Download Chapter 2 Chemistry of nucleic acid

Lecture 4
Structure and function of
nucleic acid
Contents
1. Composition of nucleic acid
2. Structure and function of
DNA
3. Structures and functions of
RNA
4. Properties of nucleic acid
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
Deoxyribonucleic acid, DNA
Nucleic acid
Ribonucleic acid, RNA
1. The components of DNA and RNA
•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
1.1 Bases
• Purines :
– Adenine (A)
– Guanine (G)
• Pyrimidines :
– Cytosine (C)
– Uracil (U)
– Thymine (T)
DNA: A,G,C,T
RNA: A,G,C,U
Thymine (T) is a 5-methyluracil (U)
1.2 Ribose (in RNA) and deoxyribose (in DNA)
•
• Ribose and deoxyribose predominantly
exist in the cyclic form.
1.3 Nucleosides =ribose/deoxyribose + bases
•The bases are covalently attached to the 1’ position
of a pentose sugar ring, to form a nucleoside
Glycosidic bond
1
R
Ribose or 2’-deoxyribose
Adenosine, guanosine, cytidine, thymidine, uridine
1.4 Nucleotides = nucleoside + phosphate
•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.
Phosphate ester bonds
Deoxynucleotides
(containing deoxyribose)
Ribonucleotides
(containing ribose)
BASES
NUCLEOSIDES
Adenine (A) Adenosine
NUCLEOTIDES
Adenosine 5’-triphosphate (ATP)
Deoxyadenosine Deoxyadenosine 5’-triphosphate
(dATP)
Guanine (G) Guanosine
Deoxyguanosine
Cytosine (C) Cytidine
Uracil (U)
Guanosine 5’-triphosphate (GTP)
Deoxy-guanosine 5’-triphosphate
(dGTP)
Cytidine 5’-triphosphate (CTP)
Deoxycytidine
Deoxy-cytidine 5’-triphosphate
(dCTP)
Uridine
Uridine 5’-triphosphate (UTP)
Thymine (T) Thymidine/
Deoxythymidie
Thymidine/deoxythymidie
5’-triphosphate (dTTP)
phosphate
nucleic acid
pentose
nucleotides
nucleosides
bases
NH2
N
O
HO
P
O
CH2
O
N
OH
OH OH
O
Composition of DNA and RNA
Nucleic
acid
base
ribose
DNA
A、G、C、T
deoxyribose
RNA
A、G、C、U
ribose
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.
Nucleic acid derivatives
Multiple phosphate nucleotides
adenosine monophosphate (AMP)
adenosine diphosphate (ADP)
adenosine triphosphate (ATP)
NH2
NH
2
NH2
HO
O
O
O
O
O
O
HO P HOO PP O
O CH
CH2
P O P O P O CH2 O
O
OH
OH
OH
OH
OH
OH
OH
OH
ADP AMP OH
ATP
N
N
N
N
N
N
OH
OH
OH
NN
N
N
N
N
2. Structure and function of DNA
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.
5’end
Phosphodiester
bond
3’ end: free hydroxyl
(-OH) group
• 3’,5’ phosphodiester bond link nucleotides
together to form polynucleotide chains
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
2.2 Secondary structure
The secondary structure is defined as
the relative spatial position of all the
atoms of nucleotide residues.
Secondary structure
— DNA double helix structure
•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
The discovery of DNA double helix
• Chargaff's Rule
(A=T, G=C in DNA)
• Franklin, Wilkins:
X-ray
Diffraction
Refined Structure
DNA double helix
•Two separate strands
•Antiparellel (5’3’
direction)
•Base pairing:
hydrogen bonding
that holds two
strands together
•Complementary
(sequence)
• Sugar-phosphate
backbones (negatively
charged): outside
• Base pairs (stack one
above the other): inside
Essential for replicating DNA
and transcribing RNA
3’
5’
3’
5’
6
7
5
8
1
9 4
3 2
4
32 1
G:C
A:T
Base pairing
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).
Groove binding
• Small molecules like drugs bind in the minor
groove, whereas particular protein motifs can
interact with the major grooves.
•
Watson, Crick, and Wilkins
shared the Nobel Prize in
medicine or physiology in 1962
for this brilliant accomplishment.
•
The discovery of the DNA
double helix revolutionized
biology: it led the way to an
understanding of gene function
in molecular terms (their work is
recognized to mark the
beginning of molecular biology).
Conformational variation in
double-helical structure
•
B-DNA
•
A-DNA
•
Z-DNA
• 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.
2.3 Tertiary structure :
•
Supercoils: double-stranded circular DNA
form supercoils if the strands are
underwound (negatively supercoiled) or
overwound (positively supercoiled).
Increasing degree of supercoiling
Relaxed
supercoiled
•
•
If the strands
are overwound,
form positively
supercoiled;
If the strands
are underwound,
form negatively
supercoiled.
• The DNA in a prokaryotic cell is
a supercoil.
• Supercoiling makes the DNA molecule more
compact thus important for its packaging
in cells.
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.
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”
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.
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.
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.
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
Classes of eukaryotic RNAs
RNA structure
• RNA molecules are largely singlestranded but there are doublestranded regions.
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)
Eukaryotic mRNA Structure
(1) Capping: linkage of 7methylguanosine to the 5’ terminal
residue.
(2) Tailing: attachment of an
adennylate polymer (poly A, 20~250
nucleotides) at the 3’ terminal.
3.2 Transfer RNA (tRNA)
• 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.
• 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
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)
•Tertiary structure of tRNA
3.3 Ribosomal RNA (rRNA)
• Components of ribosomes.
• They make up 80% of the RNA in the cell.
* 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.
The proposed
secondary structure
for E.coli 16S rRNA
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.
Three rRNA
52 proteins
Four rRNA
83 proteins
• Ribosomes are ribonucleoprotein particles for
synthesizing proteins.
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
4. Physical and Chemical
Properties of Nucleic Acids
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).
4.1 UV Absorption
• Specific absorption at 260nm.
• This can be used to identify nucleic
acid.
The UV absorption spectra of the common ribonucleotides
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.
• 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.
•
Factors affect Tm:
G-C content:
•There are three hydrogen bonds between G-C
pair. The more G-C content, the higher Tm value.
Less G+C
Higher G+C
Temperature
Tm of
two DNA
molecules with
different G+C
content
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.
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.
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