Bioc 302 Nucleic Acid Structure And Stability

Front 1

polymerization of nucleotides

Back 1

to make a dinucleotide, the phosphate is esterified to the 3'OH of another nucleotide
= phosphodiester linkage

directionality: 5' --> 3' (left to right)
can write as: 5'-ATG-3'
or: p-ATG-OH

Front 2

how does DNA half-life compare to RNA?

Back 2

RNA t1/2 = days
DNA t1/2 - thousands of years
-difference is OH in 2' position (self-destruct mechanism)
-OH is close to phosphorous atom and can cleave off (unstable)
=Alkaline Hydrolysis of RNA

Front 3

DNA structure

Back 3

-double helix
-2 polynucleotides associated by H-bonding between the bases
-2 polynucleotide ("DNA strands") are complementary
-the sequence of one determines the sequence of the other (and vice versa)
-the 2 strands are anti-parallel (they run in opposite directions)

Front 4

why does G-C and A-T?

Back 4

-if mix G&C together, exothermic rxn (spontaneous)
-in G-C complex, 3 complementary H-bonds that can form: donors & acceptors = molecular recognition (perfect fit in terms of distances and angles)
-the same for A-T (2 H-bonds)

Front 5

Forces that determine DNA structure

Back 5

1) Electrostatic (repulsion)
-work against Watson-Crik structure
2) Hydrophobic "forces"
-limit exposure or nitrogenous bases from aqueous conditions
-work for structure
3) Hydrogen bonds
-work for structure
4) Stacking interactions
-aromatic structures (nitrogenous bases) stack on top of each other
-work for structure

Front 6

How might urea affect DNA stability?

Back 6

-lots of H-bond capabilities
-can compete w H-bonds and interfere w DNA
-destabilize DNA

Front 7

How might DNSO affect DNA stability?

Back 7

-mostly non-polar (methyl groups) w polar part
-messes up hydrophobic forces
-interfere w hydrophobic effect of DNA
-destabilizes DNA

Front 8

why is binding between complementary PNA/DNA stronger than DNA/DNA?

Back 8

-backbone has no negative charge
-do not have strand-strand repulsion w DNA (stabilizes double strand PNA-DNA)
-in DNA-DNA, both are negative so get repulsion

Front 9

what is the effect of changing ionic strength on DNA stability?

Back 9

-as increase [salt] it shields neg. charge on phosphate, increasing stability
-too high [salt] -> get salting out (no more water which is needed for hydrophobic effect of DNA) -> decreasing stability

Front 10

B form of DNA

Back 10

10.5 residues per turn of helix
1 turn is 36A in length
~20A diameter
3.5A helix rise per base pair
6 degree base tilt normal to helical axis
C-2' endo sugar pucker
anti glycosyl bond conformation
right handed
has major groove and minor groove

Front 11

what is the conformation of base w.r.t. pentose ring in purines

Back 11

can do syn or anti, but anti more stable

Front 12

what is the conformation of base w.r.t. pentose ring in pyrimidines

Back 12

can only do anti

Front 13

what are the differences between the major and minor grooves?

Back 13

major groove is larger and contains more infor
-H-bond donors/acceptors, methyl groups (Thymine)
-major groove s site of most protein-DNA interactions

Front 14

what is the pattern for G-C pair in major groove?

Back 14

Acceptor, Acceptor, Donor

Front 15

what is the pattern for G-C pair in minor groove?

Back 15

Acceptor, Donor, Acceptor

Front 16

what is the pattern for A-T pair in major groove?

Back 16

Acceptor, Donor, Acceptor, Methyl

Front 17

what is the pattern for A-T pair in minor groove?

Back 17

Acceptor, Donor

Front 18

DNA denaturation

Back 18

-can be denatured by temp and pH
-disruption of H-bond and hydrophobic interactions between the bases
-results in separation of strands

ds (native) <--> ss (denatured)
-viscosity changes (ds more viscous)
-UV abs changes

Front 19

what is melting temp (Tm)

Back 19

the temperature at the midpoint in the denaturation curve

Front 20

influences on Tm

Back 20

1) increase G-C content, increase Tm
2) increase [salt] decrease Tm (salting out)
3) low (<2.3) or high (>11.5) pH, decrease Tm
4) organic compounds:
-increase [urea], decrease Tm
-increase [lysine], increase Tm
-increase [glutamate], increase Tm
(lys (-) and glu (+) have charge that can shield neg. charge on phosphate)

