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DNA and RNA
Structure and Function
RNA and DNA Chemical Structures
DNA Structural Elements
RNA Structural Elements
Cleavage of DNA and RNA by Nucleases
Nucleic Acid-Protein Complexes
1
The nucleic acids
Nucleic acids are complex structures used
to maintain genetic information.
DNA deoxyribonucleic acid
Serves as the “Master Copy” for most
information in the cell.
RNA ribonucleic acid
Several types. Overall, it acts to transfer
information from DNA to the rest of the
cell.
2
DNA and RNA composition
Primary structure of both materials is very
similar.
Each consists of a sugar/phosphate backbone
with nitrogenous bases attached.
sugar
phosphate
base
Major difference is in the type of sugar and bases used.
3
Sugars used
ribose
used in RNA
O
HOCH2
H
deoxyribose
used in DNA
OH
H
H
OH
OH
H
O
HOCH2
H
OH
H
H
OH
H
H
Not a very
big difference!
4
Nucleoside
A sugar - base combination.
Base
O
HOCH2
H
Sugar
In this case
deoxyribose
H
H
OH
H
H
-N-glycosidic
linkage
5
The nitrogenous bases
Five bases are used that fall in two classes
Purines
A double ring (6 and 5 membered) structure
Includes adenine and guanine
Used by both DNA and RNA
Pyrimidines
A six membered ring structure
Cytosine is used in both DNA and RNA
Thymine is used in DNA, Uracil used in RNA
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The nitrogenous bases
NH2
|
C
N
O
||
C
adenine
C
N
HN
guanine
N
C
CH
HC
C
N
CH
C
N
H
H2N
NH2
|
C
O
O
||
C
N
CH
HN
C
CH
C
N
H
cytosine
O
C
N
H
N
O
||
C
CH3
C
CH
N
H
thymine
O
HN
CH
C
CH
N
H
uracil
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Nucleotides
Produced if the -OH on the sugar of a
nucleoside is converted into a phosphate
ester.
NH2
|
C
deoxyadenosine monophosphate
(dAMP)
N
C
N
CH
HC
Each is named based on
sugar and base name
and then the number of
phosphates is indicated.
C
O
||
N
-O-P-O-CH
|
O-
O
2
H
N
H
H
OH
H
H
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Primary structure
NH2
|
C
O
|
- O -- P -- O -- CH
2
||
O
N
C
HC
C
N
CH
N
N
NH2
|
C
O
N
O
|
- O -- P -- O -- CH
2
||
O
CH
C
O
CH
N
O
O
||
C
HN
Phosphate bonds link
DNA or RNA
nucleotides together
in a linear sequence.
3’,5’-phosphodiester
O
C
|
- O -- P -- O -- CH H N
2
||
O
O
2
N
C
CH
C
N
N
O
||
C
HN
O
|
- O -- P -- O -- CH
2
||
O
C
C
O
CH3
CH
N
O
OH
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Primary structure
It is cumbersome to draw complete nucleic
acids so a simplified set of lines can be
used.
base
A
G
C
T (U)
1’
sugar
5’
nucleosides
10
Primary structure
Representation of an oligonucleotide
showing the 3’,5’-phosphodiester bonds.
A
G
C
T (U)
5’ end
HO
3’ end
P
P
P
OH
A very abbreviated representation is to simply list
the base sequence starting from the 5’ end - AGCT
11
DNA
In native DNA, a large number of
nucleotides are linked together.
The size and conformation of the molecule
is species dependent.
In simple prokayrotic cells, a single strand is
produced.
In more complex species, multiple strands
are used. Each is combined with proteins
called histones.
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DNA
Organism
Viruses
SV40
Adenovirus
 phage
Number of
Base Pairs
5,100
36,000
48,600
Length
(m)
1.7
12
17
Conformation
circular
linear
circular
Bacteria
E. coli
Eukaryotes
Yeast
Fruit fly
Human
4,700,000
1,400
13,500,000
165,000,000
3,000,000,000
4,600
56,000
1-2 x 106
circular
linear
linear
linear
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RNA
Approximately 5-10% of the total weight of a
cell is RNA. DNA is only about 1%
RNA exists in three major forms.
• Ribosomal RNA - rRNA. Combined with protein
to form ribosomes, the site of protein synthesis.
• Messenger RNA - mRNA. Carries instructions
from a single gene from DNA to the ribosome.
• Transfer RNA - tRNA. Forms esters with specific
amino acids for use in protein synthesis.
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DNA structural elements
Sugar-phosphate backbone
Causes DNA chain to coil around the outside of
the attached bases like a spiral staircase.
Base Pairing
Hydrogen bonding occurs between purines and
pyrimidines. This causes two DNA strands to
bond together.
adenine - thymine
guanine - cytosine
Always pair together!
Results in a double helix structure.
