch C-U deamination ss Break AP site Covalent X-linking Thymidine dimer  A HISTORY OF DNA Discovery of the DNA double helix A. Frederick Griffith – Discovers that a factor in diseased bacteria can transform harmless bacteria into deadly bacteria (1928) B. Rosalind Franklin - X-ray photo of DNA. (1952) C. Watson and Crick - described the DNA molecule from Franklin’s X-ray. (1953)  Deoxyribonucleic acid (DNA) DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. DNA is a set of blueprints needed to construct other components of cells, such as proteins and RNA molecules. The Central Dogma: DNA Encodes RNA, RNA Encodes Protein. The central dogma of molecular biology describes the flow of genetic information in cells from DNA to messenger RNA (mRNA) to protein. It states that genes specify the sequence of mRNA molecules, which in It was first stated turn specify the sequence of by Francis Crick in 1958 proteins.  Genes can be regulated at many levels DNA RNA PROTEIN TRANSCRIPTION TRANSLATION The “Central Dogma” The central dogma of molecular biology describes the flow of genetic information within a biological system. It was first stated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970 33  DNA Double Helix 5 O 3 3 O P 5 P 5 O 1 G C 3 2 4 4 2 1 3 5 O P P 5 T A 3 O O 5 P 3 P  Two long strands makes the shape of a double helix. two strands run in opposite directions to each other and are therefore anti-parallel. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of base, sugars and phosphate groups. Fig : DNA double helix  Topology Double-stranded nucleic acids are made up of complementary sequences, in which extensive Watson-Crick base pairing (canonical) results in the highly repeated and quite uniform double-helical three-dimensional structure. In contrast, single-stranded RNA and DNA molecules are not constrained to a regular double helix, and can adopt highly complex three-dimensional structures that are based on short stretches of intramolecular base-paired sequences that include both Watson-Crick and noncanonical base pairs, as well as a wide range of complex tertiary interactions.  Nucleic acid molecules are usually unbranched, and may occur as linear and circular molecules. For example, bacterial chromosomes, plasmids, mitochondrial DNA and chloroplast DNA are usually circular double-stranded DNA molecules, while chromosomes of the eukaryotic nucleus are usually linear double-stranded DNA molecules. Most RNA molecules are linear, single-stranded molecules, but both circular and branched molecules can result from RNA splicing reactions.  Structure of DNA Utilizing X-ray diffraction data, obtained from crystals of DNA, James Watson and Francis Crick proposed a model for the structure of DNA. This model predicted that DNA would exist as a helix of two complementary antiparallel strands, wound around each other in a rightward direction and stabilized by H-bonding between bases in adjacent strands. In the Watson-Crick model, Purine bases form hydrogen bonds with pyrimidines, in the crucial phenomenon of base pairing.  Experimental determination has shown that, in any given molecule of DNA, the concentration of adenine (A) is equal to thymine (T) and the concentration of cytidine (C) is equal to guanine (G). This means that A will only base-pair with T, and C with G. According to this pattern, known as Watson-Crick base-pairing, the base-pairs composed of G and C contain three H-bonds, whereas those of A and T contain two H-bonds. This makes G-C base-pairs more stable than A-T base-pairs.  The antiparallel nature of the helix stems from the orientation of the individual strands. From any fixed position in the helix, one strand is oriented in the 5'—>3' direction and the other in the 3'—>5' direction. On its exterior surface, the double helix of DNA contains two deep grooves between the ribose-phosphate chains. These two grooves are of unequal size and termed the major and minor grooves. The difference in their size is due to the asymmetry of the deoxyribose rings and the structurally distinct nature of the upper surface of a base-pair relative to the bottom surface.  DNA forms The double helix of DNA has been shown to exist in several different forms, depending upon sequence content and ionic conditions of crystal preparation. The B-form of DNA prevails under physiological conditions of low ionic strength and a high degree of hydration. Regions of the helix that are rich in pCpG dinucleotides can exist in a novel left-handed helical conformation termed Z-DNA. This conformation results from a 180 degree change in the orientation of the bases relative to that of the more common A- and B-DNA.  Parameters A Form B Form Z-Form Direction of helical rotation Right Right Left Residues per turn of helix 11 10 12 base pairs Rotation of helix per residue 33 36 -30 (in degrees) Base tilt relative to helix axis 20 6 7 (in degrees) wide and Major groove narrow and deep Flat deep narrow and Minor groove wide and shallow narrow and deep deep Orientation of N-glycosidic anti for Pyrimidines, anti anti bond syn for Purines occurs in stretches of most Non alternating Comments prevalent physiological purine-pyrimidine within cells base pairs B-form Most common DNA conformation in vivo Narrower, more elongated helix than A. Wide major groove easily accessible to proteins Narrow minor groove Favored conformation at high water concentrations (hydyration of minor groove seems to favor B-form) Base pairs nearly perpendicular to helix axis A-form Most RNA and RNA-DNA duplex in this form shorter, wider helix than B. deep, narrow major groove not easily accessible to proteins wide, shallow minor groove accessible to proteins, but lower information content than major groove. favored conformation at low water concentrations base pairs tilted to helix axis Z-form Helix has left-handed sense Can be formed in vivo, given proper sequence and superhelical