1. Biochemistry
Lecture 2
Nucleic Acids

2. Chapter 4, Unnumbered Figure, Page 74

3. Nucleobases

4. Polynucleotides
Chapter 4, Figure 4.2, Chemical structure of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)

5. Hydrolysis of RNA
FIGURE 8-7 Phosphodiester linkages in the covalent backbone of DNA and RNA. The phosphodiester bonds (one of which is shaded in the DNA) link successive nucleotide units. The backbone of alternating pentose and phosphate groups in both types of nucleic acid is highly polar. The 5′ end of the macromolecule lacks a nucleotide at the 5′ position, and the 3′ end lacks a nucleotide at the 3′ position.

6. Functions of Nucleotides and Nucleic Acids
Nucleotide Functions:
Energy for metabolism (ATP)
Enzyme cofactors (NAD+)
Signal transduction (cAMP)
Nucleic Acid Functions:
Storage of genetic info (DNA)
Transmission of genetic info (mRNA)
Processing of genetic information (ribozymes)
Protein synthesis (tRNA and rRNA)

7. Discovery of DNA Structure
One of the most important discoveries in biology
Why is this important
"This structure has novel features which are of considerable biological interest“
--- Watson and Crick, Nature, 1953
Good illustration of science in action:
Missteps in the path to a discovery
Value of knowledge
Value of collaboration
Cost of sharing your data too early

8. Covalent Structure of DNA (1868-1935)
Friedrich Miescher isolates “nuclein” from cell nuclei
Hydrolysis of nuclein:
phosphate
pentose
and a nucleobase
Chemical analysis:
phosphodiester linkages
pentose is ribofuranoside
Structure of DNA:
1929
(Levene and London)
Structure of DNA:
1935
(Levene and Tipson)

9. FIGURE 8-12 X-ray diffraction pattern of DNA
FIGURE 8-12 X-ray diffraction pattern of DNA. The spots forming a cross in the center denote a helical structure. The heavy bands at the left and right arise from the recurring bases.

10. Road to the Double Helix
Franklin and Wilkins:
“Cross” means helix
“Diamonds” mean
that the phosphate-
sugar backbone
is outside
Calculated helical
parameters
Watson and Crick:
Missing layer means
alternating pattern
(major & minor groove)
Hydrogen bonding:
A pairs with T
G pairs with C
Double helix fits the data!
Watson, Crick, and Wilkins shared
1962 Nobel Prize
Franklin died in 1958

13. FIGURE 8-13 Watson-Crick model for the structure of DNA
FIGURE 8-13 Watson-Crick model for the structure of DNA. The original model proposed by Watson and Crick had 10 base pairs, or 34 Å (3.4 nm), per turn of the helix; subsequent measurements revealed 10.5 base pairs, or 36 Å (3.6 nm), per turn. (a) Schematic representation, showing dimensions of the helix. (b) Stick representation showing the backbone and stacking of the bases. (c) Space-filling model.

14. Hydrogen Bonding!
FIGURE 8-11 Hydrogen-bonding patterns in the base pairs defined by Watson and Crick. Here as elsewhere, hydrogen bonds are represented by three blue lines.

15. Chapter 4, Figure 4.11c, Fundamental elements of structure in the DNA double helix

16. Base Stacking Stabilizes DNA
Hydrophobic interactions drive the bases to the inside of the helix
Bases stack on top of each other and are attracted by van der Waals forces
Over the entire molecule, the individually small van der Waals forces add up and stabilize the DNA structure

17. The Central Dogma

18. DNA Replication
FIGURE 8-15 Replication of DNA as suggested by Watson and Crick. The preexisting or "parent" strands become separated, and each is the template for biosynthesis of a complementary "daughter" strand (in pink).
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible
copying mechanism for the genetic material”
Watson and Crick, in their Nature paper,1953

19. Translation: Protein Synthesis

20. Chapter 4, Figure 4.23, Conformations of single-stranded nucleic acids

21. RNA Has 2° Structure
FIGURE 8-22 Typical right-handed stacking pattern of single-stranded RNA. The bases are shown in gray, the phosphate atoms in yellow, and the riboses and phosphate oxygens in green. Green is used to represent RNA strands in succeeding chapters, just as blue is used for DNA.
FIGURE 8-19a Hairpins and cruciforms. Palindromic DNA (or RNA) sequences can form alternative structures with intrastrand base pairing. (a) When only a single DNA (or RNA) strand is involved, the structure is called a hairpin.

