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MBB 222 > Lecture 9 > Flashcards

Lecture 9 Flashcards

1
Q

What is DNA

A

the genetic material of all free living organisms

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2
Q

(some ___ – which are not necessarily “living organisms”, have an RNA genome)

A

Viruses

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3
Q

What is a gene

A

The part of a DNA molecule that encodes the information required for producing a functional product (-> RNA -> protein)

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4
Q

What is the primary structure

A

the nucleotide sequence, e.g. :

5’-GCGGCAATCGTA-3’

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5
Q

What is the secondary structure

A

any regular, stable structure adopted by a segment of DNA, typically a base-paired double helix

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6
Q

What is the tertiary

A
  • the 3D fold - the complex folding of DNA into bacterial nucleoids (supercoiled) or eukaryotic chromatin
  • or RNA into tRNA and other molecules
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7
Q

The basic building block of DNA is a nucleotide, which is comprised of:

A

phosphate + sugar + base

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8
Q

Which way do DNA strands run?

A

antiparallel directionality of DNA strands… 5’ → 3’

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9
Q

What is on the 1’ carbon? 3’? 5’?

A 
1' = Purine or pyrimidine base
3' = OH group
5' = phosphate group
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10
Q

What does a nucleoside consist of?

A

base + sugar

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11
Q

The pentose sugar is ___ in RNA and ___ in DNA

A

Ribose in RNA and deoxyribose in DNA

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12
Q

Name the 2 purine and 3 pyrimidine bases

A

Purine - Adenine and guanine

Pyrimidine - Thymine and cytosine and uracil (RNA)

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13
Q

In RNA the 2’ carbon has a ___ group?

A

In RNA the 2’ carbon has a hydroxyl group (2’-OH)

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14
Q

In DNA the 2’ carbon has a ___ group?

A

a hydrogen

i.e., it has been “deoxygenated”

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15
Q

Is the 2’OH on the RNA or 2’H

A

The H at the 2’ C of DNA is less reactive than the 2’-OH of RNA,

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16
Q

The pentose in each nucleotide is attached to the base via which carbon

A

1’ carbon

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17
Q

The phosphate in each nucleotide is attached to the base via which carbon

A

5’ carbon of the pentose and is thus called the 5’ phosphate (5’-PO4).

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18
Q

The 3’ hydroxyl (3’-OH) of one nucleotide is linked to the ___ of an adjacent nucleotide to form the repeating sugar-phosphate backbone of DNA and RNA.

A

5’-PO4

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19
Q

The phosphate groups link the pentoses in
both DNA and RNA strands via a
____ bond.

A

phosphodiester linkage/bond

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20
Q

Why are DNA/RNA strands said to be asymmetric or polar

A

They have a free 5’-phosphate at one end – the “5’ end” - and a 3’-hydroxyl at the
other end – the “3’ end”.

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21
Q

At physiological pH the acidic phosphate
group of every nucleotide within the
DNA/RNA strand is ___ and thus carries a net ___ charge

A

At physiological pH the acidic phosphate

group of every nucleotide within the DNA/RNA strand is deprotonated and thus carries a net negative charge

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22
Q

The phosphate groups are acidic or basic?

A

Acidic

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23
Q

Because of these acidic phosphate groups
(and despite having nitrogenous bases),
DNA and RNA polymers are called
__ acids

A

Nucleic acids

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24
Q

What are the bases in nucleotides?

A
  • nitrogenous heterocyclic ring structures which include purines and pyrimidines.
  • called “bases” because some of their ring nitrogens can be protonated
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25
Q

The pentose forms a covalent bond with the base via a ____ linkage

A

Beta-glycosidiclinkage

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26
Q

In a b-linkage the base lies ___ the plane of the sugar

How does this differ if it was in alpha-linkage

A
  • above the plane of the sugar

- in alpha, base would lie below the plane, where the H is

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27
Q

What are the Purine deoxyribonucleotides? Give the base name and base + sugar name

A
  • adenine (base) – adenosine (with sugar)

- guanine (base) – guanosine (with sugar)

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28
Q

What are the Pyrimidine deoxyribonucleotides? Give the base name and base + sugar name