Front 21

DNA renaturation steps

Back 21

1) nucleation
-the 2 strands find a region of complementarity and form a short double helix

2) zippering
-in either direction from the paired region of complementarity, the double helix is elongated

Front 22

annealing temp

Back 22

temp where strands can find each other and get nucleation
= Tm - 25 deg C

Front 23

supercoiling

Back 23

DNA is being used to make RNA
-process of separating strands
ahead = stress is overwound
behind = stress is underwound

onderwound: > 10.5 bases/turn (looser)
overwound: < 10.5 bases/turn (tighter)
relaxed = 10.5

being over/underwound causes supercoiling b/c it wants to be 10.5 which is the most stable form

Front 24

supercoiling in prokaryotes

Back 24

keep DNA in underwound state
-easier to do strand separation needed for replication
-makes DNA more compact

Front 25

2 forms of supercoil:

Back 25

1) plectomeme
-twisted sheet

2) solemoid
-1 loop

Front 26

linking number

Back 26

the number of crosses a single strand makes across the other
-2 rings linked together, Lk = 1
-if ring is not closed, Lk = undefined

in relaxed state: Lk0 = bp/10.5

Front 27

Agarose Gel Electrophoresis

Back 27

-utilizes polysaccharide matrix w large pores
-neg. charge (from phosphates) is proportional to length of DNA
-separation based on size
-if relaxed and supercoiled DNA of same size, would get 2 bands (relaxed will travel shorter distance, smaller will travel farther)
-supercoiling reduces the effective size of the molecule, allowing it to migrate faster
-each band represents a discrete linking number

Front 28

Topoisomerases

Back 28

move DNA to relaxed state

to change Lk:
1) have to make a cut
2) change
3) seal

2 types: topo I and topo II

Front 29

Topoisomerase I mechanism

Back 29

change Lk by 1
does not req ATP
1) active site Tyr attacks a phosphodiester bond in 1 DNA strand, cleaving it & creating a covalent 5'-phosphotyrosyl protein-DNA linkage
2) enzyme changes to open conformation
3) unbroken DNA strand passes through break in first strand
4) enzyme in closed conf; liberated 3'OH attacks the 5'-phosphotyrosyl protein-DNA linkage to religate the cleaved DNA strand

Front 30

Topoisomerase II mechanism

Back 30

changes Lk by 2
requires ATP
1) multisubunit enzyme binds a segment of a DNA molecule
2) a 2nd segment of same DNA molecule is bound at the N gate
3) 2nd DNA is trapped. 1st piece cleaved on both strands to form 2 5'-phosphotyrosyl linkages to the enzyme
4) 2nd DNA segment passed through break
5) broken DNA religated & 2nd piece released through the C gate

Front 31

bacterial gyrase

Back 31

special type of topo II

fxn: underwinds bacterial DNA
(need ATP to take DNA from relaxed to underwound)

Front 32

sulfolobus acidocaldarius grows optimally at 75-80C and pH = 2-3, under strictly aerobic conditions
-DNA typically denatures at those temps, what is the mechanism to protect against denaturation?

Back 32

-high concentration of G and C (75%)
-overwinds the DNA (special gyrase that uses ATP to overwind DNA and stabilizes it at high temp)

Front 33

how is bacterial supercoiling maintained?

Back 33

bacterial supercoiling levels are determined by the balance between the activity of the topoisomerases

Front 34

how is eukaryotic supercoiling maintained?

Back 34

by binding to histones and relaxation of DNA between nucleosomes

Front 35

chromosome structure in prokaryotes

Back 35

E coli chromosome:
-4.5 mbp of DNA in a single circle
-the chromosome is highly compacted
-~500 supercoiled loops
-histone-like proteins (HU proteins)
-nucleoid

Front 36

chromosome structure in eukaryotes

Back 36

-very compact structures made of chromatin (DNA-protein complex)
-the basic unit is a nucleosome
-the nucleosome can be packaged together into higher order structures

Front 37

properties of histones

Back 37

-form octomer (2 of each histone)
-contain high amounts of Lys & Arg (basic aas have positive charge on nitrogen to go w negative charge on phosphate)
-histone is core of nucleosomes w DNA wrapped around

Front 38

Telomeres

Back 38

-then ends of linear chromosomes
-consist of tandem repeats of a short (usually 6-8NT) DNA sequence of T,G in one strand
-form "T loop" w 3' overhang
-prevents degradation on ends of chromosome