DNA and RNA are polymers composed of nucleotide units. DNA contains the sugar deoxyribose and the bases adenine, guanine, cytosine, and thymine. RNA contains the sugar ribose and replaces thymine with uracil. DNA exists as a double helix with base pairing between adenine-thymine and guanine-cytosine. There are different forms of DNA structure including A, B, and Z-DNA. RNA exists in several types including messenger RNA, transfer RNA, and ribosomal RNA that play important roles in protein synthesis.
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DNA Structure and Functions
1. Chemistry of Nucleic Acids
Dr Sreenivasa Murthy M D
Assitant Professor
Department of Biochemistry
Mandya Institute of Medical Sciences,
Mandya
2. Contents
1. Composition of nucleic acid
2. Structure and function of
DNA
3. Structures and functions of
RNA
4. Properties of nucleic acid
3. Brief history
• 1869: isolated DNA from salmon sperm (Friedrich
Miescher)
• 1944: proved DNA is genetic materials (Avery et al.)
• 1953: discovered DNA double helix (Watson and
Crick)
• 1968: decoded the genetic codes (Nirenberg)
• 1981: invented DNA sequencing method (Gilbert and
Sanger)
• 1987: launched the human genome project
• 2001: accomplished the draft map of human genome
7. Structure of DNA?
• The Genetic Material
• Crick and Watson
– Race with Linus Pauling to predict structure
• Chargaff’s rules:
– Chemical analysis:
[A] = [T]
[G] = [C]
– Constant
• for each organism
– over time
– across all tissues
10. •DNA and RNA are polymers of nucleotide
units.
• DNA (RNA) consists of 4 kinds of
ribonucleotide units linked together through
covalent bonds.
• Each nucleotide unit is composed of
a nitrogenous base
a pentose sugar
a phosphate group
1. The components of DNA and RNA
14. 1.2 Ribose (in RNA) and deoxyribose (in DNA)
•
• Ribose and deoxyribose predominantly
exist in the cyclic form.
15. •The bases are covalently attached to the 1’ position
of a pentose sugar ring, to form a nucleoside
Glycosidic bond
R Ribose or 2’-deoxyribose
1.3 Nucleosides =ribose/deoxyribose + bases
1
17. •A nucleotide is a nucleoside with one or more phosphate
groups bound covalently to the 3’-, 5’, or ( in
ribonucleotides only) the 2’-position. In the case of 5’-
position, up to three phosphates may be attached.
Deoxynucleotides
(containing deoxyribose)
Ribonucleotides
(containing ribose)
Phosphate ester bonds
1.4 Nucleotides = nucleoside + phosphate
22. 1.5 Some important nucleotides
• dATP, dGTP, dCTP, dUTP
– Raw materials for DNA biosynthesis.
• ATP, GTP, CTP, UTP
– Raw materials for RNA biosynthesis
– Energy donor
– Important co-enzymes
• Cycling nucleotides—cAMP, cGMP
– Secondary messengers in hormones action.
23. Nucleic acid derivatives
Multiple phosphate nucleotides
adenosine monophosphate (AMP)
adenosine diphosphate (ADP)
adenosine triphosphate (ATP)
N
O
CH2
O
OH
OH
N
N
N
NH2
P
O
OH
O
H
AMP
N
O
CH2
O
OH
OH
N
N
N
NH2
P
O
OH
O
P
O
OH
O
H
ADP
N
O
CH2
O
OH
OH
N
N
N
NH2
P
O
OH
O
P
O
OH
O
P
O
OH
O
H
ATP
33. 2.1 Primary structure
Definition: the base sequence (or the
nucleotide sequence) in
polydeoxynucleotide chain.
The smallest DNA in nature is virus DNA.
The length of φX174 virus DNA is 5,386
bases (a single chain).
The DNA length of human genome is
3,000,000,000 pair bases.
2. Structure and function of DNA
34. • 3’,5’ phosphodiester bond link nucleotides
together to form polynucleotide chains
5’end
3’ end: free hydroxyl
(-OH) group
Phosphodiester
bond
35. The structure of a DNA chain can be
concisely represented
• An even more abbreviated notation for
this chain is
– pApCpGpTpA
– pACGTA
• The base chain is written in the 5’ →3’
direction
36. 2.2 Secondary structure
The secondary structure is defined as
the relative spatial position of all the
atoms of nucleotide residues.
37. •Watson and Crick , 1953
•The genetic material of
all organisms except for
some viruses.
