Biochemistry Lecture 2 Nucleic Acids

Chapter 4, Unnumbered Figure, Page 74

Nucleobases

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

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.

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)

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

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)

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.

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

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.

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.

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

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

The Central Dogma

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

Translation: Protein Synthesis

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

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.

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

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

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

Highly Structured Ribosomal RNA

Chapter 4, Figure 4.31, Denaturation of DNA

Using DNA Structure

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)

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.

Bio-mimicry is a powerful motivation Velcro: inspired by burrs Conformation Switching Probes Marvin J S et al. PNAS 1997;94:4366-4371 Randomize peptides, express, replicate successful

Optical Conformation Switching TF Switch Sensors

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

TF Beacon Actual Performance

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

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.

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

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.

Pyrimidine Dimers from UV http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf

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