Chapter 19 (part 2)

1. Chapter 19 (part 2) Nucleic Acids

2. DNA • 1o Structure - Linear array of nucleotides • 2o Structure – double helix • 3o Structure - Super-coiling, stem-loop formation • 4o Structure – Packaging into chromatin

3. 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

4. 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

6. 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)

7. 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

10. 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'

13. DNA Secondary structure • DNA is double stranded with antiparallel strands • Right hand double helix • Three different helical forms (A, B and Z DNA.

14. 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

15. A-DNA B-DNA Z-DNA

16. Z-DNA • Found in G:C-rich regions of DNA • G goes to syn conformation • C stays anti but whole C nucleoside (base and sugar) flips 180 degrees

17. 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

18. 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 H-bonds of A:T pair.

19. DNA 3o Structure • Super coiling • Cruciform structures

20. 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

21. Each super coil compensates for one + or – turn of the double helix

22. Cruciforms occur in palindromic regions of DNA • Can form intrachain base pairing • Negative supercoiling may promote cruciforms

23. DNA and Nanotechnology

24. DNA and Nanotechnology

25. DNA 4o Structure • In chromosomes, DNA is tightly associated with proteins

26. 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

27. 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

28. 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

29. Solenoid Structure of Chromatin

31. 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

32. 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.

33. RNA can have extensive 2o structure