DNA Chemisty And Analysis Flashcards
(64 cards)
DNA and RNA
-composed of nucleotides linked in linear chains (polymers of nucleotides)
Nucleotide
- Nitrogenous base- purine or pyrimidine
- Sugar moiety-ribose or deoxyribose
- Phosphate
Nucleotide-phosphate in group
Nucleoside- base of sugar minus phosphate
Rare or minor bases
-methylation, acetylation, and hydroxymethylation
Transfer RNA contains high percentage of minor bases
Ribosomal RNA contains high percentages of methylated bases
Methylation
- methyl group acts as silencer of gene expression
- -5-methylcytosine is the most common modified base in DNA, most often CpG sites are modified
- cytosine loses amino group and becomes Uricil (deamination). Would be damaging to DNA, this is why DNA has Thyamine
DNA contains:
A. Nitrogenous bases: adenine, guanin, cytosine, and thymine.
B. Sugar moiety: 2-deoxyribose
C. Phosphate
RNA contains:
A. Nitrogenous bases: adenine, guanine, cytosine, and uracil
B. Sugar moiety-ribose (hydroxyl group at 2nd position)
C. Phosphate
Linkage of Nucleic acids
- mononucleotides are joined by phosphodiester bridges between the 5’-hydroxyl group of one nucleotide and the 3’-hydroxyl group of the adjacent nucleotide.
- bases are not directly attached covalently to one another
Nuclei acid sequences read:
-left to right in the 5’ to 3’ direction
Acid hydrolysis
- products of strong acid hydrolysis of DNA or RNA are purine bases, pyrimidine nucleosides, deoxyribose or ribose and phosphate.
- Purine nucleoside are acid labeled while pyramiding nucleosides are stable in acid (will lose purines)
Base hydrolysis
- RNA proceeds through a 2’, 3’-cyclic phosphate intermediate.
- DNA doesn’t contain a 2’-hydroxyl group, it is resistant to base hydrolysis (important method for separating DNA from RNA)
- -DNA is stable in base and RNA is not stable.
- product of base hydrolysis of RNA are 2’ and 3’ nucleoside monophosphates.
DNA Double Helix
- prokaryote and eukaryotic DNA exists as a double helix
- right handed helix (alpha-helix)
- phosphodiester bonds and the deoxyribose moieties form the backbone of each strand and nitrogenous bases project into the interior
- nitrogenous bases:planes of bases are parallel to each other and perpendicular to the long axis of the double helix. van der Waals forces greatly stabilize the helix
Polar groups
- phosphates project to the outside where they interact with proteins (ones carrying + charge at physiological pH)
- negatively charged phosphates (at physiological pH) are complexed with cations such as Na, K, and CA in vivo-leads to stabilization
Nonpolar groups
-interior of the molecule is very hydrophobic-water is excluded
Base pairs
-hydrogen bonds between base pairs hold strands together
A-T and G-C
-number of A=T
-number of G=C
Frequency of base pairs
A=T does not necessarily equal G=-C
-GC has three hydrogen bonds compared to AT which has two, so GC is stronger
Antiparallel
-one strand runs in the 5’ to 3’ direction while the other strand runs in the 3’ to 5’ direction
X-ray crystallography of DNA and distances
Distance b/t stacked nitrogenous bases (0.34 nm) and the distance for one complete turn of the double helix (3.4 nm)
- 10 base pairs for each complete turn of the double helix.
- B form is most abundant
- 10A= 1nm
Grooves
- grooves in the DNA (particularly the major groove) provide surfaces to which regulatory proteins can bind.
- major grooves regulate genes (turn on and off)
- major groove is almost twice as wide as the minor groove (22A versus 12A)
Reasons for DNA stability
- hydrophobic and electronic interaction b/t the stacked bases of each strand.
- hydrogen bonds b/t strands
- hydrophobic nitrogenous bases are shielded from the aqueous environment
- negatively charged phosphate groups and polar sugar moieties are exposed to the aqueous environment.
Melting
- heating DNA disrupts hydrogen bonds and the strands separate.
- AT melt at lower temp than GC
Hypochromic effect
- melting of DNA increases ultraviolet light absorbance
- stacked bases in double stranded DNA decreases the UV absorption relative to free nucleotides in solution.
Annealing
- opposite of melting
- hydrogen bond formation allows double strand DNA to form two complementary single strands
- slow step
Conformations of double helical DNA
–changes in the shape of DNA may function in regulation of gene expression, since conformational changes in DNA can affect the binding of proteins to DNA, and protein binding is essential to regulation.
B-DNA
- most common conformation
- discovered by Watson and Crick
- right turn