A
  • cytosine (base) – cytidine (with sugar)

- thymine (base) – thymidine (with sugar)

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29
Q

Give the nucleotide names for the deoxyribonucleotides

A
  • deoxyadenosine 5’- monophosphate, deoxyguanosine 5’- monophosphate, deoxythymidine 5’- monophosphate, deoxycytidine5’-monophosphate
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30
Q

2 ways ribonucleotides differ from deoxyribonucleotides

A
  • have a hydroxyl at the ribose 2’ carbon

* have uracil base instead of thymine

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31
Q

How do uracil and thymine differ

A

(uracil is like thymine but without the

methyl group)

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32
Q

What is uridine?

A

Uracyl + ribose

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33
Q

What are the nucleotide names for ribonucleotides?

A 
- adenosine
5’- monophosphate
- guanosine
5’- monophosphate
- uridine
5’- monophosphate
- cytidine
5’- monophosphate
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34
Q

The nucleotide sequence is written and read from the ___’ to the ___’ end

A

5’- to the 3’- end

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35
Q

If the nucleotide sequence only has a few AA’s, what is it called?

A

oligonucleotide

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36
Q

two DNA strands interact via ___ bonds between the

___

A

two DNA strands interact via hydrogen bonds between the

bases: base-pairing

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37
Q

together the strands form a twisted ladder, with the sugarphosphate backbone and the base pairs forming which parts of the ladder

A

sugarphosphate backbone forming the sides of the ladder and the base pairs forming the rung

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38
Q

• two strands of DNA are wound around the same

axis to form a ____-handed double helix

A

Right-handed

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39
Q

What direction do the DNA strands run?

A

the strands run in opposite directions, one 3’ to 5’ and the other 5’ to 3’: antiparallel

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40
Q

How do the bases line up in terms of the center of the double helix?

A

the bases are “offset” from the center of the double helix (i.e., if you look end-on, you’ll see that the bases don’t cross through the central axis of the double strand)

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41
Q

___ means hydrogen bonding between the

bases in the two DNA strands

A

base-pairing

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42
Q

• pairing is specific based on H-bond complementarity: guanine forms ___ H-bonds with cytosine, adenine forms ___ H-bonds with thymine (or uracil in RNA, A=U)

A
  • G-C = 3 H bonds

- A-T = 2 H bonds

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43
Q

• Watson and Crick determined the double helix

structure of DNA that is made possible because of

A

the specific base pairing

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44
Q

• the specific pairing of bases (G-C, A-T) permits the duplication of genetic information because each strand is a ___ for its complementary strand

A

template

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45
Q

The G-C interaction, with 3 H-bonds, is ____ than the A-T interaction, with 2 H-bonds.

A

stronger

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46
Q

In addition to H-bonding between base pairs, the double helix is held together by a second force between the bases: ____

A

base stacking

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47
Q

Base stacking between the hydrophobic bases

does what to the double helix?

A

Base stacking between the hydrophobic bases minimizes their contact with water and stabilizes
the double helix.

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48
Q

Base stacking is a form of what force?

A

van der Waals force

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49
Q

Are bases stacked directly on top of one another?

A
  • bases are slightly offset so they are not directly on

top of one another

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50
Q

What direction do bases lie to the axis of the helix

A
  • the bases actually lie in a plane almost

perpendicular to the axis of the helix

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51
Q

How many base pairs per turn in DNA?

A

10 base pairs/turn

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52
Q

The major and minor grooves are equivalent or non-equivalent

A

non-equivalent

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53
Q

Why are the major and minor grooves non-equivalent

A

Because the two glycosidic bonds are not diametrically opposite each other (i.e., are offset relative to the axis of the double helix), each base pair has a larger side that defines the major groove and a smaller side that defines the minor groove.

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54
Q

The grooves are lined by what bonds

A

Hydrogen bonds

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55
Q

that functional groups on the bases are more accessible to proteins in the which groove

A

major groove, rather than minor

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56
Q

• Watson and Crick based their double helix model on ?

A

the X-ray diffraction pattern of DNA fibers in the B form

57
Q

What is B form? Where found? Left or right handed? Most stable when?