22. Chapter 4, Figure 4.24, How self-complementarity dictates the tertiary structure of a tRNA molecule

23. Chapter 4, Figure 4.25, The tertiary structure of a transfer RNA as determined by X-ray diffraction

24. FIGURE 8-25 Three-dimensional structure in RNA
FIGURE 8-25 Three-dimensional structure in RNA. (a) Three-dimensional structure of phenylalanine tRNA of yeast (PDB ID 1TRA). Some unusual base-pairing patterns found in this tRNA are shown. Note also the involvement of the oxygen of a ribose phosphodiester bond in one hydrogen-bonding arrangement, and a ribose 2′-hydroxyl group in another (both in red). (b) A hammerhead ribozyme (so named because the secondary structure at the active site looks like the head of a hammer), derived from certain plant viruses (derived from PDB ID 1MME). Ribozymes, or RNA enzymes, catalyze a variety of reactions, primarily in RNA metabolism and protein synthesis. The complex three-dimensional structures of these RNAs reflect the complexity inherent in catalysis, as described for protein enzymes in Chapter 6. (c) A segment of mRNA known as an intron, from the ciliated protozoan Tetrahymena thermophila (derived from PDB ID 1GRZ). This intron (a ribozyme) catalyzes its own excision from between exons in an mRNA strand (discussed in Chapter 26

25. Highly Structured Ribosomal RNA

26. Chapter 4, Figure 4.31, Denaturation of DNA

27. Using DNA Structure

28. Why detect Transcription Factor targets?
Transcription factors are medically relevant
~10% of human genes
Crucial roles in development and cell life cycle
Misregulation and mutation cause disease
Critically, most cancers involve TF overactivity
Darnell, Nature Reviews Cancer 2, 740 (2002)

29. Traditional methods for Transcription Factor detection
Expression Microarrays
Western Blots
Gel Shift Assays
The challenge: Most of these methods are indirect, slow (hours), or can’t differentiate active and inactive protein.

30. Bio-mimicry is a powerful motivation
Velcro: inspired by burrs
Conformation Switching Probes
Marvin J S et al. PNAS 1997;94:
Randomize peptides, express, replicate successful

31. Optical Conformation Switching TF Switch Sensors

32. Rationally Tuning TF Sensors
KS = 10
[ ]
[ ]
[ ]
From different lecture from Biochemistry papers, we found that the most popular model to describe the structure-switching sensors is the population-shift model.
Give an example (like with my hand and a tennis ball): switch typically exist into two states
Although many results gathered by the Scientific community suggest the validity of this model, no studies had ever try to to test this model experimentally. That’s what we decided to do…
KS =
KD =
KS = 1
[ ]
[ ]
% switches open
KS = 0.01
KS = 0.001
KS = 0.1
KS [target]
KD (1+ KS) + KS [target]
% switches open =
Target [M] 32

33. TF Beacon Actual Performance

34. Quantitative Detection in 4 easy steps
HeLa nuclear extract has substantial optical background
Addition of exogenous TBP gives well-behaved signal
But surprisingly, apparent sensitivity is increased
Addition of a DNA that sequesters TBP reduces initial signal
Endogenous TBP is present, and directly detected by sensor
Detects 5.7 ± 1.6 nM TBP in 250μg/ml extract

35. Molecular Mechanisms of Spontaneous Mutagenesis
Deamination
Very slow reactions
Large number of residues
The net effect is significant: 100 C  U
events /day in a mammalian cell
Depurination
N-glycosidic bond is hydrolyzed
Significant for purines: 10,000 purines
lost/day in a mammalian cell
Cells have mechanisms to correct most of these modifications.

36. FIGURE 8-30a Some well-characterized nonenzymatic reactions of nucleotides. (a) Deamination reactions. Only the base is shown.

37. UV Absorption of Nucleobases
FIGURE 8-10 Absorption spectra of the common nucleotides. The spectra are shown as the variation in molar extinction coefficient with wavelength. The molar extinction coefficients at 260 nm and pH 7.0 (ε260) are listed in the table. The spectra of corresponding ribonucleotides and deoxyribonucleotides, as well as the nucleosides, are essentially identical. For mixtures of nucleotides, a wavelength of 260 nm (dashed vertical line) is used for absorption measurements.

38. Pyrimidine Dimers from UV

39. FIGURE 8-31b Formation of pyrimidine dimers induced by UV light
FIGURE 8-31b Formation of pyrimidine dimers induced by UV light. (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA