Download 第三章 核酸的结构和功能

Chapter 3
Structures and
Functions of
Nucleic Acids
Nucleic acid
A biopolymer composed of nucleotides
linked in a linear sequential order through
3’,5’ phosphodiester bonds
Classification of nucleic acid
• Ribonucleic acid (RNA) is composed of
ribonucleotides.
– in nuclei and cytoplasm
– participate in the gene expression
• Deoxyribonucleic acid (DNA) is
composed of deoxyribonucleotides.
– 90% in nuclei and the rest in
mitochondria
– store and carry genetic information;
determine the genotype of cells
Interesting history
• 1944: proved DNA is genetic materials (Avery et al.)
• 1953: discovered DNA double helix (Watson and
Crick)
• 1968: decoded the genetic codes (Nirenberg)
• 1975: discovered reverse transcriptase (Temin and
Baltimore)
• 1981: invented DNA sequencing method (Gilbert and
Sanger)
• 1985: invented PCR technique (Mullis)
• 1987: launched the human genome project
• 1994: HGP in China
• 2001: accomplished the draft map of human genome
Section 1
Chemical Components of
Nucleic Acids
§ 1.1 Molecular Constituents
Nucleic acid can be hydrolyzed into
nucleotides by nucleases. The hydrolyzed
nucleic acid has equal quantity of base,
pentose and phosphate.
phosphate
nucleic acid
pentose
nucleotides
nucleosides
bases
Base: Purine
NH2
N
N
7
8
9
NH
5
4
6
3
N
1N
2
NH
N
N
Adenine (A)
O
N
NH
NH
N
Guanine (G)
NH2
Base: Pyrimidine
O
5
4
N
3
2
NH
6 1
NH
NH
O
Uracil (U)
NH2
O
H3 C
N
NH
Cytosine (C)
NH
O
NH
O
Thymine (T)
Pentose
HO
CH 2
5´
O
OH
HO
CH 2
OH
O
1´
4´
3´
OH
2´
OH
-D-ribose
OH
-D-2-deoxyribose
Ribonucleoside
NH2
N
HO
CH2
1
O
N
1´
O
glycosidic bond
OH OH
Purine N-9 or pyrimidine N-1 is connected
to pentose (or deoxypentose) C-1’ through a
glycosidic bond.
Ribonucleotide
NH2
phosphoester bond
N
O
HO
P
O
CH2
O
N
O
OH
OH OH
A nucleoside (or deoxynucleoside) and a
phosphoric acid are linked together through
the 5’-phosphoester bond.
Nomenclature
base
nucleoside
guanine
guanosine
cytosine
cytidine
adenine
adenosine
uracil
uridine
nucleotide
guanosine monophosphate
(GMP)
cytidine monophosphate
(CMP)
adenosine monophosphate
(AMP)
uridine monophosphate
(UMP)
(NMP)
Nomenclature
base
nucleoside
nucleotide
guanine
deoxyguanosine
deoxyguanosine monophosphate
(dGMP)
cytosine
deoxycytidine
deoxycytidine monophosphate
(dCMP)
adenine
deoxyadenosine
deoxyadenosine monophosphate
(dAMP)
thymine
deoxythymidine
deoxythymidine monophosphate
(dTMP)
(dNMP)
Composition of DNA and RNA
Nucleic
acid
base
ribose
DNA
A、G、C、T
deoxyribose
RNA
A、G、C、U
ribose
Nucleic acid derivatives
Multiple phosphate nucleotides
adenosine monophosphate (AMP)
adenosine diphosphate (ADP)
adenosine triphosphate (ATP)
NH
NH
22
NH
2
N
N
N
O
HO
O
O
PP OO CH
OO P
CH
P HOO PP O
CH222
H
O
O
O
OH
OH
OH
NN
N
OO
O
N
N
N
OH
OH
OH
OH
OH
OH
ADP
AMP
ATP
OH
OH
OH
NN
Nucleic acid derivatives
Cyclic ribonucleotide: 3’,5’-cAMP, 3’,5’cGMP, used in signal transduction
NH2
N
O
CH2
P
O
N
O
cAMP
O
OH
OH
N
N
Nucleic acid derivatives
Biologically active systems containing
ribonucleotide: NAD+, NADP+, CoA-SH
Phosphoester bond formation
The -P atom of the triphosphate group of a
dNTP attacks the C-3’ OH group of a nucleotide
or an existing DNA chain, and forms a 3’phosphoester bond.