A
  • B-helix, B-DNA
  • In cells
  • Right handed
  • Most stable under physiological conditions
58
Q

when does DNA assume A-form?

A

when DNA is dehydrated (relative humidity is reduced to less than 75%) DNA assumes what form

59
Q

A form DNA, like B-DNA, is a ___-handed double helix made up of ___ strands held together by ___ base pairing

A
  • RIght handed
  • Anti-parallel
  • Watson and Crick base-pairing
60
Q

Which is wider and shorter, A-helix or B-helix

A

• the A-helix is wider and shorter than the B-helix

61
Q

Which has deeper major groove and more shallow minor groove, A-helix or B-helix

A

A-helix

62
Q

In A-helix are the base pairs tilted or straight

A

tilted

63
Q

Where are A-helix frequently found in?

A

DNA-RNA hybrids

and in double-stranded RNA (e.g., tRNA)

64
Q

How does Z-form DNA compare to A and B forms?

A

left-handed and “stretched out compared to A and B forms

65
Q

Nucleotide bases absorb UV light at __ nm?

A

260 nm

66
Q

A260 can be used to determine DNA ___ and ___

A

Concentration and structure

67
Q

Since purine and pyrimidines are highly conjugated, what does this allow

A

resonance among rings give most of the bonds a partial double-bond character, allows UV
absorption (just like for aromatic amino acids)

68
Q

A260 changes depending on the ____

A

on the structure of the DNA: single-stranded or double-stranded

69
Q

double-stranded DNA (dsDNA) helix can be

disrupted by ___, resulting in ____ as the product. After this conversion, ___ and ___ are disrupted

A
  • Heating
  • ssDNA
  • Base-paring and base stacking are disrupted
70
Q

What is reanneal? Why does this happen

A

When the DNA is cooled, the strands
reanneal (come back together) due to base
complementarity: ssDNA -> dsDNA

71
Q 
stacked bases in nucleic acids (i.e., in a double helix) absorb \_\_\_\_ UV light than
unstacked bases (i.e. single strands)
A

Less

72
Q

What is hypochromisn

A

absorbance is “quenched” when strands come together

73
Q

double-stranded DNA is held together by ____ bonds and ____ interactions

A
  • Hydrogen bonds

- Base stacking interacting

74
Q

DNA can be melted apart by____ or by adding ____

A

raising the temp or by adding chaotropic (denaturing) agents like urea, and by removing salt

75
Q

What does highly cooperative mean in terms of DNA?

A

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

76
Q

What is Tm

A

melting temp. – point at which strands are ½ denatured

77
Q

DNA melting (denaturation) and reannealing (renaturation) can be followed by studying the ____

A

hyperchromatic shift

78
Q

Melting occurs at a specific Tm depending on what 3 things

A

the nucleotide sequence,

length, and salt concentration

79
Q

Why do AT-rich regions melt first?

A

A=T has only 2 H-bonds so has a weaker interaction than GºC with 3 H-bonds; the more GC pairs, the higher the Tm

80
Q

Why is Tm is also proportional to [salt] and sequence length

A

high salt concs and longer sequences stabilize the duplex and increase the Tm. Salt ions shield the negatively-charged phosphates on the DNA backbones, which can repel
each other when unshielded.

81
Q

So, how do cells contain/package/handle their DNA?

A

Bacterial DNA can be compacted by a process called supercoiling into a nucleoi. DNA is condensed, organized and segregated with the help of topoisomerase enzymes, nucleoid associated proteins and Structural Maintenance of Chromosome (SMC) complex

82
Q

Supercoiling resuts from slighty ___ or ____ the DNA double-helix prior to joining both ends of each strand. Enzymes called ____ perform this task

A
  • Overwinding (DNA can only be supercoiled by breaking the strands,
    twisting them around each other and religating them) or under winding
  • Topoisomerases
83
Q

Supercoiling can only exists in what kinds of molecules

A

Supercoiling can only exist in a molecule where both strands of DNA are closed circles or otherwise fixed at one end

84
Q

What happens when one stand breaks?

A

If one strands breaks the DNA rapidly loses its supercoiling

i.e., it relaxes

85
Q

DNA molecules in different coiled forms are called?

