NUCLEIC ACIDS Genetic material of all known organisms DNA: deoxyribonucleic acid RNA: ribonucleic acid (e.g., some viruses)

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1 NUCLEIC ACIDS Genetic material of all known organisms DNA: deoxyribonucleic acid RNA: ribonucleic acid (e.g., some viruses) Consist of chemically linked sequences of nucleotides Nitrogenous base Pentose- 5-carbon sugar (ribose or deoxyribose) Phosphate group The sequence of bases provides the genetic information

2 Nitrogen bases Two types of bases Purines are fused five- and six-membered rings Adenine A DNA RNA Guanine G DNA RNA Pyrimidines are six-membered rings Cytosine C DNA RNA Thymine T DNA Uracil U RNA

3 Nitrogen bases

4 Ribose

5 Nucleoside, nucleotides and nucleic acids phosphate phosphate sugar base sugar base sugar base nucleoside nucleotides phosphate sugar base phosphate sugar base nucleic acids The chemical linkage between monomer units in nucleic acids is a phosphodiester

6 Nucleic Acids Levels of structure 1 structure: the order of bases on the polynucleotide sequence; the order of bases specifies the genetic code 2 structure: the three-dimensional conformation of the polynucleotide backbone 3 structure: supercoiling 4 structure: interaction between DNA and proteins

7 DNA is a polymer of 2 -deoxyribonucleotides RNA is a polymer of ribonucleotides Formed by Polymerase and Ligase activities A phosphodiester group has a pk a of about 1, and so will always be ionized and negatively charged under physiological conditions (ph ~7). Nucleic acids require counterions such as Mg 2+, polyamines, histones or other proteins to balance this charge. The phosphate groups of DNA and RNA are negatively charged

8 Secondary structure: the ordered arrangement of nucleic acid strands the double helix model of DNA 2 structure was proposed by James Watson and Francis Crick in 1953 Double helix:a type of 2 structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis structure based on X-Ray crystallography DNA - 2 Structure

9 A HISTORY OF DNA Discovery of the DNA double helix A. Erwin Chargaff - studied DNAs from various sources and analyzed the distribution of purines and pyrimidines in them. The distribution of the bases adenine (A), guanine (G), thymine (T), and cytosine (C) varied among species. Total purines (A and G) and the total pyrimidines (T and C) were always equal. %A = %T, and %G = %C B. Rosalind Franklin - X-ray photo of DNA. (1952) C. Watson and Crick - described the DNA molecule from Franklin s X-ray. (1953)

10 5 DNA: Helix Antiparallel Chains 5 p OH3 3 OH p5 Two strands of the DNA double helix are antiparallel and complementary to each other. A nucleic acid polymer has a free 5 -phosphate group at one end and a 3 OH group at the other end.

11 In general, DNA is double-stranded. Double-stranded (ds) DNA takes the form of a right handed helix with approximately 10 base pairs per turn of the helix.

12 The B-form DNA helix has a diameter of about 20 Å Standard DNA double helix under physiological conditions ~20 Å Base pairs fill the center of the helix; the phosphates ( ) are on the outside. A base pair is more exposed to the solvent on one side (the major groove ) than the other (the minor groove ).

13 DNA double helix major groove 12 Å one helical turn 34 Å minor groove 6 Å backbone: deoxyribose and phosphodiester linkage bases

14 Tertiary Structure of DNA: Supercoils. Each cell contains about two meters of DNA. DNA is packaged by coiling around a core of proteins known as histones. The DNA-histone assembly is called a nucleosome. Histones are rich is lysine and arginine residues. Pdb code 1kx5

15 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

16 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

17 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

18 The two strands of the double helix separate reversibly at high temperatures % Denatured % GC Temperature / o C 110 The temperature at which this denaturation or melting occurs depends on the ph and salt concentration, and increases with the GC content of the DNA. (The curves drawn here are schematic.) If the temperature is lowered, the strands recombine. The rate of reassociation is inversely proportional to the complexity of the DNA.

19 Denaturation of DNA Double helix unwinds when DNA is denatured Can be re-formed with slow cooling and annealing

20 Summary of the main structural features of B-form DNA Right-handed helix Two antiparallel strands held together by Watson-Crick hydrogen bonds Pitch (repeat length) = 34 Å (3.4 nm) 36 o rotation between residues Helix diameter of 20 Å (2.0 nm) Wide major groove, narrow minor groove Chargaff s Rules: A = T; G C Charged phosphates Bases in anti configuration The strands separate at high temperatures The solution structure is dynamic

21 Cells contain a variety of types of RNA with different functions Principle kinds of RNA in E. coli Type Sed. Coef. Mol. Wt. Residues % of total RNA mrna ,000-1,000, ~2 trna ~4 23,000-30, rrna 5 35, ,000 1, ,100,000 2,904 Eucaryotic cells contain an additional type, small nuclear RNA (snrna)

