Unit 8.2 Flashcards
What were Chargaff’s rules?
- base composition of DNA varies between species
- different tissues of the same species have the same base composition, regardless of age, nutritional state, or environment.
- %A = %T, %G=%C, %A + %G= %T + %C
Describe B-form DNA structure
- Double right-handed (clockwise turn) helix
- sugar-phosphate backbone points outward, bases on the interior
- 20 A diameter
- anti-parallel polynucleotide chains (5’ > 3’
3’ > 5’ ) - bases are nearly perpendicular to the axis
- approx. 10 bp/turn, 3.4 A base separation
- has major and minor grooves. proteins and other molecules to bind at the major grooves
What interactions stabilize the double helix?
- Hydrogen bonds
- Van der waals interactions (base pairing)
- hydrophobic interactions
- only if you have complementary bases. No complementary bases- no H-bonds= no double strand
How many H-bonds are formed between A&T, G&C?
- A&T forms 2 hydrogen bonds
- G&C forms 3 hydrogen bonds
these pairs bond at the most stable angle for stacking
Why are hydrogen bonds not enough to stabilize the DNA helix?
- Hydrogen bonds also competes in water
major groove side vs. minor groove side
- proteins bind major groove side b/c it gives more information about what bases are present and what order compared to the minor groove side.
- do not have to denature the protein to tell where the bases are
List the characteristics of A-form DNA
- has C-3 endo deoxyribose shape
- angles of the bases are tilted due to the C-3 endo shape.
- more compressed than B-form
- antiparallel chains
- This form is seen when DNA is dehydrated/ crystalline, RNA-DNA hybrids, and RNA-RNA double strands
List the characteristics of Z-form DNA
- uses C-2 endo shape for pyrimidines
- uses C-3 endo shape for purines
- a left-handed helix
- zigzag backbone due to the alternating C-2 and C-3 endo
- seen in vitro induced by high-salt concentrations. (high salt concentrations reduce repulsions b/w phosphate groups)
- promoted by underwinding of DNA
- anti for pyrimidines and syn for purines
List the alternate DNA structures and what they are supported by
Watson and Crick
1. Hairpins (1 side)
2. Cruciforms (both sides)
Hogsteen pairing
1. H-DNA (triplexes) has waston-crick pairing too
2. quadruplexes
3. tetraplexes
Most dominant form of DNA?
B-form
Compare DNA double helix to Protein alpha helix
DNA Double helix:
- the hydrogen bonds in the double helix are perpendicular to the helix axis.
-the double helix is flexible and bendable
Alpha helix
- hydrogen bonds in the alpha helix are parallel to the helix axis
- alpha helix is stiff and less bendable
Convert 1 kilobase pair to base pairs
1 Kbp = 1000 bp
What is a codon?
a sequence of 3 nucleotides in a DNA (or mRNA) that specifies a particular amino acid or termination of protein synthesis
What is complementarity?
- each strand serves as a template for a new ‘daughter’ strand with a complementary sequence
what are the Biological consequences of DNA structure?
- the base sequence provides a code for the storage and transmission of genetic information
- the complementarity allows for the maintenance and transmission of information
What is DNA tertiary structure?
- supercoiling
- the double helix coils around the axis
- this happens when there’s torsional stress
What are the reasons behind torsional stress in supercoiling of DNA helix
- local unwinding
- usually during replication
Where is DNA supercoiling seen?
- in circular (or covalently closed) DNA
- in linear DNA molecules with fixed ends
True or false. Most DNA in the cell is supercoiled?
True
- supercoil compacts the molecule
- it facilitates strand separation
- alters access of proteins
What manages supercoiled DNA? and How do they do this?
Topoisomerases
- These are enzymes that convert one topoisomer to another
- these enzymes release or introduce torsional stress by cleaving and resealing the DNA strand
- can happen with single-strand or double-strand cuts
What are the DNA supercoiling parameters?
- Lk: linking number
- Tw: twisting number
- Wr: Writhing number
define the linking number, twisting number, and writhing number.
Lk: the number of times a stand, when laying flat touches a surface.