•The foundation of the
molecular biology.
James D. Watson
Francis H.C. Crick
Secondary structure
— DNA double helix structure
38. The discovery of DNA double helix
• Chargaff's Rule
(A=T, G=C in DNA)
• Franklin, Wilkins:
X-ray
Diffraction
Refined Structure
39. DNA conformations
A- DNA B-DNA Z-DNA
Helix Right-handed Right-handed Left-handed
Width Widest Intermediate Narrowest
Planes of
bases
planes of the base
pairs inclined to
the helix axis
planes of the base
pairs nearly
perpendicular to
the helix axis
planes of the base
pairs nearly
perpendicular to the
helix axis
Central axis 6A hole along helix
axis
tiny central axis no internal spaces
Major groove Narrow and deep Wide and deep No major groove
Minor groove Wide and shallow Narrow and deep Narrow and deep
40. Right-handed helix
intermediate
planes of the base pairs nearly
perpendicular to the helix axis
tiny central axis
wide + deep major groove
narrow + deep minor groove
B-DNA
41. DNA conformations
Right-handed helix
Widest
planes of the base pairs
inclined to the helix axis
6A hole along helix axis
narrow + deep major
groove
Wide + shallow minor
groove
A- DNA
42. Left-handed helix
Narrowest
planes of the base pairs
nearly perpendicular to the
helix axis
no internal spaces
no major groove
narrow + deep minor
groove
Z-DNA
DNA conformations
45. Direction of Replication
The enzyme helicase unwinds several sections of parent DNA
At each open DNA section, called a replication fork, DNA
polymerase catalyzes the formation of 5’-3’ester bonds of the
leading strand
The lagging strand, which grows in the 3’-5’ direction, is
synthesized in short sections called Okazaki fragments
The Okazaki fragments are joined by DNA ligase to give a
single 3’-5’ DNA strand
47. •Two separate strands
•Antiparellel (5’3’
direction)
•Base pairing:
hydrogen bonding
that holds two
strands together
•Complementary
(sequence)
Essential for replicating DNA
and transcribing RNA
5’
3’
3’
5’
• Sugar-phosphate
backbones (negatively
charged): outside
• Base pairs (stack one
above the other): inside
DNA double helix
49. B form of DNA double
helix
• Right-handed helix;
•The diameter of the
double helix:2 nm
• The distance
between two base
pairs: 0.34 nm;
• Each turn of the
helix involves 10 bases
pairs, 3.4 nm.
Stable configuration
can be maintained by
hydrogen bond and base
stacking force
(hydrophobic interaction).
50. Groove binding
• Small molecules like drugs bind in the minor
groove, whereas particular protein motifs can
interact with the major grooves.
52. • B-form: the duplex structure proposed by Watson and Crick is
referred as the B-form DNA.
•It is the standard structure for DNA molecules.
•A-form: at low humidity the DNA molecule will take the A-form:
•The A-form helix is wider and shorter, with a shorter more compact
helical structure, than the B-form helix.
• Z-form: the Z-form DNA is adopted by short oligonucleotides.
•It is a left-handed double helix in which backbone phosphates zigzag.
53. 2.3 Tertiary structure :
• Supercoils: double-stranded circular DNA
form supercoils if the strands are
underwound (negatively supercoiled) or
overwound (positively supercoiled).
Relaxed supercoiled
Increasing degree of supercoiling
54. • If the strands
are overwound,
form positively
supercoiled;
• If the strands
are underwound,
form negatively
supercoiled.
55. • The DNA in a prokaryotic cell is
a supercoil.
• Supercoiling makes the DNA molecule more
compact thus important for its packaging
in cells.
56. 2.4 Eukaryotic DNA
• DNA in eukaryotic cells is highly
packed.
• DNA appears in a highly ordered form
called chromosomes during metaphase,
whereas shows a relatively loose form
of chromatin in other phases.
• The basic unit of chromatin is
nucleosome.
• Nucleosomes are composed of DNA
and histone proteins.
57. Nucleosome
• The chromosomal DNA
is complexed with five
types of histone.
•H1, H2A, H2B, H3 and
H4.
•Histons are very basic
proteins, rich in Arginine
and Lysine.
•Nucleosomes: regular association of DNA with
histones to form a structure effectively compacting
DNA. ”beads”
58. Beads on a string
• 146 bp of
negatively
supercoiled DNA
winds 1 ¾ turns
around a histone
octomer.
