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Nucleic Acids Nucleic Acids Are Essential For Information Transfer in Cells • Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule • The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein. Central Dogma of Biology Nucleic Acids • First discovered in 1869 by Miescher. • Found as a precipitate that formed when extracts from nuclei were treated with acid. • Compound contained C, N, O, and high amount of P. • Was an acid compound found in nuclei therefore named nucleic acid Nucleic Acids • 1944 Oswald, Avery, MacLeod and McCarty demonstrated that DNA is the molecule that carrier genetic information. • 1953 Watson and Crick proposed the double helix model for the structure of DNA Nucleic Acids • Nucleic acids are long polymers of nucleotides. • Nucleotides contain a 5 carbon sugar, a weakly basic nitrogenous compound (base), one or more phosphate groups. • Nucleosides are similar to nucleotides but have no phosphate groups. Pentoses of Nucleotides • D-ribose (in RNA) • 2-deoxy-D-ribose (in DNA) • The difference - 2'OH vs 2'-H • This difference affects secondary structure and stability Nitrogenous Bases Bases are attached by b-Nglycosidic linkages to 1 carbon of pentose sugar – (Nucleoside) Nucleosides • Base is linked via a b-Nglycosidic bond • The carbon of the glycosidic bond is anomeric • Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine • Conformation can be syn or anti • Sugars make nucleosides more water-soluble than free bases Anti- conformation predominates in nucleic acid polymers Nucleotides • Phosphate ester of nucleosides The plane of the base is oriented perpendicular to the plane of the pentose group Other Functions of Nucleotides • Nucleoside 5'-triphosphates are carriers of energy • Bases serve as recognition units • Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction • ATP is central to energy metabolism • GTP drives protein synthesis • CTP drives lipid synthesis • UTP drives carbohydrate metabolism • Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to form nucleic acid (polynucleotide) polymers Nucleic Acids • Nucleic acid backbone takes on extended conformation. • Nucleotide residues are all oriented in the same direction (5’ to 3’) giving the polymer directionality. • The sequence of DNA molecules is always read in the 5’ to 3’ direction Bases from two adjacent DNA strands can hydrogen bond •Guanine pairs with cytosine •Adenine pairs with thymine Base pairing evident in DNA compositions H-bonding of adjacent antiparallel DNA strands form double helix structure Properties of DNA Double Helix • Distance between the 2 sugar-phosphate backbones is always the same, give DNA molecule a regular shape. • Plane of bases are oriented perpendicular to backbone • Hydrophillic sugar phosphate backbone winds around outside of helix • Noncovalent interactions between upper and lower surfaces of base-pairs (stacking) forms a closely packed hydrophobic interior. • Hydrophobic environment makes H-bonding between bases stronger (no competition with water) • Cause the sugar-phosphate backbone to twist. View down the Double Helix Hydrophobic Interior with base pair stacking Sugar-phosphate backbone Structure of DNA Double Helix • Right handed helix • Rise = 0.33 nm/nucleotide • Pitch = 3.4 nm / turn • 10.4 nucleotides per turn • Two groves – major and minor • Within groves, functional groups on the edge of base pairs exposed to exterior • involved in interaction with proteins. Factors stabilizing DNA double Helix • Hydrophobic interactions – burying hydrophobic purine and pyrimidine rings in interior • Stacking interactions – van der Waals interactions between stacked bases. • Hydrogen Bonding – H-bonding between bases • Charge-Charge Interactions – Electrostatic repulsions of negatively charged phosphate groups are minimized by interaction with cations (e.g. Mg2+) DNA • 1o Structure - Linear array of nucleotides • 2o Structure – double helix • 3o Structure - Super-coiling, stemloop formation • 4o Structure – Packaging into chromatin Determination of the DNA 1o Structure (DNA Sequencing) • Can determine the sequence of DNA base pairs in any DNA molecule • Chain-termination method developed by Sanger • Involves in vitro replication of target DNA • Technology led to the sequencing of the human genome DNA Replication • DNA is a double-helical molecule • Each strand of the helix must be copied in complementary fashion by DNA polymerase • Each strand is a template for copying • DNA polymerase requires template and primer • Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA • DNA polymerases add