Structure of Nucleic Acids Objectives: I. Describe the flow of information in the cell - The Central Dogma of Biochemistry II. Describe the primary structure of Deoxyribonucleic acid (DNA) and Ribonucleic Acid (RNA). A. Identify the nucleotides that are present in each. 1. Differences and similarities 2. Distinguish between the numbering schemes for the two ring systems. B. Type and direction of chemical bonds between the nucleotide monomers. III. Describe the secondary structure of DNA. A. Chargaff’s Rules. B. Hydrogen bonding between adenine & thymine and between guanine and cytosine bases. 1. Can other pairs form that are stabilized by hydrogen bonded? C. Describe the Watson & Crick Model of DNA Secondary Structure. 1. Describe the forces that stabilize the double helical structure. a) Hydrogen bonds between complimentary bases; A-T & G-C. b) Base stacking 2. Describe the major and minor groove. a) Describe the structure and possible functions of these grooves. 3. Identify the direction of the individual DNA strands in the double helical molecule. D. Describe the other possible secondary structures that the DNA double helix can assume 1. Possible functions? E. Define the melting temperature (Tm) of double stranded DNA IV. Describe the higher order structures of DNA. A. Describe the structure of Chromatin. 1. What are histones? 2. What comprises the Core Histone Particle? 3. What comprises the Nucleosome Core Particle? 4. What is linker DNA? B. Describe the 30 nm Solenoid Structure. 1. How is it formed? C. Describe the RNA-Protein Scaffold (Nuclear Scaffold or Nuclear Matrix). D. Describe a Rosette E. Describe a Miniband Unit. V. Other Nucleic Acids A. Describe the types of RNA 1. hnRNA, mRNA, tRNA, rRNA, sRNA, RNAi B. Can these molecules assume secondary structures? VI. Be able to write the complimentary nucleotide sequence to a given nucleotide sequence using the accepted conventions. VII. Nucleases A. Distinguish between Ribonucleases (RNases), Deoxyribonucleases (DNases), Endonucleases, and Exonucleases. B. Describe a Restriction Endonuclease. 1. What are their normal function within a bacterial cell? 1 ©Kevin R. Siebenlist, 2019 2. What function do Molecular Biologists use these enzymes for? 3. What is a sticky end? GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT GCAT Background A cell requires three things for growth, maintenance, and reproduction. It requires energy to drive metabolic processes, precursors for energy generation and for the synthesis of complex biopolymers, and information to maintain, coordinate, and control the entire process. The precursor molecules as well as some of the energy generating processes and biosynthetic processes have been discussed. In this section the third type of information carrying molecule will be examined. Allosteric enzymes and hormones are two types of information molecules that have been examined. In this section the primary cellular information molecule DEOXYRIBONUCLEIC ACID (DNA) and the working copy of this information in the form of RIBONUCLEIC ACID (RNA) will be examined. DNA and RNA are long linear polymers of deoxyribonucleotides or ribonucleotides, respectively. The archive copy of the cellular blueprint is carried on DNA. This information is duplicated (replicated) and passed on to each daughter cell during cell division. When needed to direct some cellular process, the information is copied (transcribed) into a molecule of RNA. RNA is the “working copy” of the cellular information. Some of the RNA molecules are used to direct the synthesis of proteins. The information on RNA is translated into the primary structure of proteins. Proteins, as allosteric enzymes, hormones, receptors, etc., control the moment to moment activity of the cell. The flow of information from DNA to RNA and then to the primary structure of a protein is the CENTRAL DOGMA of BIOCHEMISTRY. In this section of the course: 1. The structure of nucleic acids, DNA and RNA, will be described. 2. The process by which DNA is duplicated (REPLICATION) before cell division will be examined. REPLICATION produces two exact copies, two daughter strands, of DNA from the original DNA molecule, from the one parent strand. 3. Methods for DNA repair will be discussed. An accurate functional copy of the information on DNA must be maintained to assure proper cellular function. DNA is the only biomolecule with repair mechanisms. 4. The method for copying the information stored on DNA into a working copy of RNA will be discussed. This process is TRANSCRIPTION. 5. The control of transcription will be touched upon. This is the CONTROL of GENE EXPRESSION. 