Nucleic acid chain extension
A nucleic acid chain, having a phosphate
group at 5’ end and a -OH group at 3’ end,
can only be extended from the 3’ end.
Phosphodiester bonds
Alternative
phosphodiester
bonds and
pentoses
constitute the 5’3’ backbone of
nucleic acids.
Section 2
Structures and Functions
of Nucleic Acids
§ 2.1 Primary Structure
• The primary structure of DNA and RNA is
defined as the nucleotide sequence in the 5’ –
3’ direction.
• Since the difference among nucleotides is the
bases, the primary structure of DNA and RNA
is actually the base sequence.
• The nucleotide chain can be as long as
thousands and even more, so that the base
sequence variations create phenomenal
genetic information.
A
5' P
C
P
T
P
P
T
C
G
P
P
A
P
P
5' pApCpTpGpCpTpApApC-OH 3'
5' ACTGCTAAC 3'
C
A
P
OH 3'
§ 2.2 Secondary structure
The secondary structure is defined as the
relative spatial position of all the atoms of
nucleotide residues.
§ 2.2.a Chargaff’s rules
• The base composition of DNA generally
varies from one species to another.
• DNA isolated from different tissues of the
same species have the same base
composition.
• The base composition of DNA in a given
species does not change with its age,
nutritional state, and environmental
variations.
• The molarity of A equals to that of T, and the
molarity of G is equal to that of C.
Molarity of bases
A
G
C
T
A/T
G/C
G+C
Pu/Py
E. coli
26.0
24.9
25.2
23.9
1.09
0.99
50.1
1.04
Tuberc
ulosis
15.1
34.9
35.4
14.6
1.03
0.99
70.3
1.00
Yeast
31.7
18.3
17.4
32.6
0.97
1.05
35.7
1.00
Cow
29.0
21.2
21.2
28.7
1.01
1.00
42.4
1.01
Pig
29.8
20.7
20.7
29.1
1.02
1.00
41.4
1.01
Human
30.4
19.9
19.9
30.1
1.01
1.00
39.8
1.01
Historic X-ray diffraction picture
Building a milestone of life
James Watson
and Francis
Crick proposed a
double helix
model of DNA in
1953.
It symbolized the
new era of
modern biology.
§ 2.2.b Double helix of DNA
• Two DNA strands coil together around the
same axis to form a right-handed double
helix (also called duplex).
• The two strands run in opposite directions,
i.e., antiparallel.
• There are 10 base pairs or 3.4nm per turn
and the diameter of the helix is 2.0nm.
Antiparallel
Backbone and bases
The hydrophilic
backbone is on
the outside of the
duplex, and the
bases lie in the
inner portion of
the duplex.
Base interactions
• The two strands of DNA are stabilized by the
base interactions.
• The bases on one strand are paired with the
complementary bases on another strand
through H-bonds, namely G≡C and A=T.
• The paired bases are nearly planar and
perpendicular to helical axis.
• Two adjacent base pairs have base-stacking
interactions to further enhance the stability of
the duplex.
Watson-Crick base pair
Watson-Crick base pair
Base-stacking interaction
Major and minor grooves
Groove binding
Small molecules like drugs bind in the minor
groove, whereas particular protein motifs can
interact with the major grooves.
§ 2.2.c Polymorphisms of DNA
• DNA can resume different forms
depending upon their chemical
microenvironment, such as ionic strength
and relative humidity.
• B-form DNA is the predominant structure
in the aqueous environment of the cells.
• A-form and Z-form are also native
structures found in biological systems.
Structural features of DNAs
Feature
A-DNA
B-DNA
Z-DNA
Helix rotation
Right-handed
Right-handed
Left-handed
Base pair per turn
11
10
12
Pitch
2.46nm
3.4nm
4.56nm
Helical diameter
2.55nm
2.0nm
1.84nm
Rise per base pair
0.26nm
0.34nm
0.37nm
Glycosyl formation
Anti-
Anti-
Anti- at C,
syn- at G
Rotation between adjacent
base pair
33º
36º
-60º per
dimer
Relative humidity
75%
92%
Triplet DNA
Hoogsteen base pair
The third strand is using Hoogsteen Hbonds to pair with bases on the first strand.
G-quartet DNA
• The telomere of DNA
is a G-righ sequence,
such as
T
TG
T
T
G
5’ (TTGGGG)n 3’
• 4 G residues
constitute a plane
which is stabilized by
Hoogsteen H-bonds.