15
Base pairing and
hydrogen bonding
H-N
N
N
N-H
guanine
N
cytosine
N
N
N-H
H 3C
H
thymine
N
H
|
N- H
N
N
adenine
N
N
N
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Hydrogen bonding
Each base wants to
form either two or three
hydrogen bonds.
That’s why only certain
bases will form pairs.
C
G
T
A
G
C
C
G
A
T
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The double helix
The combination of
the stairstep sugarphosphate backbone
and the bonding
between pairs results
in a double helix.
One
complete
turn is
3.4 nm
Distance between
bases = 0.34 nm
2 nm
between
strands
10.5 bases/turn
18
Double helix
The original Watson and Crick model for the
double helix, B-DNA, is one of several
conformations.
B-DNA - believed to be the predominate form under
physiological conditions. It has 10.5 bases per
turn.
A-DNA - formed when B-DNA is chemically treated.
It has 11 bases per turn.
Z-DNA - left handed helix with 12 bases per turn. It
may play a role in regulation of gene expression.
19
Physical and biological
properties of the double helix
Inspection of the DNA double
helix indicates a mechanism
for its replication and
transfer of information.
3’
3’
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Physical and biological
properties of the double helix
It is possible to overcome the hydrogen
bonding and van der Waal forces that hold
the strands together - denaturing.
heat, acids, bases or organic solvents
The strands tend to reassociate if the
source of denaturing is removed.
Example - heating DNA will cause it to
unwind. It will recoil when cooled annealing.
21
Tertiary structure of DNA
Studies of native, intact DNA have revealed
two distinct forms - linear and circular.
The circular form is the result of covalent
joining of the two ends of a linear double
helix.
It can exist in two conformations
Relaxed
Supercoiled
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Tertiary structure of DNA
Relaxed
Supercoiled
23
Supercoiled DNA
• The supercoil is not simply a coil of the
circular form, there is extra twisting.
• While it is less stable than the relaxed form,
there is evidence to show that it exists in vivo.
• Topoisomerases - enzymes that catalyze
changes in the topology of DNA have been
isolated.
• This form may play a regulatory role in DNA
replication or represent a more compact form
for storage.
24
RNA structural elements
Two fundamental differences between the
primary, covalent structures of RNA and
DNA.
• RNA contains ribose rather than 2-deoxyribose.
• RNA uses uracil instead of thymine.
The extra hydroxyl group in RNA makes it
more susceptible to hydrolysis.
This may be the main reason that DNA is
the ultimate repository of genetic
information.
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RNA structural elements
• RNA always exists as single-stranded
molecules.
• It does not take on an extended,
secondary structure like DNA.
• The strands tend to fold into a uniform,
periodic pattern.
• Several structural elements are observed.
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RNA structural elements
Hairpin turns
Loops in the chain that bring together
complementary base pairs. If long enough, a
double helix region is observed.
Right-handed double helix
Result from intrastrand folding.
Internal loops and bulges
Relatively common in RNA molecules. These are
structural features that disrupt the formation of
continuous double helix regions.
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RNA structural elements
Helical
segments
Bulge
Unpaired
loop at tip
Internal
loop
28
tRNA structure
The smallest type
of RNA. It consists
of 74-93 nucleotides.
These molecules
are the carriers of
the 20 amino acids
with at least one tRNA
for each.
They often contain
several unusual
purines or pyrimidine
bases -modifications
of the basic four.
29
tRNA structure
HO-
All tRNA have a
common 2o and
3o structure.
3’
A
C
C
A
5’
G
A
U
G
C
U
G
C
G
G
U
G
C
G
C
G
U
C
G
G
C
U
U
G
C
A
G
G
G
U
C
A
anticodon
G
G
C
C
U
U
A
G
U
C
G
C
C
C
C
G
G
C
G
C
U
G
C
G
U
A
C
G
C
G
C
G
A
U
G
U
G
C
G
G
C
30
rRNA
These molecules have 2o and 3o structure
similar to tRNA. However, they are much
larger.
16S rRNA
E. Coli
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Cleavage of DNA
and RNA by nucleases
Characterization of nucleic acid structure
and function often requires breaking
these large molecules into fragments.
• Primary structural analysis of DNA/RNA
• Site specific cleavage for recombinant
DNA manipulation.
Historically, hydrolysis by acids (for DNA),
bases (for RNA) and enzymes has been
used.
32
Cleavage of DNA
and RNA by nucleases
Nucleases
• Enzymes that catalyze the hydrolysis of
phosphodiester bonds.
• Normally used to catalyze degradation of
damaged or aged nucleic acids.
• Some work on both DNA and RNA and
others are substrate specific.
DNases - deoxyribonucleases, act on DNA.
RNases - ribonucleases, act on RNA.
33
Cleavage of DNA
and RNA by nucleases
Nucleases
• Exonucleases - catalyze the removal of
terminal nucleotides, 3’ and 5’ types.