- in relaxed DNA Lk= #bp / (#bp/turn)
- positive for right handed helix
Tw: # of twists or turns of the helix
Wr: the number of supercoils
What happens when you unwind DNA helix causing torsional stress?
- you get a negative superhelix
- it’s the same number of bp, same sequence, but different degree of supercoiling
relaxed DNA vs Highly coiled DNA on agarose gel
- supercoils are highly compact and so DNA must be linearized to see how long they actual are.
- linearizing the DNA decreases the linking number
What is a nick?
- A cut in DNA
Type I topoisomerases vs. type II topoisomerases
- type I
- makes single-strand breaks to relax DNA/release supercoils.
- involves reversible transesterification reactions
- type II
- makes double-strand breaks to release supercoils.
- requires ATP for conformational change. not to cleave DNA
- some introduce negative supercoils (e.g. bacterial gyrase)
What is the clinical correlation for bacterial topoisomerases?
- bacterial topoisomerases are targets for antibiotics.
- bacteria cannot handle the torsional stress it cannot replicate
What does Novobiocin do? What do Nalidixic acid and Ciprofloxacin do? Why is this important in cancer treatment?
- novobiocin prohibits the taking of the second DNA strand
- Nalidixic acid and Ciprofloxacin prevent the religation of the double strand. this causes a build-up in ds breaks in the bacteria leading to cell death.
- important in cancer treatment because it leads to cell death during S-phase where replication occurs rapid for cancer cells.
Describe the characteristics of RNA
- contains Uracil, not thymine during de-novo synthesis
- contains ribose, not deoxyribose
- has lower chemical stability compared to DNA b/c of the 2’ carbon hydroxyl group that can react with a phosphate group
- secondary structure is a single strand
- local double-strand formation through stretches of base pairing
- many different conformations possible
- stability of the double helical regions are similar to DNA
Describe RNA secondary structure
- right-handed helix with base stacking in ssRNA
- partial double-stranded secondary and tertiary conformations
- secondary structures are antiparallel with conformation similar to the A-form of DNA
what are common RNA secondary structures?
- Hairpins
- stem-loop
What are common RNA tertiary structures?
- pseudoknot (stem-loop)
Can you calculate base percentages of RNA? why or why not?
You cannot calculate the base percentages of RNA because it is single stranded. It has to be calculated through chemical analysis
What is denaturation?
the partial loss of secondary/tertiary structure
What is renaturation?
reforming the secondary/teritary structure
Hybridization vs. annealing
Hybridization is specific terminology for nucleic acids, it also means renaturation
annealing is the terminology used for binding a longer nucleic acid to a shorter one. but also means renaturation
List the methods for denaturation/ disruption of noncovalent bonds
- heat (most common)
- extreme ph
- certain chemicals in vitro
- helicase (in vivo)
ssDNA vs. dsDNA in spectroscopy
- ssDNA absorbs more UV light than dsDNA because more base stacking in dsDNA
What is hyperchromicity?
increase of UV light absorption in ssDNA or dsDNA
What can be concluded from 50% denaturation?
the melting temperature (Tm)
What determines melting temp?
Base composition:
- the # of non-covalent bonds present
- the more non-covalent bonds that have to be broken the higher the melting Tm
- the higher the G-C content the higher the melting temp
- can use the melting temp to figure out the G-C content
Length of template:
- more base pairs= more interactions
- 4C(#G+C) +2C(#A+T)
ion concentration
- ions reduce repulsive forces between phosphates, thus low ion concentration lowers Tm
pH extremes:
- protonation of bases at very low ph
- deprotonation of bases at very high pH
What is the relationship between G-C content and melting temp (Tm)
- linear relationship
- directly proportional
What are the means for denaturing nucleic acids?
- increase temperature
- H-bond disrupting agents (mostly used for electrophoresis)
urea + formamide - Extreme pH at low temp
- Low ionic concentration
methods for renaturing/hybridizing nucleic acids
- Decreasing temperature
- Neutralizing pH
- adjusting ion concentration
Explain the significance of complementarity in reforming h-bonds
- it does not matter what the source of the bases are (DNA, RNA, eukaryote, prokaryote, etc) as long as the bases are complementary they will reform hydrogen bonds