tension, but function remains obscure. Narrower, more elongated helix than A or B. Major "groove" not really groove Narrow minor groove Conformation favored by high salt concentrations, some base substitutions, but requires alternating purine-pyrimidine sequence. Base pairs nearly perpendicular to helix axis GpC repeat, not single base-pair – P-P distances: vary for GpC and CpG – GpC stack: good base overlap – CpG: less overlap. Zigzag backbone due to C sugar conformation compensating for G glycosidic bond conformation If you hold it pointing away from you and it twists clockwise moving away, it is right-handed, otherwise it is left-handed. These models are mirror images and can not be converted one into the other by rotation. The helix of normal DNA is right-handed. Structure of B-DNA Structure of Z-DNA Genomic imprinting is the epigenetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. If the allele inherited from the father is imprinted, it is thereby silenced, and only the allele from the mother is expressed.  Thermal Properties of DNA As cells divide it is a necessity that the DNA be copied (replicated), in such a way that each daughter cell acquires the same amount of genetic material. In order for this process to proceed the two strands of the helix must first be separated, in a process termed denaturation. This process can also be carried out in vitro. If a solution of DNA is subjected to high temperature, the H-bonds between bases become unstable and the strands of the helix separate in a process of thermal denaturation.  The base composition of DNA varies widely from molecule to molecule and even within different regions of the same molecule. Regions of the duplex that have predominantly A-T base-pairs will be less thermally stable than those rich in G-C base-pairs. In the process of thermal denaturation, a point is reached at which 50% of the DNA molecule exists as single strands. This point is the melting temperature (Tm), and is characteristic of the base composition of that DNA molecule. The Tm depends upon several factors in addition to the base composition. These include the chemical nature of the solvent and the identities and concentrations of ions in the solution. Absorption of ultraviolet light: When using spectrophotometric analysis to determine the concentration of DNA or RNA, the Beer-Lambert law is used to determine unknown concentrations without the need for standard curves. In essence, the Beer Lambert Law makes it possible to relate the amount of light absorbed to the concentration of the absorbing molecule. The following absorbance units to nucleic acid concentration conversion factors are used to convert OD to concentration of unknown nucleic acid samples: A260 dsDNA = 50 µg/ml A260 ssDNA = 37 µg/ml A260 ssRNA = 40 µg/ml Hyperchromicity is the increase of absorbance (optical density) of a material. The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level. The opposite, a decrease of absorbance is called hypochromicity.  When thermally melted DNA is cooled, the complementary strands will again re-form the correct base pairs, in a process is termed annealing or hybridization. The rate of annealing is dependent upon the nucleotide sequence of the two strands of DNA.  Complementary Base Pairs Two H bonds for A-T Three H bonds for G-C 54 BASE-PAIRINGS H-bonds G C T A  Eukaryotic Chromosome Structure Eukaryotic DNA is found packaged with protein, forming a substance called chromatin. There's a good reason for this: the amount of DNA in a single human cell, lined up end to end, would stretch nearly two meters! This DNA has to be compacted enough to fit into a single nucleus, and packaging the DNA into chromatin accomplishes this.  The essential unit of DNA packaging is the nucleosome. A nucleosome consists of a small amount of DNA wrapped up with protein. The proteins that interact with DNA to form chromatin comprise a family of basic (positively charged) proteins called histones. There are five different types of histone protein: H1, H2A, H2B, H3, and H4. Of these, two molecules each of H2A, H2B, H3, and H4 combine to form a histone octamer.  DNA wraps around the octamer, making 1 3/4 turns around the protein complex. The amount of DNA associated with the histone octamer is 146 bp. The octamer plus the DNA comprise what is called the nucleosome core. A small stretch of DNA (60 bp) runs between adjacent nucleosome cores, and is known as the linker. A single nucleosome consists of one core plus a linker. The total amount of DNA involved in a single nucleosome is approximately 206 bp.  Chromatin vs Chromosome In the nucleus, the DNA double helix is packaged by special proteins (histones) to form a complex called chromatin. The chromatin undergoes further condensation to form the chromosome. So while the chromatin is a lower order of DNA organization, chromosomes are the higher order of DNA organization. An organism’s genetic content is counted in terms of the chromosome pairs present. e.g. humans have 23 pairs of chromosomes. Gene expression Examples of altered bases Deamination Events +H2O -NH3 +H2O -NH3 +H2O -NH3 +H2O -NH3  Mutation In biology, mutations are changes to the nucleotide sequence of the genetic material of an organism. Mutations can be caused by copying errors in the genetic material during cell division, by exposure to ultraviolet or ionizing radiation, chemical mutagens, or viruses, or can occur deliberately under cellular control during processes such as hypermutation. In multicellular organisms, mutations can be subdivided into germ line mutations, which can be passed on to descendants, and somatic mutations, which are not transmitted to descendants in animals. A new mutation that was not inherited from either parent is called a de novo mutation.  Types of DNA Damage Summarised G A T C ds DNA Break Mismatch C-U deamination ss Break AP site Covalent X-linking Thymidine dimer