22 Transfer RNA, trna: the smallest kind of the three RNAs a single-stranded polynucleotide chain between nucleotide residues carries an amino acid at its 3 end intramolecular hydrogen bonding occurs in trna trna

23 rrna Ribosomal RNA, rrna: a ribonucleic acid found in ribosomes, the site of protein synthesis only a few types of rrna exist in cells ribosomes consist of 60 to 65% rrna and 35 to 40% protein in both prokaryotes and eukaryotes, ribosomes consist of two subunits, one larger than the other analyzed by analytical ultracentrifugation particles characterized by sedimentation coefficients, expressed in Svedberg units (S)

24 mrna Messenger RNA, mrna: a ribonucleic acid that carries coded genetic information from DNA to ribosomes for the synthesis of proteins present in cells in relatively small amounts and very short-lived single stranded biosynthesis is directed by information encoded on DNA a complementary strand of mrna is synthesized along one strand of an unwound DNA, starting from the 3 end

25 DNA molecules are packaged in the cell as structures called chromosomes. Bacteria have a single chromosome. Eukaryotes have multiple chromosomes. A single chromosome contains thousands of genes, each encoding a protein. All of an organism s chromosomes make up the genome. Humans have 46 chromosomes. The human genome has about 3 billion nucleotide base pairs.

26 Semiconservative Replication of DNA

27 Figure 3.2 The Meselson Stahl experiment. The demonstration of semiconservative replication in E. coli. Cells were grown in a 15N-containing medium for several replication cycles and then were transferred to a 14N-containing medium. At various times over several replication cycles, samples were taken; the DNA was extracted and analyzed by CsCl equilibrium density gradient centrifugation. Shown in the figure are a schematic interpretation of the DNA composition after various replication cycles, photographs of the DNA bands, and densitometric scans of the bands.

28 The Chemistry of DNA replication DNA Synthesis by DNA polymerase

29 Replication of E. coli DNA Is semi-conservative Is bidirectional Proceeds from a specific point of origin Proceeds in a 5-3 direction

30 Synthesis of Leading and Lagging Strands of DNA

31 Initiation of Okazaki Fragments with RNA Primers

32 Removal of RNA Primers and Joining of Okazaki Fragments

33 Removal of RNA Primers and Joining of Okazaki Fragments

34 Action of Helicases and Single-Stranded DNA-Binding Proteins

35 Action of Topoisomerases during DNA Replication

36 Direction of synthesis on leading strand Two dimensional view of a replication fork

37 Origin of replication (e.g., the prokaryote example): Begins with double-helix denaturing into single-strands thus exposing the bases. Exposes a replication bubble from which replication proceeds in both directions. ~245 bp in E. coli

38 Proofreading by the 3 5 exonuclease activity of DNA polymerases during DNA replication.

39 Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA Schematic representation of DNA Polymerase III Structures of DNA polymerase during polymerizing and editing E: exonucleolytic; P: polymerization

40 DNA replication proteins

41 There are many different types of DNA polymerase Polymerase Polymerization (5-3 ) Exonuclease (3-5 ) Exonuclease (5-3 ) #Copies I Yes Yes Yes 400 II Yes Yes No? III Yes Yes No to 5 exonuclease activity = ability to remove nucleotides from the 3 end of the chain Important proofreading ability Without proofreading error rate (mutation rate) is 1 x 10-6 With proofreading error rate is 1 x 10-9 (1000-fold decrease) 5 to 3 exonuclease activity functions in DNA replication & repair.

42 Eukaryotic enzymes: Five DNA polymerases from mammals. 1. Polymerase α (alpha): nuclear, DNA replication, no proofreading 2. Polymerase β (beta): nuclear, DNA repair, no proofreading 3. Polymerase γ (gamma): mitochondria, DNA repl., proofreading 4. Polymerase δ (delta): nuclear, DNA replication, proofreading 5. Polymerase ε (epsilon): nuclear, DNA repair (?), proofreading Different polymerases for nucleus and mtdna Some polymerases proofread; others do not. Some polymerases used for replication; others for repair.

43 Roles of DNA Polymerases in E. coli and Mammalian Cells

44 Origin of Replication in E. coli

45 Replication Origins in Eukaryotic Chromosomes

46 DNA polymerases can only synthesize DNA only in the 5 to 3 direction and cannot initiate DNA synthesis These two features pose a problem at the 3 end of linear chromosomes Problem at ends of eukaryotic linear Chromosomes

47 Action of Telomerase

48 Action of Telomerase

49 Step 1 = Binding The bindingpolymerizationtranslocation cycle can occurs many times Step 2 = Polymerization This greatly lengthens one of the strands Step 3 = Translocation The complementary strand is made by primase, DNA polymerase and ligase RNA primer