• H1 histone binds
to the DNA
spacer.
59.
60. The importance of packing of DNA
into chromosomes
Chromosome is a compact form of the DNA
that readily fits inside the cell
To protect DNA from damage
DNA in a chromosome can be transmitted
efficiently to both daughter cells during
cell division
Chromosome confers an overall organization
to each molecule of DNA, which facilitates
gene expression as well as recombination.
61. 2.5 Functions of DNA
• The carrier of genetic information.
• The template strand involved in replication
and transcription.
Gene: the minimum functional unit in DNA
Genome: the total genes in a living cell or
living beings.
63. DNA conformations
A- DNA B-DNA Z-DNA
Helix Right-handed Right-handed Left-handed
Width Widest Intermediate Narrowest
Planes of
bases
planes of the base
pairs inclined to
the helix axis
planes of the base
pairs nearly
perpendicular to
the helix axis
planes of the base
pairs nearly
perpendicular to the
helix axis
Central axis 6A hole along helix
axis
tiny central axis no internal spaces
Major groove Narrow and deep Wide and deep No major groove
Minor groove Wide and shallow Narrow and deep Narrow and deep
64. • B-form: the duplex structure proposed by Watson and Crick is
referred as the B-form DNA.
•It is the standard structure for DNA molecules.
•A-form: at low humidity the DNA molecule will take the A-form:
•The A-form helix is wider and shorter, with a shorter more compact
helical structure, than the B-form helix.
• Z-form: the Z-form DNA is adopted by short oligonucleotides.
•It is a left-handed double helix in which backbone phosphates zigzag.
68. Ribonucleic Acid (RNA)
There are several important differences between RNA and
DNA:
- the pentose sugar in RNA is ribose, in DNA it’s deoxyribose
- in RNA, uracil replaces the base thymine (U pairs with A)
- RNA is single stranded while DNA is double stranded
- RNA molecules are much smaller than DNA molecules
69. DNA RNA
Double stranded helical structure Single stranded
Deoxyribose (No alkali hydrolysis) Ribose ( Susceptible to alkali hydrolysis
due to the presence of 2’ OH group)
A, G, C, T A, G, C, U
Obeys Chargaff rule Doesnot Obey Chargaff rule
Genetic repository Protein biosynthesis
No enzymatic activity Catalytic activity (Ribozyme)
Large Smaller 100 -500 bp
70. Types of RNA:
Ribosomal RNA (rRNA),
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Hetrogeneous nuclear RNA (hn RNA)
Small nuclear RNA (Sn RNA)
71. 3. Structures and functions of RNA
Conformational variability of RNA is important
for the much more diverse roles of RNA in
the cell, when compared to DNA.
Types :
• mRNA: messenger RNA, the carrier of genetic
information from DNA to translate into protein
• tRNA: transfer RNA , to transport amino acid to
ribosomes to synthesize protein
• rRNA: ribosomal RNA, the components of
ribosomes
• hnRNA: Heterogeneous nuclear RNA
• snRNA: small nuclear RNA
74. Ribosomal RNA and Messenger RNA
Ribosomes are the sites of protein synthesis
- they consist of ribosomal DNA (65%) and proteins
(35%)
- they have two subunits, a large one and a small one
Messenger RNA carries the genetic code to the
ribosomes
- they are strands of RNA that are complementary to the
DNA of the gene for the protein to be synthesized
75. Transfer RNA
Transfer RNA translates the genetic code from the messenger
RNA and brings specific amino acids to the ribosome for protein
synthesis
Each amino acid is recognized by one or more specific tRNA
tRNA has a tertiary structure that is L-shaped
- one end attaches to the amino acid and the other binds to the
mRNA by a 3-base complimentary sequence
76. RNA structure
• RNA molecules are largely single-
stranded but there are double-
stranded regions.
77. 3.1 Messenger RNA( mRNA)
• Function: the carrier of genetic
information from DNA for the
synthesis of protein.
• Comprises only about 5% of the
RNA in the cell.
• Composition: vary considerably in
size (500-6000 bases in E. coli)
78. Eukaryotic mRNA Structure
(1) Capping: linkage of 7-
methylguanosine to the 5’ terminal
residue.
(2) Tailing: attachment of an
adennylate polymer (poly A, 20~250
nucleotides) at the 3’ terminal.