A

Topoisomers

86
Q

DNA supercoiling helps to compact DNA but it is also a consequence
of _____

A

DNA unwinding during replication and transcription (RNA synthesis)

87
Q

Most cellular DNA is overwound or underwound?

A

Underwound, - DNA has fewer helical turns than would be expected for the B form

88
Q

relaxed DNA has ___ base pairs per turn

A

~ 10.5

89
Q

a segment of DNA in a closed circular
molecule is relaxed with ___ bases in
8 helical turns

A

84

90
Q

removal of one turn (unwinding) induces ___

A

structural strain

91
Q

To accommodate the strain from removal of one turn (underwinding), the DNA can either ___ or _____

A
  • become supercoiled

- or break H bonds

92
Q

Why is the strain generally accommodated by formation of supercoil rather than breaking H bonds

A

It disperses the
strain throughout the molecule. This requires less energy than breaking the H bonds that
stabilize paired bases.

93
Q

Cells actively underwind their DNA using ____, and the resulting strained state represents a form of ___ energy

A
  • Topoisomerases

- Stored energy

94
Q

The underwound state can be maintained only if?

A

the DNA is a closed circle or if it is bound and stabilized by proteins so that the strands are not free to rotate about each other

95
Q

This underwound, supercoiled state

allows for what of the DNA?

A

This underwound, supercoiled state

allows compact packaging of the DNA

96
Q

What is Lk

A
  • linking number
  • the number of times one strand crosses
    over the other.
97
Q

Does the Lk change when DNA is bent or deformed?

A

No as long as both DNA strands
remain intact. i.e., it is a topological
property of the DNA

98
Q

How can Lk be changed

A

Lk can only be changed by breaking one or both strands and re-joining (re-ligating) them.

99
Q

Are Lk for a closed-circular DNA always an integer?

A

Yes

100
Q

By convention, if the links between two DNA strands are arranged so that the strands are interwound in a right-handed helix, Lk is + or -. Linking numbers in a left-handed helix are + or -?

A
  • Right-handed helix = pos

- Left handed helix = neg

101
Q

The double helix is a right-handed helix so cellular DNA has a +/- linking number?

A

+

102
Q

The interwinding of the supercoils can be ____ (underwound, right-handed helix) or ____ (overwound, left-handed helix)

A
  • Negative = underwound, right-helix

- Positive = overwound, left helix

103
Q

For relaxed DNA, Lk = ?

A
  • # of base pairs divided bythe # of base pairs per helical turn in relaxed DNA
    (i.e., it is equal to the number of turns).
  • So for a circular
    piece of relaxed DNA with 2100 base pairs,
    Lk = 2100 bp/10.5 bp per turn = 200 (turns)
104
Q

Does Underwinding or overwinding changes the linking number?

A

Yes

105
Q

For the relaxed DNA molecule,

underwinding or overwinding by two helical turns (i.e., to Lk = 198 or 202) will produce what kind of supercoiling

A
  • Underwinding = negative supercoiling

- Overwinding = positive supercoiling

106
Q

Negative supercoiling results in a ___ handed helix whereas positive supercoiling is ___-handed.

A
  • Negative supercoiling results in righthanded helix

- Positive supercoiling results in lefthanded helix

107
Q

Can relaxed or supercoiled DNA lie flat on a planar surface

A
  • Supercoiled
108
Q

Why is supercoiled DNA called the tertiary structure?

A

Supercoiled DNA represents higher

order folding of secondary structure, or helix

109
Q

A supercoiled DNA molecule is more or less compact than a relaxed DNA molecule
of the same length

A

More

110
Q

Supercoiling allows small bacterial cells

to ____ long lengths of DNA

A

Package

111
Q

Supercoiled DNA is more compact: it moves ___ than relaxed DNA when subjected to
centrifugation or gel electrophoresis.

A

Faster

112
Q

The only way to change Lk for a circular molecule is to ?

