2 - Nucleotides Nucleic Acids Flashcards
1
Q
What does the term amphipathic describe?
A.Something similar to frogs and other
amphibians.
B.Something shaped like a half-circle.
C.Something that has both polar and non-polar properties.
D.Something that “hates” water i.e. is poorly soluble in water
A
C.Something that has both polar and non-polar properties.
2
Q
Amphipathic
A
Has both polar and non polar properties
3
Q
Nucleic acids are made form monomers called
A
Nucleotides
4
Q
What are nucleic acids
A
DNA and RNA
5
Q
Which of the following is NOT a component of a nucleotide? A. A purine or pyrimidine base B. A carboxyl group C. A pentose sugar D. A phosphate group
A
B. A carboxyl group
6
Q
Nucleotide structure
A
- Phosphate group
- sugar / ribose
- base (either purine or pyramidine)
7
Q
Nucleoside structure
A
- sugar
- base
8
Q
A nucleoside consists of a nitrogenous jbase attached to a sugar(ribose or deoxyribose) with the help of a
A
covalent bond.
9
Q
Difference between purine and pyramidine
A
- Pyramidine has 6 membered ring
- purine has 6 membered + 5 membered ring
10
Q
Which N does ribose attach to on purine and pyramidine
A
N9 for purine
N1 for pyramidine
11
Q
The ring for bases is
A
Rigid and small with very little bond rotational.
- Very planar structures.
12
Q
The sides of the planar rings on bases are very
A
Hydrophobic because of electron delocalization
13
Q
Bases can undergo structural rearrangements known as
A
tautomerization
14
Q
Tautomerization is essentially a rearrangement of the
A
atoms position, not like resonance because atoms are moved. Not electrons.
15
Q
Can bases absorb UV light?
A
Yes, at 260nm
16
Q
How do bases absorb UV light
A
Because of electron delocalization
17
Q
Can nucleotides and nucleic acids absorb light?
A
Yes
18
Q
Getting a wavelength ratio of
A260/A280= 1.9 means that
A
The sample is pure DNA/RNA or pure bases.
19
Q
A260nm can be used to measure the concentration of
A
nucleic acids in solution.
20
Q
Absorbance of UV light and concetration of nucleic acids are
A
Directly proportional
21
Q
If the A260:A280 ratio in your sample is 1.4, what does that mean?
A
Your sample contains proteins
22
Q
If absorbance ratio decreases that means
A
Samples contaminated with proteins
23
Q
The bases are ….. in water at pH 7
A
hydrophobic and relatively insoluble
24
Q
Can bases form H-bonds?
A
Yes because of the polar side groups.
25
How many H-bonds can bases form? Dependant on what?
Depends on:
- number of side groups attached
- number of atoms on base that H bond
- number of H bonds possible (theoretical max)
26
The base attached to the sugar through
C1
27
What kind of bond is used to bond base to the sugar?
N-B-glycerol bond
Attaches to N1 in pyramidine
Attaches to N9 in purines
28
The phosphate group is attached to the sugar at
C5’ via a phosphodiester bond
29
Phosphate groups only attach to sugars in
Nucleotides
30
Phosphoanhydride bonds
P-O-P bonds that hold the phosphate together within themselves.
31
Differences between phosphodiester bond and phosphoanhydride bond?
Phosphoanhydride bonds is within the phosphate group, whether it be mono, do, tri phosphate. [P-O-P]
Phosphodiester bond connected the phosphate group to the sugar / ribose via C5’ [P-O-C]
32
A nucleotide is a nucleoside that has
1,2,or 3 phosphates
33
• Deoxynucleoside =
2’deoxyribose + base + 1,2, or 3 phosphate groups
34
Nucleic acid primary strcure
Sequence of nucleotides joined by phosphodiester bonds
- monomers joined by covalent bonds
35
What kind of bonds are phosphodiester bonds?
Covalent
So are phosphoanhydride bonds
36
Difference between phosphodiester bond and phosphoester bond
phosphodiester:
C-O-P-O-C
phosphoester:
O-P-O-C
37
Phosphodiester bonds are used to link
Sugars of neighbouring Nucleotides together.
38
Phosphodiester bond is in what direction
3’ to 5’. Cuz the one of one joins the start of the other (3’—>5’)
39
Is phosphodiester bonds charged?
Yes -1 charge per residue. So for 1000 residues the nucleic acid will have a net charge of -1000
40
The ends of nucleic acids are
Distinct and not identical
41
If your were to flip the entire phosphate sugar backbone, the geometry would be
Completely different, you cant flip it!