• Endonucleases - catalyze removal of
internal phosphodiester bonds.
Type a - act on the 3’ hydroxyl group of a
nucleotide with the phosphorous group.
Type b - act on the 5’ hydroxyl group of a
nucleotide with the phosphorous group.
34
Cleavage of DNA
and RNA by nucleases
Endonucleases
Type a
G
C
A
T
5’ end
snake venom
phosphodiesterase
HO
3’ end
P
P
P
OH
Type b
While these enzymes are useful for cutting DNA and
RNA into manageable sizes, they are not very specific.
35
Properties of nucleases
Enzyme
Substrate Type
Specificity
Rattlesnake venom
phosphodiesterase
DNA,RNA exo(a)
3’ end, no base
specificity.
Spleen
phosphodiesterase
DNA,RNA exo(b)
5’ end, no base
specificity.
Pancreatic
ribonuclease A
RNA
endo(b)
3’ side preference
for pyrimidines.
Spleen
DNA
deoxyribonuclease II
endo(b)
Internal ester
bonds. No base
specificity
36
DNA restriction enzymes
Restriction endonucleases
• Restriction enzymes.
• Most specific enzymes for DNA cleavage.
• Recognize specific base sequences.
These enzymes are produced in bacterial
cells and act to degrade or “restrict”
foreign DNA molecules.
Host DNA is protected because some bases
near the cleavage sites are methylated.
37
DNA restriction enzymes
Several hundred restriction enzymes have
been isolated and characterized.
Nomenclature.
• Three letter abbreviation representing the
source species.
• A letter representing the strain.
• A roman number for the order of discovery.
EcoRI - The first restriction enzyme
isolated from E. coli, strain R.
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DNA restriction enzymes
These enzymes are
all DNA specific of
the type endo (a).
The red line indicates
the point of cleavage
for the recognition
sequence.
Enzyme
Sequence
EcoRI
5’ - GAATTC
BaII
5’ - TGGCCA
TaqI
5’ - TCGA
HinfI
5’ - GANTC
39
DNA restriction enzymes
Catalysis of DNA into smaller fragments can
be used for:
• Making it easier to assay DNA
• Identification
Results in a unique set of fragments
for different DNA molecule.
Each individual will produce a unique
set of fragments - DNA fingerprints.
40
Nucleic acid - protein complexes
These are examples of ‘supramolecular
assemblies’ - organized clusters of
macromolecules.
Significant examples are nucleoproteins
- complexes of protein and nucleic acids.
Examples
Viruses
Chromosomes
snRNPs
Ribosomes
Ribonucleoprotein enzymes
41
Viruses
Stable, infective particles composed of a
nucleic acid (DNA or RNA) and protein.
The protein subunits
form a protective shell
around the nucleic acid
core.
Larger, complex viruses
also
have an additional
envelope of
glycoproteins and membrane lipids.
42
Viruses
Diagram of the human immunodeficiency virus (HIV).
RNA
Glycoproteins
Protein
subunits
Reverse
transcriptase
43
Chromosomes
Eukaryotic chromosomes consists of DNA
strands coiled around protein - histones.
44
Chromosomes
The normal number of chromosome pairs
varies among the species.
Animal
Man
Cat
Mouse
Rabbit
Honeybee,
male
female
Pairs
23
30
20
22
8
16
Plant
Onion
Rice
Rye
Tomato
White pine
Adder’s
tongue fern
Pairs
8
14
7
12
12
1262
45
Chromosomes
• DNA is tightly wrapped around histones which
are relatively small proteins.
•
The resulting complex is called chromatin.
• Eight histones are used to form the basic core
for the DNA to wrap around.
• The spooled complexes are called nucleosomes
with about one million per human chromosome.
• The nucleosomes continue to wind, forming
chromatids.
• The chromatids combine resulting in a
chromosome unit.
46
Histone
47
Coiling of DNA
One coil
30 rosettes
Two chromatids
are produced from
2 x 10 coils.
They are not shown
in this figure.
One loop
5 x 107 bp
30 nm Fiber
‘Beads-on-a
-string’ form
of chromatin
DNA
48
snRNPs
• A new class of ribonucleoprotein
complexes
• Active clusters of small nuclear
ribonucleic acids (snRNAs) and protein.
• They are only found in eukaryotic cells.
• They catalyze specific splicing that
removes exon sequences to produce
mature mRNA.
• Analogous cytoplasmic particles are
scRNPs.
49
snRNPs
50
Other complexes
Ribosomes
Serve as the platform for protein
synthesis. They consist of about 65%
RNA and 35% protein. There are two
major subunits that dissociate during the
translation process.
Ribonucleoprotein enzymes
An important class of enzyme. It includes
ribonuclease P and telomerase.
51
Ribonuclease A
52