Lecture 1_Nucleic Acids -DNA and RNA_Final PDF

Dr Icolyn Amarakoon Email: [email protected] OFFICE LOCATION: LEVEL 2 Block A- Offices behind the Molecular Biology lab {through the glass doors and it’s the 1st door on the right}  LECTURE SERIES OUTLINE (3) Nucleic acids (DNA and RNA) Structures and Functions Nucleic acids...

Dr Icolyn Amarakoon Email: [email protected] OFFICE LOCATION: LEVEL 2 Block A- Offices behind the Molecular Biology lab {through the glass doors and it’s the 1st door on the right}  LECTURE SERIES OUTLINE (3) Nucleic acids (DNA and RNA) Structures and Functions Nucleic acids metabolism Biosynthesis and Degradation of the nucleotides Associated disorders in the metabolic processes Pharmaceutical applications of nucleotide monomers  TEXTS Lippincott's Illustrated Reviews: Biochemistry by Richard A. Harvey, Pamela C. Champe, Denise R. Ferrier, et al. Lehninger Principles of Biochemistry, by David L. Nelson; Michael M. Cox Biochemistry by Jeremy Berg; Gregory Gatto Jr.; Justin Hines; John L. Tymoczko; Lubert Stryer  LEARNING OUTCOMES At the end of this lecture you should be able to…… Describe the structural components of nucleic acids Discuss the main functions of DNA and RNA Demonstrate how the structures of DNA and RNA account for their functions Explain how DNA molecules, packaged within the nucleus  Why study nucleic acids? DNA is the blueprint for the individuality of an organism. The organism relies upon the information stored in its DNA for the management of every biochemical process. The life, growth and unique features of the organism depend on its DNA. 5  SIGNIFICANCE Life depends on the ability of cells: to store, retrieve and translate the genetic instructions required to make and maintain a living organism. This hereditary information is passed on from a cell to its daughter cells at cell division, from one generation of an organism to the next through the organism's reproductive cells. 6  NUCLEIC ACIDS The two main nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules that record and transmit our genetic information structure and function of our bodies (via proteins and enzymes) pass information from generation to generation  The Central Dogma of Molecular biology  The flow of genetic information : DNA → RNA → Protein DNA REPLICATION TRANSCRIPTION TRANSLATION A gene is expressed in two steps: DNA is transcribed to RNA 8 RNA is translated into protein STRUCTURES AND FUNCTIONS OF DNA & RNA 9  COMPONENTS OF NUCLEIC ACIDS Ribonucleic Acid: RNA Deoxyribonucleic Acid: DNA is the genetic material that is the hereditary material in transcribes DNA's instructions and translates humans these instructions to proteins DNA and RNA are polymers [polynucleotides] 10 Monomer unit is a nucleotide.  Nucleotide A nucleotide consists of: a 5-carbon sugar (pentose) – deoxyribose for DNA and ribose for RNA a purine or a pyrimidine base containing nitrogen attached to the sugar and a phosphate group. 11 5-Carbon Sugar (Pentose) 12  Nitrogen bases  Purines have 2 ring structure 13  Nitrogen bases  Pyrimidines have 1 ring structure 14 NUCLEOTIDE FORMATION COMPONENTS OF NUCLEIC ACIDS NUCLEOSIDE NUCLEOTIDE FUNCTIONS OF NUCLEOTIDES IN CELLS Precursors of DNA and RNA Carriers of chemical energy (ATP and GTP) Components of co-factors (NAD+, FAD, Co-enzyme A) Components of activated biosynthetic intermediates (UDP-glucose - precursor of glycogen) Cellular secondary messengers (cAMP and cGMP)  NOMENCLATURE SUFFIX:’osine’ for purine bases;’idine’ for pyrimidine bases PREFIX: ‘deoxy’ for DNA.  BASE COMPOSITION OF DNA Chemical basis of base pairing in DNA came from the analysis of DNA by Erwin Chargaff in 1940’s. four bases do not occur in equimolar amounts and these varied from species to species. Certain bases were always found in 1:1 ratio number of pyrimidines always equaled the number of purines  Chargaff’s Rules (1952) # purines = # pyrimidines % amino bases (A & C) = % keto bases (G & T) Equivalence between the amounts of A & T, and between the amounts of G and C ( A = T and G = C). Wide variations in the molar proportions of bases although DNA from different organs and tissues of any one species are essentially the same. A+T/G+C (base ratio) may vary widely between species, and remains constant for any one species.  