6. The TRANSLATION of the information on messenger RNA into the primary structure of a protein will be examined. The control points of translation in the eukaryotic system will also be explored. Nucleotide Primary Structure A quick review. Nucleic acids are long linear polymers of nucleotides. When nucleic acids are hydrolyzed, nucleoside monophosphates result. The monomeric unit of nucleic acids are nucleoside monophosphates. Nucleoside triphosphates are the precursors for the synthesis nucleic acids. The energy released by the 2 ©Kevin R. Siebenlist, 2019 hydrolysis of the two phosphoanhydride bonds is used to form the 3´,5´-phosphodiester bond between the monomer units and to “drive” the reaction to completion. O NH O O O N O O P O P O P O CH 2 O O O O O N NH O OH 5’ end N O P O N NH2 O O CH NH2 2 O H3C NH N O OH RNA N O O O O O P O N O O P O P O P O CH2 O CH O O 2 O NH2 O O O N N NH N O O OH N O P O N NH2 O P O N N O CH2 NH2 O CH O 2 O NH2 N N N O O OH DNA N O O P O O P O N N O CH2 O CH NH2 O NH2 2 O N N N O O OH 3’ 5’ N phosphodiester O P O N O P O N O bond O CH2 O CH O NH2 2 O N N O OH OH O P O N N O CH NH2 2 O N O 3’ end O P O N O O CH 2 O OH DNA, Deoxyribonucleic acids, contain 2´-deoxyribonucleotides, whereas RNA, Ribonucleic acid, contain 3 ©Kevin R. Siebenlist, 2019 ribonucleotides. The backbone of the linear DNA and RNA polymers is comprised of repeating sugar and phosphate residues held together by 3´,5´ phosphodiester bonds. The heterocyclic bases on DNA are Adenine (A), Guanine (G), Cytosine (C), and Thymidine (T). In RNA thymidine is replaced by Uracil (U). Information of DNA and RNA is carried by the unique linear sequence of bases on the long linear polymer. At one end of each strand there is a 5´ triphosphate group that is not involved in a phosphodiester bond. This is the 5´ end of the molecule. At the other end there is a free, unreacted 3´ hydroxyl group. This is the 3´ end of the molecule Nucleotide Secondary Structure DNA was first isolated and purified by Friedrich Miescher in 1868. In 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrated that DNA carried the genetic information for the cell. The bases, Adenine, Guanine, Cytosine, Thymine, and Uracil, can form hydrogen bonds between themselves and each other. In 1947 J.M. Gulland demonstrated that Adenosine formed two very stable hydrogen bonds with Thymidine or Uracil and that Guanosine formed three very stable hydrogen bonds with Cytidine. In the early 1950’s Erwin Chargaff observed that DNA isolated from a given species of organism contained equal molar amounts of the bases Adenine and Thymidine. The molar ratio of Cytosine to Guanine was also equal, and the ratio of purines to pyrimidines was 1:1 even when the amount of G and C was vastly different from the amount of A and T. X-ray diffraction patterns of DNA were obtained in the very early 1950’s by Maurice Wilkins, Rosalind Franklin, and Linus Pauling. In 1953 James D. Watson and Francis H.C. Crick proposed a model for the secondary structure of DNA. Their model was based upon X-ray diffraction patterns obtained by Rosalind Franklin and her group, upon the stable hydrogen bonding between A&T and G&C, and upon the A/T & G/ C ratios noted by Chargaff. According to the Watson & Crick model, DNA as isolated from the cell is a double stranded molecule. The sugar phosphate backbone of the polymer is on the outside of the molecule with the sugars linked to one another by 3´,5´-phosphodiester bonds. In the interior of the molecule are the heterocyclic bases. The bases on one strand interact specifically with the bases on the opposite strand. Adenine hydrogen bonds / base pairs with Thymidine and Guanine hydrogen bonds / base pairs with Cytosine. The two stranded DNA molecule is twisted into a right handed helix. Double stranded DNA is held together / stabilized by several weak intermolecular forces. One of the forces is the hydrogen bonding between the bases. Adenine forming 2 hydrogen bonds with Thymidine and Guanine forming 3 hydrogen bonds with Cytosine. This double helical arrangement allows the strongest possible hydrogen bonds to form between the heterocyclic bases. BASE STACKING is the second stabilizing force. In BASE STACKING the delocalized pi (π) electrons of the aromatic heterocyclic bases overlap and interact with each other. The aromatic, π, electrons are delocalized (free to move) along the entire length of the DNA double helix. Base pairing stabilizes the DNA helix across the strands and stacking maximizes interactions between the bases up and down the DNA strand. There are electrostatic interactions along the backbone of the molecules.