1. Structure of Informational Molecules: DNA and RNA
Stryer Short course
Chapter 33

2. Nucleic Acid Structure
Nucleobase
Nucleoside
Nucleotide
Nucleic acid
Chromatin
Chromosome

3. Polymeric Structure Polymer ideal for informational molecule
Ribose and deoxyribose
Numbering system

4. Directionality 5’  3’ directionality by convention
3’  5’ phosphodiester linkage

5. Base Structure
Purines and pyrimidines
Aromatic
Tautomers

6. Nucleosides Ribonucleosides and deoxyribonucleoside
Purine = osine; pyrimidine = idine (watch cytosine)

7. Nucleotides Phosphorylated on 2’, 3’, or 5’ 5’ unless noted
Letter abbreviations
Draw these: dA
ADP
ppAp

8. Nucleotides
pA is normally called _______ or ____________

9. Problem
List 4 ways that ATP differs from 3’-dGMP.

11. Polynucleotides Phosphate diesters polyanion
Abbreviation is pdApdGpdTpdC
Tetranucleotide
Oligonucleotide
Exonucleases and endonucleases

12. Double Helix B-DNA Chargoff’s Rule Antiparallel
Right handed twist ladder

13. Complementary Base Pairs
Mismatching may occur with tautomers

14. Double Helix Structure
Dimensions-10 bp/turn
Major/minor grooves
Sugar phosphate backbone toward solvent
Base pairs stacked, perpendicular
Edges of bases exposed in grooves for recognition

15. Weak Forces Stabilize Double Helix
Stacking interactions (vdW forces)
Hydrophobic effect
Charge-charge
Hydrogen bonding
Little contribution to stability
Large contribution to selectivity

16. Denaturation
Melting point
Melting curve
UV-absorption
cooperative

17. Problem
True or False: Because a G:C base pair is stabilized by three hydrogen bonds, whereas an A:T base pair is stabilized by only two hydrogen bonds, GC rich DNA is harder to melt than AT-rich DNA.

18. A/T Rich and G/C Rich strands
GC rich strands harder to denature due to STACKING (not H-bonds)
Cooperativity due to initial unstacking, which exposes bases to water, which destabilizes H-bonds, which leads to further denaturation

19. Helical Forms B- form is major
A-form is similar to RNA/RNA and hybrid DNA/RNA structures
Z-DNA not understood, but shows flexibility of structure

20. Major/Minor Groove in B-DNA
Many pictures show ladder with backbone at 180o
Actually a distorted ladder with poles closer to each other, on one side

21. Semiconservative Replication
Meselson and Stahl
Density gradient equilibrium centrifugation

22. Explain the Results

23. Bacterial DNA Closed, circular DNA Supercoiling
Topology and topoisomerases

24. Eukaryotic DNA
Highly compacted (by factor of 104) into chromatin (DNA/protein complex)

25. RNA Structure

26. RNA Structure, Stability, and Function
Structural difference of 2’ hydroxyl
H-bonding in RNA structure
Reactions of catalytic RNA (rare)
Hydrolysis
Structure dictates role difference in DNA/RNA

27. Why does DNA not contain U?
DNA damage from UV light, hydrolysis, oxidation
If DNA contained U, it would be unable to recognize a hydrolyzed cytosine
In RNA, damage not as important, and T production is costly

28. Recombinant DNA Techniques
Optional Lecture

29. DNA Sequencing DNA Polymerase: 5’  3’ Sanger method
dideoxynucleotides

32. Pyrosequencing Attach DNA to a solid surface
Run dNTPs over DNA one at a time
If reaction occurs, PPi is produced
Linked to a luciferase
Light detected

33. Polymerase Chain Reaction
PCR
Denature
Anneal primer
Polymerase
Repeat
Taq polymerase
Exponential production

34. Recombinant DNA technology
Allows incorporation of gene(s) into other DNA
Cut with exonucleases, anneal, and ligate
Recombinant DNA serves as a cloning vector
Incorporate into cells
Select cells that have been transformed

35. Catalytic Hydrolysis: Nucleases
Enzymes can catalyze hydrolysis
Very important reactions!
Nucleases
RNase vs DNase
Single/double strand
Exonuclease vs Endonuclease
Orientation of hydrolysis

36. Endonuclease

37. Restriction Enzyme Endonucleases recognize palindromes
Sticky ends and blunt ends

38. Problem
Restriction enzymes are used to construct restriction maps of DNA. These are diagrams of specific DNA molecules that show the sites where the restriction enzymes cleave the DNA. To construct a restriction map, purified samples of DNA are treated with restriction enzymes, either alone or in combination, and then the reaction products are separated by agarose gel electrophoresis. Use the results of this gel to construct a restriction map for this sample of DNA.

39. Making a Cloning Vector

40. Making a Cloning Vector
ampR is gene for ampicillin resistance
LacZ encodes galactosidase

41. Selecting Transformed Bacteria
Some plasmids are recombinant, and some are not
Some cells accept a plasmid, some accept recombinant plasmid, and some don’t accept any
Transformed cells selected by growing on a petri dish with ampicilin and galactose derivative
Explain

42. Site-directed Mutagenesis
Point mutations
Examine importance of a residue
Modify protein function