1. Three dimensional structure of DNA A double helix has major groove and minor grooveA double helix has major groove and minor groove Within each groove base pairs are exposed and are accessible to interactions with other moleculesWithin each groove base pairs are exposed and are accessible to interactions with other molecules DNA-binding proteins can use these interactions to “read” a specific sequenceDNA-binding proteins can use these interactions to “read” a specific sequence B-DNA is a right-handed helix, diam. = 2.37nm Rise (distance between stacked bases) =0.33nmRise (distance between stacked bases) =0.33nm Pitch (distance to complete one turn) = 3.40 nmPitch (distance to complete one turn) = 3.40 nm 10.4 base pairs per turn10.4 base pairs per turn Chapter 19 Nucleic Acids II

2. Weak Forces Stabilize the Double Helix (1)Hydrophobic effects. Burying purine and pyrimidine rings in the double helix interior (2) Stacking interactions. Stacked base pairs form van der Waals contacts (3) Hydrogen bonds. Hydrogen bonding between base pairs. (4) Charge-charge interactions. Electrostatic repulsion of negatively charged phosphate groups is decreased by cations (e.g. Mg 2+ ) and cationic proteins

3. Weak Forces Stabilize the Double Helix (1) Hydrophobic effects. Burying purine and pyrimidine rings in the double helix interior (2) Stacking interactions. Stacked base pairs form van der Waals contacts (3) Hydrogen bonds. Hydrogen bonding between base pairs. (4) Charge-charge interactions. Electrostatic repulsion of negatively charged phosphate groups is decreased by cations (e.g. Mg 2+ ) and cationic proteins ds DNA predominates in vivo Double-stranded DNA is thermodynamically more stable than the separated strands (under physiological conditions)

4. Denaturation of DNA Denaturation - Complete unwinding and separation of the 2 strands of DNA …….(only in vitro) Heat or chaotropic agents (e.g. urea) can denature DNA Double-stranded (DS) DNA (pH 7.0), absorbance max 260nm Denatured DNA absorbs 12% -40% more than DS DNA Local unwinding can occur in vivo Allows easy measurement of ‘DNA melting’

5. Heat denaturation of DNA Melting point (T m ) - temperature at which 1/2 of the DNA has become single stranded Melting curves can be followed at Abs 260nm cooperative

6. DNA Can Be Supercoiled Overwound or underwound DNA….compensates by supercoiling to restore 10.4 base pairs per turn of helix Over/under winding DNA (stress) can be caused by various ‘activities’ Transcription local unwinding of dsDNA and helical stress Replication

7. Topoisomerases - enzymes that can alter the topology of DNA helixes by: (1) Cleaving one or both DNA strands (2) Unwinding or overwinding the double helix by rotating the strands (3) Rejoining ends to create (or remove) supercoils Overwound or underwound DNA….compensates by supercoiling to restore 10.4 base pairs per turn of helix Topoisomerase

8. Cells Contain Several Kinds of RNA Cells Contain Several Kinds of RNA Ribosomal RNA (rRNA) - an integral part of ribosomes, accounts for ~80% of RNA in cellsRibosomal RNA (rRNA) - an integral part of ribosomes, accounts for ~80% of RNA in cells Transfer RNA (tRNA) - carry activated amino acids to ribosomes for polypeptide synthesis (small molecules 73- 95 nucleotides long)Transfer RNA (tRNA) - carry activated amino acids to ribosomes for polypeptide synthesis (small molecules 73- 95 nucleotides long) Messenger RNA (mRNA) - carry sequence information to the translation complexMessenger RNA (mRNA) - carry sequence information to the translation complex Small RNA - have catalytic activity or associate with proteins to enhance activitySmall RNA - have catalytic activity or associate with proteins to enhance activity Four major classes

9. DNA mRNA RNA Protein Non-coding tRNA and Ribosomal RNA catalytic antisense RNA interference Guide RNA Small RNAs 1 23 4 Cells Contain Several Kinds of RNA Cells Contain Several Kinds of RNA (Junk DNA) Essential for protein function Four major classes 80% of RNA in cell

10. Chromatin - DNA plus various proteins that package the DNA in a more compact formChromatin - DNA plus various proteins that package the DNA in a more compact form The packing ratio: difference between the length of the metaphase DNA chromosome and the extended B form of DNA is 8000-foldThe packing ratio: difference between the length of the metaphase DNA chromosome and the extended B form of DNA is 8000-fold DNA Is Packaged in Chromatin in Eukaryotic Cells How to pack long DNA molecule (genome) into nucleus

11. Active and silent chromatin

12. DNA mRNAProtein Hb Liver cell RBC Cheek cell Regulation of gene expression Regulation of gene expression Insulin receptor amalase

13. DNA mRNAProtein Hb Liver cell RBC Cheek cell Regulation of gene expression Regulation of gene expression Insulin receptor amalase XX heterochromatin euchromatin

14. Nucleosomes Histones - the major proteins of chromatinHistones - the major proteins of chromatin Eukaryotes contain five small, basic histone proteins containing many lysines and arginines: H1, H2A, H2B, H3, and H4Eukaryotes contain five small, basic histone proteins containing many lysines and arginines: H1, H2A, H2B, H3, and H4 Positively charged histones bind to negatively-charged sugar- phosphates of DNAPositively charged histones bind to negatively-charged sugar- phosphates of DNA Extended Chromatin Beads-on-a-string Histone-DNA complex: nucleosome