Structure of DNA Watson and Crick in 1953 proposed that the DNA molecule extended chain having a highly ordered structure and is composed of: Two complementary polymeric chains forming a regular right-handed double helix The two stands run in opposite directions (antiparallel alpha-helices), and are of opposite polarity The rails of the ladder runs in opposite direction and contain alternating units of deoxyribose sugar and phosphate. The sugar and phosphate groups are always linked together by 3’ - 5’ phosphodiester linkages.  Secondary Structure of DNA Watson & Crick proposed that: The bases are arranged at right angles to the long axis of the polynucleotide chain. Each step is composed of a pair of nucleotides - a base pair held together by weak hydrogen bonds. The order of the purine and pyrimidine bases along the chain is highly irregular, varying from one molecule to the other. The chain is not straight but is wound helically around a central axis, one full turn (the pitch) of the helix extending 3.4 nm (34 Å), and there are 10 bases per turn  Structure of DNA Watson & Crick proposed that: The bases are separated by a spacing of 0.34 nm (3.4 Å). The width of the double helix is 2 nm (20 Å). The chains are complementary, the sequence of bases on one strand is the exact complement of the other strand. Adenine always pair with thymine and cytosine always pair with guanine.  DISCOVERY OF DNA STRUCTURE Watson and Crick Franklin and Wilkins 24  DNA & RNA backbone DNA/RNA backbone: a polymer with an alternating sugar-phosphate sequence. The deoxyribose sugars are joined at both the 3'- hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds 25 DNA & RNA BACKBONE 26  DNA Helix DNA is a double stranded macromolecule. Two polynucleotide chains, held together by weak bonds, form a DNA molecule 27  Features of the DNA Double Helix Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral The two polynucleotide chains run in opposite directions 28  Features of the DNA Double Helix The sugar-phosphate backbone of the two DNA strands spirals around the helix axis The bases are on the inside of the helix, stacked like the steps of a spiral staircase. 29  THE DOUBLE HELIX C G 5 end C G Hydrogen bond 3 end G C G C T A 3.4 nm T A G C G C C G A T 1 nm C G T A C G G C C G A T A T A T 3 end T A 0.34 nm 5 end (a) Key features of (b) Partial chemical structure DNA structure  BASE PAIRS RULES Adenine always base pairs with Thymine (or Uracil if RNA) A’ forms 2 hydrogen bonds with T on the opposite strand Cytosine always base pairs with Guanine. G’ forms 3 hydrogen bonds with C on the opposite strand There is exactly enough room for one purine and one pyrimidine base between the two polynucleotide strands of DNA. 31  Bases Pair in a Specific Way Purines are larger structures than pyrimidine, if two purine are paired their dimensions are too great to fit the constant diameter of the double helix (2 nm) while the dimensions of the two pyrimidines are too small The specificity of position of the H atoms that can participate in bonding. It is essential that the hydrogen bonds have relatively stable positions to have the biological functioning of DNA. Structure of DNA 5'-ACGTAACGTT-3' 3'-TGCATTGCAA-5' The standard A=T and G≡C base pairs have very similar geometries and there is exactly enough room for one purine and one pyrimidine base between the strands of DNA. incorrectly paired bases can exclude them from the active site (Blue shade) 34  DNA Structural Conformations Major forms of DNA : the A-form DNA the B-form, [Watson and Crick model] the Z-form DNA.  DNA Structural Conformations A-form DNA:-right handed helix, 11bp/turn, dehydrated form of B, base are 20% tilt to axis, DNA/RNA hydrids The B-form, [Watson and Crick model]: right handed helix, 10 bp/turn, base perpendicular to helical axis The Z-form DNA: left handed helix, 12 bp/turn, poly GC regions DNA Conformation: B-form (Watson and Crick) Primary form of DNA in cell Helix has 2 grooves Major groove Minor groove Provides binding sites for regulatory proteins  Stability of the double helix The two strands of double- stranded DNA are held together by a number of interactions hydrogen bonds stacking interactions of bases hydrophobic effects [3-D structure of DNA]  FACTORS AFFECTING DNA STABILITY Heat and pH Extremes of heat (80oC) and pH denatures DNA Disruption of hydrogen bonds and base stacking. No covalent bonds in the DNA are broken, but strand separation. Nucleic acids high in G-C have a higher temperature of melt Deamination Loss of an amino group C converted to U in DNA Important that DNA has T instead of U so that it can recognize when cytosine is deaminated and repair the system  FACTORS AFFECTING DNA STABILITY Radiation Ionizing radiation (X-rays and