79. 3.2 Transfer RNA (tRNA)
• Primary Structure :
– 74~95 bases, the smallest of the three major
RNA.
– Modified bases: pseudouridine (ψ)
methylguanosine
dihydrouridine (D)
– The sequence CCA at the 3’ terminus
• They make up 15% of the RNA in the cell.
• Function: Transport amino acids to ribosomes for
assembly into proteins.
• There are at least 20 types of tRNA in one cell.
80. Secondary structure: cloverleaf
• Four loops and four
arms
– Amino acid arm
(7bp): to bide amino
acid
– D loop(8-14bp) and
D arm(3-4bp):
– Anticoden loop(5bp)
and arm(7bp): to
recognize amino acid
coden on the mRNA.
– TψC loop(7bp) and
arm(5bp)
– Variable loop(4-5bp
or 13-21bp)
82. * The species of rRNA
•Eukaryotes
• 5S rRNA
• 28S rRNA
• 18S rRNA
• 5.8S rRNA
•Prokaryotes
• 5S rRNA
• 23S rRNA
• 16S rRNA
• S represents Svedberg units, they represent
measures of sedimentation rate.
3.3 Ribosomal RNA (rRNA)
• Components of ribosomes.
• They make up 80% of the RNA in the cell.
84. Ribosomes
• Ribosomes are cytoplasmic structures that
synthesize protein, composed of RNA (2/3)
and protein (1/3).
• The ribosomes of prokaryotes and
eukaryotes are similar in shape and function.
The difference between them is the size
and chemical composition.
85. Three rRNA
52 proteins
Four rRNA
83 proteins
• Ribosomes are ribonucleoprotein particles for
synthesizing proteins.
86. Other RNAs
• Small nuclear RNA (snRNA)
– Involved in mRNA processing
• Small nucleolar RNA (snoRNA)
– Play a key role in the processing of rRNA
molecules
• Small cytoplasmic RNA (scRNA)
– Involved in the selection of proteins for export
• Catalytic RNA or Ribozyme
• Small interfering RNA (siRNA)
– Interfere with the expression of a specific
gene
• RNomics
88. General properties
• Acidity
– Amphiphilic molecules; normally acidic because
of phosphate.
• Viscosity
– Solid DNA: white fiber; RNA: white powder.
Insoluble in organic solvents, can be precipitate
by ethanol.
• Optical absorption
– UV absorption due to aromatic groups.
• Thermal stability
– Disassociation of dsDNA (double-stranded DNA)
into two ssDNAs (single-stranded DNA).
89. 4.1 UV Absorption
• Specific absorption at 260nm.
• This can be used to identify nucleic
acid.
The UV absorption spectra of the common ribonucleotides
90. 4.2 Denaturation
• Concept:
• The course of hydrogen bonds broken,
3-D structure was destroyed, the double
helix changed into single strand irregular
coil.
• Results:
(1) the value of 260nm absorption is increased;
(2) biological functions are lost.
91. • Heat denaturation and Tm
• When DNA were
heated to certain
temperature, the
absorption value at
260nm would increased
sharply,which indicates
that the double strand
helix DNA was
separated into single
strand.
•Tm (melting temperature of DNA):
• The temperature of UV absorption increase to an
half of maximum value in DNA denaturation.
92. • Factors affect Tm:
G-C content:
Higher G+C
Less G+C
Temperature
Tm of
two DNA
molecules with
different G+C
content
•There are three hydrogen bonds between G-C
pair. The more G-C content, the higher Tm value.
93. 4.3 Renaturation of DNA
• When slowly cooling down (Annealing)
the denatured DNA solution, the single
strand DNA can reform a double strands
helix to recover its biological functions.
94. Molecule hybridization
• During the course of
lowing down denaturing
temperature, between
different resource DNAs
or single stand DNA and
RNA with
complementary bases
will repair into a double
strands to form a hybrid
DNA or DNA-RNA . This
course is called molecule
hybridization.
95. Points
• The components of DNA and RNA
– Nucleotide: base (A,G,C,T,U), pentose sugar
(Ribose and deoxyribose), phosphate group
• Structure and function of DNA
– Primary structure: 3’,5’ phosphodiester bond
– Secondary structure: DNA double helix
– Tertiary structure: supercoil
– Eukaryotic chromosomes: nucleosome
• Structures and functions of RNA
– mRNA, tRNA, rRNA
• Properties of nucleic acid
– UV absorption, denaturation and renaturation,
molecule hybridization