42
The … is what determines the unique structure of 5’.
phosphoester bond that sticks out above the plane at 5’
43
RNA and DNA both have a hydroxyl group at
3’ on the ribose/sugar
44
Do bases form hydrophobic bonds at
| their van der Waal’s radii?
Yes
45
Is the backbone hydrophobic or hydrophilic
The backbone is hydrophilic and forms
| hydrogen bonds with water
46
Does the backbone form H-bonds?
Yes
47
Are bases hydrophobic or hydrophilic ?
Hydrophobic, they form H-bonds because of the side groups
48
The spiral staircase structure of the nucleic acid is subject to the
Hydrophobic effect
49
The primary structure of RNA is less stable than that of DNA. Why?
Because RNA contains ribose and DNA contains deoxyribose.
50
Hydrolysis of phosphodiester bond is done through the breaking of whihc bonds
The double bond between P=O and single bond between P-O
- known as alkaline hydrolysis
51
For RNA, the phosphodiester bond undergoes ______ when being broken down?
Autohydrolysis
52
What catalyzes the hydrolysis of phosphodiester bond?
The 2’ hydroxyl group —> giving 2’,3’-cyclic monophosphate derivative
53
Secondary structure of nucleic acids
Double helix structure of DNA and RNA
54
What are the base compositions of DNA
A=T
G=C
A/T > G/C (eukaryotes)
A/T < G/C (prokaryotes)
55
What is the double helix (secondary structure) of nucleic acids dependant on?
Base pairing
56
What is base pairing held by
H-bonds
57
Normal base paring is called
Watson and crick base pairing
58
How many base pair H-bonds between A and T / G and C
A/T = 2 h bonds
G/C = 3 h bonds (stronger)
59
What kind of bonds are between the polar substituents on the bases within the double helix?
H bonds
60
What are buried in the centre of the helix?
H bonds (between bases)
61
How many base pairs per 360 degree turn?
10 base pairs
62
Major groove on double helix important for
Protein binding
63
Minor groove on double helix is
- deeper and fewer sides of bases exposed
| - contributes to H-bond formation
64
Features of double helix
- double stranded NA
- made of 2 molecules of nucleic acids
- antiparallel
- helical
- complementary
65
What forces stabilize the double helix?
Non covalent (H bonds and van derwall)
66
Folding / fomration of double helix is driven by
Hydrophobic effect
67
Single stranded helical strcure has the same
Secondary strcure as the double helical nucleic acids.
68
Single stranded helical strcure form spontaneous base pairing is driven by the
Hydrophobic effect
69
Secondary structure of single stranded RNA is stabilized by
The same non covalent forces that stabilize DNA
- H bonds
- base stacking (driven by hydrophobic effect)
70
Both DNA / RNA, single or double stranded both have folded secondary structure. Which is stabilized by :
1. H-bonds between bases, which are either inter or intra strand H-bonds, depending on whether is single or double stranded.
2. Base stacking energies (can der walls interactions)
71
Denatuation done via
Heat —-> melting
72
Why is it easy to melt/ denature secondary structure
Because its stabilized by non covalent bonds like h bonds and van der waal interactions
73
Double and single stranded DNA both peak at around
Which is higher?
260nm
Single stranded is higher
74
Single stranded nucleic acid is
Hyperchromic: more absorptive then double stranded nucleic acid
75
Double stranded nucleic acid is
hypochromic: less absorptive compared to single stranded nucleic acid
76
Does ss or ds NA absorb more UV
Ss cuz its hyperchromic
77
The reason why the single stranded DNA / NA is hypochromic opposed to double stranded hyperchromic NA is because the
secondary folded structure of DNA’s electrons are less excitable. Their excitability is “quenched”
78
hyperchromic
When DNA is unfolded, the electrons are more excitable and therfore absorbed more UV light
79
hypochromic
Because of the stable base stacking in the secondary folded double helix, electrons excitability is decreased
80
DNA melting
Double stranded becomes single stranded
81
Melting temp (Tm)
Centre of the curve
- half are still helical
- half are melted
82
Zone of melting
Wider zone = broader the melting curve
Narrow zone = spiked melting curve
83
Zone of melting depends on:
- base sequence
- length (#of base pairs),
longer = broader curve
Shorter = narrower curve
- more varied base sequence = broader curve
84
Renaturation of double stranded DNA (or ds RNA)
After they have been denatured,
1) cool very slowly to allow nucleation (proper alignment) to occur:
- separated complementary strands form helix
- longer the NA, the more slowly you need to cool
2) zippering (fast):
- once aligned, they zip together into helical
85
Uncontrolled cooling leads to partial
Renaturation
86
Cool to quickly
- only partial realignment
- partial nucleation
- results having both ds and ss NA.