gamma rays) Causes opening of ring structure and fragmentation of bases as well as breaks in covalent backbone UV radiation from solar system (skin cancer) Radioactive elements eg. Plutonium (KGB agent poisoning) Radiotherapy of cancer and other diseases FACTORS AFFECTING DNA STABILITY CHEMICALS Nitrous acid deaminating agent Precursors (Nitrate and nitrite salts) Bisulphite Found in preservatives FACTORS AFFECTING DNA STABILITY Oxidative damage Hydrogen peroxide, Hydroxyl ions Superoxide radicals Arise during irradiation or as a by product of aerobic metabolism Most damage done by OH- ions Human cell subjected to thousands of damaging oxidative reactions Implications for cancer Antioxidants - (Vitamins A, C and E, and the minerals copper, zinc and selenium.)  RNA: Ribonucleic Acid Chemically, RNA is very similar to DNA. There are some main differences: – RNA uses ribose instead of deoxyribose in its backbone. – RNA uses the base Uracil (U) instead of Thymine (T). U is also complementary to A. – RNA tends to be single-stranded. Functional differences between RNA and DNA – DNA single function, RNA many functions Example of types of RNA: mRNA, tRNA, rRNA 43  RNA STRUCTURE: Messenger RNA (mRNA) Synthesized during transcription-the sequence of bases in one strand of the DNA is enzymatically transcribed into the form of a single strand of mRNA with complementary base sequence. Differs greatly in molecular weight and in base sequence Total cellular mRNA = 5%  tRNA STRUCTURE Small molecules that act as the carriers of specific amino acids during protein synthesis. Clover leaf structure Total cellular tRNA = 15% At least 20 types of tRNA in a cell To recognise and read the codon of the mRNA to fetch correct amino acid  transfer a specific amino acid to the growing polypeptide chain at the ribosomal site 45  RNA : Ribosomal RNA (rRNA) & Other RNAs Ribosomal RNA (rRNA) Other RNAs Small nuclear RNA (snRNA) 2% Most abundant RNA. Involved in post-transcriptional modification of Form part of the site for proteins protein synthesis. Catalytic RNA or Ribozyme Total cellular rRNA = 80%. microRNA (miRNA) functions in RNA silencing and post- transcriptional regulation of gene expression Small interfering RNA (siRNA) Interfere with the expression of a specific gene  What is a Gene? A gene is the basic physical and functional unit of heredity. Genes, which are made up of DNA, act as instructions to make molecules called proteins. Every person has two copies of each gene, one inherited from each parent 47 What is a chromosome? In the nucleus the DNA molecule is packaged into thread-like structures called chromosomes. Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the cell is not dividing. 48  CHROMOSOMES Chromosomes contain DNA, histones and other proteins that affect gene expression (which proteins and how many proteins are synthesized from a given gene).  DNA-Protein Complexes DNA complexes with specific DNA binding proteins to form compact molecules called chromatin. The most prominent DNA binding proteins are the histones. Histones are small, positively charged arginine-lysine rich proteins that aggregate, around which DNA supercoils.  CHROMOSOMES, CHROMATID,CHROMATIN Each chromosome made with two identical part called chromatid. Two chromatid link each other at centromere Chromatin is comprised of histone & DNA. 147 bps of wrapped DNA around 8 core histones to form the basic chromatin unit, the nucleosome  NUCLEOSOME 8 histones in core DNA wrapped twice around the core one histone holding the nucleosome together a DNA linker continuing towards the next nucleosome  Nucleosomes Nucleosomes help to supercoil the DNA: essential to pack genetic material into the nucleus to organise DNA to allow cell division to occur (most DNA supercoiling occurs at this time) to control DNA expression - supercoiled DNA cannot be transcribed allow cells to specialise by permanently supercoiling DNA (heterochromatin) transcription of active chromatin (Euchromatin) can be promoted or inhibited by the associated histones  APPLICATIONS OF NUCLEIC ACID Relationship Testing (DNA Paternity)  APPLICATIONS OF NUCLEIC ACID Diagnostics Viral STDs (eg. HPV DNA high risk screen) Chromosomal abnormities (during pregnancies) [Trisomy Testing]  DNA Structure (Pharmaceutical Application) Daunorubicin and Doxorubicin Cisplatin Antitumor drugs Treats bladder and lung tumors Treats leukemia Binds tightly to DNA Intercalates between the Distortion and malfunction of DNA bases of DNA prevents proper replication of DNA