15. Nucleosomes Histones - the major proteins of chromatinHistones - the major proteins of chromatin Eukaryotes contain five small, basic histone proteins containing many lysines and arginines: H1, H2A, H2B, H3, and H4Eukaryotes contain five small, basic histone proteins containing many lysines and arginines: H1, H2A, H2B, H3, and H4 Positively charged histones bind to negatively-charged sugar- phosphates of DNAPositively charged histones bind to negatively-charged sugar- phosphates of DNA Nucleosome “beads” are DNA-histone complexes on a “string” of double-stranded DNANucleosome “beads” are DNA-histone complexes on a “string” of double-stranded DNA Each nucleosome is composed of:Each nucleosome is composed of: Histone H1(1 molecule) Histone H1(1 molecule) Histones H2A, H2B, H3, H4 (2 molecules each) Histones H2A, H2B, H3, H4 (2 molecules each) ~200 bp of DNA ~200 bp of DNA

16. Diagram of nucleosome structure Packaging of DNA in nucleosomes reduces DNA length ~tenfoldPackaging of DNA in nucleosomes reduces DNA length ~tenfold Each nucleosome is composed of:Each nucleosome is composed of: Histone H1(1 molecule)Histone H1(1 molecule) Histones H2A, H2B, H3, H4 (2 molecules each) ~200 bp of DNA +charge groove

17. Nucleosome

18. DNA is packaged further by coiling of the “beads-on-a-string” into a solenoid structure.DNA is packaged further by coiling of the “beads-on-a-string” into a solenoid structure. Achieves another fourfold reduction in chromosome length. (4 X 10 = 40 fold)Achieves another fourfold reduction in chromosome length. (4 X 10 = 40 fold) Model of the 30nm chromatin fiber shown as a solenoid or helix formed by individual nucleosomesModel of the 30nm chromatin fiber shown as a solenoid or helix formed by individual nucleosomes Nucleosomes associate through contacts between adjacent histone H1 moleculesNucleosomes associate through contacts between adjacent histone H1 molecules 30 nm Chromatin Fiber

19. protein scaffolds in chromatin Chromatin fibers attach to scaffoldsChromatin fibers attach to scaffolds Holds DNA fibers in large loopsHolds DNA fibers in large loops This accounts for an additional 200-fold condensation in DNA length. (200 X 40 = 8000 fold)This accounts for an additional 200-fold condensation in DNA length. (200 X 40 = 8000 fold) Histones have been Removed to visualize scaffolds Protein scaffold Loops attached to scaffold

20. 10x 4x 200x

22. Nucleases - hydrolyze phosphodiester bonds RNases (RNA substrates) DNases (DNA substrates)Nucleases - hydrolyze phosphodiester bonds RNases (RNA substrates) DNases (DNA substrates) Exonucleases start at the end of a chainExonucleases start at the end of a chain Endonucleases hydrolyze sites within a chainEndonucleases hydrolyze sites within a chain Nucleases and Hydrolysis of Nucleic Acids May cleave either the 3’- or the 5’- ester bond of a 3’-5’ phosphodiester linkage Required for synthesis/repair of DNA synthesis/degradation of RNA

23. OH group of RNA OH OH OH OH Can form H-bonds in RNA mol Can participate in certain chemical rxns Diff chemical reactivity than DNA

24. Alkaline Hydrolysis of RNA 2’ OH acts as a catalyst Demonstrates the diff in chem Reactivity of DNA vrs RNA Due to 2’ OH Unstable incubation with NaOH Intramolecular transesterification

25. RNase A Uses three fundamental catalytic mechanisms: Proximity effect: position phosphodiester between 2 His residues Acid-base catalysis: (His-119 and His-12) Transition state stabilized (by Lys-41)

28. Restriction Endonucleases Enzymes that recognize specific DNA sequences and cut both strands Bacteria can restrict the invasion of foreign (bacteriophage) DNA protect their own DNA by covalent modification of bases at the restriction site (e.g. methylation) Substrate for EcoR1 Palindromic seq Bacteria contain restriction enzymes and methylases

29. Bacteria can restrict the invasion of foreign (bacteriophage) DNA Restriction Endonucleases

30. Enzymes that recognize specific DNA sequences and cut both strands Bacteria can restrict the invasion of foreign (bacteriophage) DNA protect their own DNA by covalent modification of bases at the restriction site (e.g. methylation) Substrate for EcoR1 Palindromic seq Bacteria contain restriction enzymes and methylases

31. Methylation and restriction at the EcoR1 site Palindromic seq Unmethylated Substrate for EcoR1 Foreign DNA

32. Restriction Endonucleases Type I - catalyze both the methylation of host DNA and cleavage of unmethylated DNA at a specific recognition sequence Type II - cleave double-stranded DNA only, at or near an unmethylated recognition sequence More than 200 type I and type II enzymes are knownMore than 200 type I and type II enzymes are known Most recognize “palindromic sequences” (read the same in either direction)Most recognize “palindromic sequences” (read the same in either direction)

33. GGGCCC CCCGGG GGGCC C CCGGG

34. EcoR1 restriction enzyme Dimerizes and binds one face Makes base contacts in major groove