Central Dogma
- DNA is the genetic material of all free living organisms
- the part of a DNA molecule that encodes the information required for producing a functional product is called a gene
examples of DNA and RNA structure
primary structure - the nucleotide sequence
secondary structure - any regular, stable structure adopted by a segment of DNA, typically a base-paired double helix
tertiary structure - the 3D fold, the complex folding of DNA into bacterial nucleotides (supercoiled) or eukaryotic chromatin, or RNA into tRNA and other molecules
quaternary structure - ribosomes, spliceosomes, RISC
DNA primary structure
- the nucleotide building block
- comprised of Phosphate + pentose sugar + base (purine or pyrimidine)
- nucleotide sequence
- phosphate is always on 5’
- since it only has a few residues (nucleotides) it is an oligonucleotide
Explain the characteristics of the pentose sugar in ribose RNA and deoxyribose in DNA
- has a 5’ and 3’ end
- in RNA the 2’ carbon has a hydroxyl group (2’-OH) - important in RNA splicing reactions
- in DNA the 2’ carbon has a hydrogen
- the pentose in each nucleotide is attached to the base via its 1’ carbon
- the phosphate is attached to the 5’ carbon of the pentose
- the 3’OH of one nucleotide is linked to the 5’PO4 of an adjacent nucleotide to form the repeating sugar-phosphate backbone of DNA and RNA
How can you distinguish between purines and pyrimidines
- purines are longer
- pyrimidines are smaller
- bases are highly conjugated aromatic ring structures
- called bases because some of their ring nitrogen’s can be protonated
What type of bond does the pentose form with the base
- covalent bond via a beta-glycosidic linkage
Deoxyribonucleotides
purine - adenine (base) - adenosine (with sugar)
- guanine (base) - guanosine (with sugar)
pyrimidine - cytosine (base) - cytidine (with sugar)
- thymine (base) - thymidine (with sugar)
- base + sugar = nucleoside
- base + sugar + phosphate = nucleotide
Ribonucleotides
- differ from the deoxyribonucleotides
- have a hydroxyl at the ribose 2’ carbon (are not deoxy)
- have uracil base instead of thymine (uracil is like thymine but without the methyl group)
- uridine = uracyl + ribose (nucleoside)
Characteristics of DNA and RNA primary structures
- phosphate groups link the pentoses in both DNA and RNA strands via a phosphodiester linkage/bond
- DNA/RNA strands are said to be asymmetric or polar, with a free 5’-phosphate at one end (5’ end) and a 3’ hydroxyl at the other end (3’ end)
- the phosphate group is an acid
- at physiological pH the phosphate group of every nucleotide within the DNA/RNA strand is deprotonated and carries a net negative charge
- because of these acidic phosphate groups (and despite having nitrogenous bases) DNA and RNA polymers are called nucleic acids
How do bases interact and where
- interact by H-bond
- interact in the interior/core of double helix
- two anti-parallel strand can form base pairs
DNA secondary structure - the double helix
- two DNA strands interact via hydrogen bonds between the bases: base-pairing (bp)
- together the strands form a twisted ladder, with a sugar-phosphate backbone forming the side of the ladder and the base pairs forming the rungs
- the strands run in opposite directions, one 3’ to 5’ and the other 5’ to 3’ antiparallel
DNA base-pairing
- H-bond between the bases in the two DNA strands
- pairing is specific based on H-bond complementarity: guanine forms 3 H-bonds with cytosine, adenine forms 2 H-bonds with thymine
- high GC% is more stable 3 vs 2 H-bonds, more stable base interactions = more difficult to separate N strands
- these 2 types of base pairs predominate in double stranded DNA and RNA because they are the most complementary
- base pairing specificity - watson-crick base pairing (based on x-ray diffraction data)
- specific base pairing permits the duplication of genetic information because each strand is a template for its complementary strand
G-C vs A-T
-GC interaction is stronger
- GC = 10.8
- AT = 11.1
How are double helices held together
- base stacking
- base stacking between the hydrophobic bases minimizes their contact with water and stabilizes the double helix
- base stacking is a form of van der waals forces
- bases are slightly offset so they not directly on top of one another
- the bases lie in a plane almost perpendicular to the axis of the helix
Major and minor groove
- the hydrophobic deoxyribose-phosphate backbones are exposed to the surrounding water and the hydrophobic bases are stacked on the inside of the helix nearly perpendicular to the backbone
- the offset pairing of the two strands forms a major groove (deep) and a minor groove (shallow) on the surface of the duplex
- base pairs more exposed to solvent on the major groove side than on the minor groove side
Different forms of double helix
A form - dsRNA, DNA/RNA hybrid (right-handed) - bases more filled (more compact)
B form - dsDNA (right-handed), most common/dominant in all living forms
Z form - dsDNA, alternating pur/pyrim (left-handed)
- the structure of DNA proposed by W and C represents the sodium salt of DNA in a fiber produced at very high relative humidity
- if the relative humidity surrounding the DNA fiber is reduced to 75%, the sodium salt of DNA assumes the A form
Nucleotide bases absorb UV light
- at 260 nm
- purines and pyrimidines are highly conjugated
- resonance among rings give most of the bonds a partial double-bond character, allows UV absorption
- nucleic acids are characterized by strong absorbance at a wavelength near 260nm
- aromatic aa absorb at 280 nm
How can you distinguish between single-stranded and double-stranded DNA
- can use A260
- dsDNA helix can be disrupted by heating
- ssDNA can be disrupted by melting, base pairing and base stacking are disrupted (adding energy to disrupt weak bonds)
- melting occurs at a specific temp, Tm, which depends on the nucleotide sequence
- when the DNA is cooled, the strands reanneal (come back together) due to base complementarity (ssDNA -> dsDNA)
- stacked bases in nucleic acids (in a double helix) absorb less UV light than unstacked bases - absorbance is “quenched” when strands come together
- melting and annealing can be followed by A260
Hyperchromic shift
- DNA melting (denaturation) and re-annealing (renaturation) can be followed by studying the hyperchromic shift
- dsDNA is held together by hydrogen bonds and base stacking interactions
- DNA strands can be melted apart by raising the temp or adding chaotropic (denaturing) agents like urea, and by removing salt
- both the formation and disruption of the DNA double helix are highly cooperative - the strands hold fast until the melting point Tm, and then rapidly let go
What is the relationship between salt concentration and melting temp
- the stacking energy is more negative (more stable) for GC pairs, so Tm is higher
- the AT-rich regions melt first
- Tm is proportional to [salt] and sequence length
- high salt concentrations and longer sequences stabilize the duplex and increase the Tm
- salt ions shield the negatively-charged phosphates on the DNA backbones, which repel each other when unshielded, thereby stabilizing the ds structure
- [salt] increases, Tm increases