- more likely to occur with longer strands to be unable to return to ds.
87
Melting curve and base composition.
Melting curve vary depending on the
Base composition
88
Poly A/T VS.
Natural A/T and G/C. VS
poly G/C
On melting curve
Poly A/T: curve is to the left with lower melting temp of 70C.
Natural A/T and G/C: normal curve at 90C Tm
Poly G/C: higher Tm of 110C
89
G/C have greater
Base stacking forces and H-bonds then A/T. That’s why it has a greater Tm for denaturation.
90
Regions of DNA Rich in AT Base Pairs
Denature Most Readily
91
• For A/T rich sequences, its far more easier to convert
Double stranded —-> single stranded NA.
92
There are regions of the DNA that denature more quickly at lower temperatures.
A/T regions unwind more easily. Important for transcription and replication. Therefore at the initiation sequences they are rich in A/T’s.
93
If the concentration of salt is increased, Tm of a ds DNA will:
Increase
94
Why does an increase in salt increase Tm for ds DNA?
Because the more salt concentration, the more energy required to unwind the helix, causing a greater Tm.
95
More salt, the more
Stable the helix is
96
Increasing salt causes increase in Tm because it
decreases electrostatic repulsion that is caused by the (-) charge of the backbone. Na+ ions go to phosphodiester backbone and overcome the electrostatic repulsion.
97
in a test tube, salt
stabilized NA secondary structure, since all NA in secondary structures have (-) charged phosphodiester backbone.
98
If the pH is increased or decreased notably from pH 7, Tm of a ds DNA will:
Decrease
99
If you change pH, H-bonding groups become protonated (gain an H+), or deprotonated ( lose an H+).
What effect will that have on H-bonds and H-bonding groups?
When you make the H-bonds protonated and deprotonated, you change the structure of H-bonding groups, you change its ability to make the H-bonds. This then reduced H-bonding. Negatively impact the base pairing of a double stranded NA.
The melting temperature Tm decreases if you change the pH in either direction. Because the base pairing is reduced, because you lose or gain H-bonding groups that should or should be there. Prevention prompt base pairing.
100
The melting temperature Tm decreases if you change the pH in
either direction.
101
The denaturation and renaturation behaviour of nucleic acids enables.
hybridization
102
Hybridization:
based on the ability of double stranded NA to nature, denature, and then renature.
103
75% complentary ds VS 100% complentary ds
75% complentary ds
- lower base pairing
- lower Tm
- less stable
100% complentary ds
- higher base pairing
- higher Tm
- more stable
104
Melting is done through
Heat and changing ph
105
In hybridization, you can have two kinds of ds hybrids
1) DNA:DNA or RNA:RNA
| 2) DNA:RNA
106
Hybridization involves a process called
annealing: when the strands fold back together.
107
• Annealing depends on the degree of
complementarity.
• Degree of complementarity effects Tm. (Direct variables)
108
If you have two strands with no complementarity, they
won’t form a stable double helix as there’s no H-bonding stabilizing them. 100% complementarity gives the maximum H-bonding possible.
109
The hybrid annealing formation is effected by:
* The concentration of salt
* pH
* Temperature that you do it at. (Melting temp for that sequence)
110
If you want 50% complentarity, then you need to
Tm will be low, so you need to Lower temp
111
To stabilize the helix hybrid
- lower temp
- moderate ph
- higher salt concetration (favors stability of helix)
112
You have a mixture of DNA fragments and you want only the MOST complementary fragments to hybridize. Which conditions would be best?
A. Higher temperature and lower salt
B. Lower temperature and higher salt
C. Higher temperature and higher salt
D. Lower temperature and lower salt E. High or low pH
A. Higher temperature and lower salt
113
Why does higher temp and lower salt cause for hybridization of the most complentry strands?
Because it destabilizes all the low complementary strands and weeds them out, allowing only the strands with the highest complementarity to hybridize.
114
Can RNA secondary structure melt?
Yes because secondary structure depends on base stacking (van der waal) and on base paring (H bonds).
Both are weak so can melt