A

cut it then twist and rejoin the ends

113
Q

topoisomerases are enzymes that play an especially important role in processes such as

A

Replication and DNA packaging

114
Q

What are Topoisomerases

A

enzymes that catalyze interconversion of topoisomers (changes superhelicity)

115
Q

Topoisomerases change the

linking number of DNA by catalyzing what three-step process

A

1) the cleavage of one or both strands of double-stranded DNA
2) the passage of a segment of
DNA through this break
3) resealing of the DNA breaks

116
Q

How do topoisomerases play important roles
in replication, transcription and
recombination for both prokaryotes
and eukaryotes

A

help relieve

torsional stress induced by unwinding the DNA during these events

117
Q

How do topoisomerases utilize the key tyrosine residue

A

Topoisomerases utilize a key tyrosine residue that covalently attaches to a phosphate in the sugar-phosphate backbone that is transiently broken.

118
Q

Describe Topo I (4)

A
  • nicks one strand of DNA - the other strand then passes through the ss break, and the broken strand re-seals
  • changes the linking number (DLk) by increments of 1
  • thermodynamically favorable process driven by release of energy of supercoiling
  • E. coli type I topoisomerases generally relax DNA by removing negative supercoils (increasing Lk)
119
Q

How was topo II (DNA gyrase in bacteria)

A
  • breaks both strands, and then re-seals both strands • DLk in increments of 2
  • can relax negative supercoils
  • in E. coli, Topo II (DNA gyrase) can also introduce negative supercoils using energy from ATP hydrolysis (decrease Lk)
120
Q

The degree of supercoiling of DNA is maintained by regulation of the net activity of

A

topoisomerase types I and II

121
Q

Topoisomerases are the molecular targets of _____ and _____

A

antibacterial and anti-cancer drugs

122
Q

Without topoisomerases, cells

cannot ___ or ____ or ____

A

replicate or package their DNA, or express their genes so they die

Topoisomerases have therefore
become important targets for inhibitors

123
Q

Pharmaceutical agents that specifically inhibit ___ are effective in treating infectious bacteria and malignant cells

A

topoisomerases

124
Q

The quinolones inhibit?

A

bacterial DNA gyrase, a Type II topoisomerase.

125
Q

The fluoroquinolone Ciprofloxacin (Cipro) is one of the few antibiotics effective in treating?

A

anthrax infections; useful for other bacterial infections as well

126
Q

How and why do anticancer drugs target topoisermerases

A

Anticancer drugs also target topoisomerases, which are elevated in tumor cells. Agents targeted to these enzymes are thus more toxic to tumor cells than to normal cells. They act by inhibiting Topo I or Topo II.

127
Q

Like proteins, DNA fragments can be separated based on their size using ___

A

Electrophoresis

128
Q

DNA fragment migration in electrophoresis depends on?

A

Their degree of supercoiling

129
Q

Why is DNA naturally negatively charged?

A

Because of the negatively-charged phosphates in the sugar-phosphate backbone

130
Q

Which section will DNA migrate to in electrophoresis in an buffered agarose gel

A

Anode (+)

131
Q

Do you need to add SDS during electrophoresis for DNA

A

No

132
Q

Do smaller or larger fragments migrate more quickly?

A

Smaller

133
Q

The DNA bands can be visualized how?

A

Under UV light by staining with ethidium bromide

134
Q

What is ethidium bromide

A

a dye that binds the DNA by intercalating between the bases

135
Q

Why do supercoiled DNA runs more quickly than relaxed DNA

A

Because it is more compact

136
Q

How does treatment with Topo I change DNA?

A

All DNA circles run on this agarose gel are the same length but differ in Lk after treatment with Topo I. DNA in lane 3 has been treated with Topo I for longer than that in lane 2. With long enough Topo I treatment, all DNA circles will eventually become relaxed.

137
Q

each base pair is more exposed to solvent on which groove more so

A

Major groove

138
Q

on either side (especially the major groove side) there is potential for the base pairs to H-bond with what?

A

compounds in the surrounding environment, like water or proteins

139
Q

fxnal groups of base pairs that can be accessed from grooves: “exocyclic groups” -NH2, -C=O; ring N – these groups are not involved in base pairing and are exposed in the grooves… what does this allow

A

serve as contact points for proteins searching for specific sequences on the DNA – recognize a DNA sequence based on the base pair functional groups exposed in the groove

MBB 222 (12 decks)

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