nucleotides in 5'-3' direction Chain Termination Method • Based on DNA polymerase reaction • 4 separate rxns • Each reaction mixture contains dATP, dGTP, dCTP and dTTP • Each reaction also contains a small amount of one dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP). • Each of the 4 dideoxynucleotides are labeled with a different fluorescent dye. • Dideoxynucleotides missing 3’-OH group. Once incorporated into the DNA chain, chain elongation stops) Chain Termination Method • Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally • Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule • Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide O O N NH N NH N NH2 N N HO NH2 O H HO H N H O H H N H P NH2 O N H O NH2 N H O N O- N H H N O P N O- N O O H O H H O O H H H N O NH O O PH N O- N H P N O- NH2 O O O O O P O H O- OH H H H OH H H H H O H NH H OH OH P H N H OH O O H N NH2 O N NH N HO NH2 N NH2 O H H N H O O N H H P N O- NO CHAIN ELONGATION N O O H H H O H H N OH O P O P PH N O- O O O O O- H H OH H H H O NH OH N NH2 Chain Termination Method • Run each reaction mixture on electrophoresis gel • Short fragments go to bottom, long fragments on top • Read the "sequence" from bottom of gel to top • Convert this "sequence" to the complementary sequence • Now read from the other end and you have the sequence you wanted - read 5' to 3' DNA Secondary structure • DNA is double stranded with antiparallel strands • Right hand double helix • Three different helical forms (A, B and Z DNA. Comparison of A, B, Z DNA • A: right-handed, short and broad, 2.3 A, 11 bp per turn • B: right-handed, longer, thinner, 3.32 A, 10 bp per turn • Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn A-DNA B-DNA Z-DNA Z-DNA • Found in G:Crich regions of DNA • G goes to syn conformation • C stays anti but whole C nucleoside (base and sugar) flips 180 degrees DNA sequence Determines Melting Point • Double Strand DNA can be denatured by heat (get strand separation) • Can determine degree of denturation by measuring absorbance at 260 nm. • Conjugated double bonds in bases absorb light at 260 nm. • Base stacking causes less absorbance. • Increased single strandedness causes increase in absorbance DNA sequence Determines Melting Point • Melting temperature related to G:C and A:T content. • 3 H-bonds of G:C pair require higher temperatures to denture than 2 Hbonds of A:T pair. DNA o 3 Structure • Super coiling • Cruciform structures Supercoils • In duplex DNA, ten bp per turn of helix (relaxed form) • DNA helix can be over-wound. • Over winding of DNA helix can be compensated by supercoiling. • Supercoiling prevalent in circular DNA molecules and within local regions of long linear DNA strands • Enzymes called topoisomerases or gyrases can introduce or remove supercoils • In vivo most DNA is negatively supercoiled. • Therefore, it is easy to unwind short regions of the molecule to allow access for enzymes Each super coil compensates for one + or – turn of the double helix •Cruciforms occur in palindromic regions of DNA •Can form intrachain base pairing •Negative supercoiling may promote cruciforms DNA o 4 Structure • In chromosomes, DNA is tightly associated with proteins Chromosome Structure • Human DNA’s total length is ~2 meters! • This must be packaged into a nucleus that is about 5 micrometers in diameter • This represents a compression of more than 100,000! • It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments Nucleosome Structure • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins • % major histone proteins: H1, H2A, H2B, H3 and H4 • Histone octamers are major part of the “protein spools” • Nonhistone proteins are regulators of gene expression •4 major histone (H2A, H2B, H3, H4) proteins for octomer •200 base pair long DNA strand winds around the octomer •146 base pair DNA “spacer separates individual nucleosomes •H1 protein involved in higher-order chromatin structure. •W/O H1, Chromatin looks like beads on string Solenoid Structure of Chromatin RNA • Single stranded molecule • Chemically less stable than DNA • presence of 2’-OH makes RNA more susceptible to hydrolytic attack (especially form bases) • Prone to degradation by Ribonucleases (Rnases) • Has secondary structure. Can form intrachain base pairing (i.e.cruciform structures). • Multiple functions Type of RNA • Ribosomal RNA (rRNA) – integral part of ribosomes (very abundant) • Transfer RNA (tRNA) – carries activated amino acids to ribosomes. • Messenger RNA (mRNA) – endcodes sequences of amino acids in proteins. • Catalytic RNA (Ribozymes) – catalzye cleavage of specific RNA species. RNA can have extensive 2o structure