T
G
T
T
5'
T
G
3'
G
T
T
G-quartet of DNA
Four strands are
arranged in
either parallel or
antiparallel
manner.
§ 2.3 Supercoil Structure
§ 2.3.a Supercoil structure
• The two termini of a linear DNA could be
joined to form a circular DNA.
• The circular DNA is supercoiled, and
supercoil can be either positive or
negative.
• Only the supercoiled DNA demonstrate
biological activities.
EM image of supercoiled DNA
Circular DNAs in nature, in general, are
negatively supercoiled.
§ 2.3.b Prokaryotic DNA
• Most prokaryotic DNAs are supercoiled.
• Different regions have different degrees of
supercoiled structures.
• About 200 nts will have a supercoil on average.
§ 2.3.c 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
• DNA: ~ 200 bps
• Histone: basic
proteins with
many Lys and Arg
residues
– H2A (x2),
– H2B (x2),
– H3 (x2),
– H4 (x2)
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 total length
of 46 human
chromosomes is
about 1.7 m, and
becomes 200 nm
long after 5 times
condensation.
§ 2.4 Functions of DNA
DNA is fundamental to individual life in
terms of
• They are the material basis of life
inheritance, providing the template for
RNA synthesis.
• They are the information basis for
biological actions, carrying the genetic
information.
• DNA is able to replicate itself in a high
fidelity to ensure the genetic information
transfer from one generation to the next.
• DNA can be used as a template to
synthesize RNA (transcription), and RNA
is further used as the template to
synthesize proteins (translation).
• DNA posses the inherent and the mutant
properties to create the diversity and the
uniformity of the biological world.
Gene and genome
• A gene is defined as a DNA segment
that encodes the genetic information
required to produce functional biological
products.
• A gene includes coding regions as well
as non-coding regions.
• Genome is a complete set of genes of a
given species.
Section 3
Structures and Functions
of RNA
Classification
• mRNA (messenger RNA): template for
protein synthesis
• tRNA (transfer RNA): AA carrier
• rRNA (ribosomal RNA): a component of
ribosome for protein synthesis
• hnRNA (heterogeneous nuclear RNA):
precursor of mRNA
• snRNA (small nuclei RNA): small RNAs for
processing and transporting hnRNA
Classes of eukaryotic RNAs
Unique features
• RNA is single stranded, in general.
• RNA has self-complementary intrachain
base paring.
• The double helical regions of RNA are of
the A-form.
• RNA is susceptible to hydrolysis.
§ 3.1 Messenger RNA
mRNA is the template for protein
synthesis, that is, to translate each
genetic codon on mRNA into each AA in
proteins. Each genetic codon is a set of
three continuous nucleotides on mRNA.
• mRNAs constitute 5% of total RNAs.
• mRNAs vary significantly in life spans.
• hnRNA is the precursor of mRNA.
mRNA structure
3'-poly A tail
5'-cap
AUG
UAA
AAA.....AAA
coding region
5' non-coding region
3' non-coding region
mRNA maturation
• hnRNA contains introns and exons.
• Exons are the sequences encoding
proteins, and introns are non-coding
portions.
• Splicing process of hnRNA removes
introns and makes mRNA become
matured.
• The matured mRNA has special structure
features, including 5’-cap and 3’-poly A
tail.
5’-cap
OH OH
H
H
H
H2N
N
HN
N
H 5'
O CH2
N
O
CH3
NH2
N
N
O
O
O
5'
O P O P O P O CH2
N
O N
O
O
O
H3' 2'H
H
H
OCH3
O
-
mRNA chain
5’-cap addition
5’-cap addition
• Methylation can occur at different sites
on G or A.
• 5’-cap can be bound with CBP, benefiting
transporting from nucleus to cytoplasm.
• 5’-cap can be recognized by translation
initiation factor.
• It protects the 5’-end from exonucleases.
Poly A tail
• 20-200 adenine nucleotides at 3’ end
• a un-translated sequence.
• Related with mRNA degradation that
begins with poly A tail shortening.
• Associate with poly A tail binding proteins
for protection
Poly A tailing
hnRNA splicing
intron
exon
hnRNA
mRNA
Matured mRNA of eukaryote
3'-poly A tail
5'-cap
AUG
UAA
AAA.....AAA
coding region
5' non-coding region
3' non-coding region
§ 3.2 Transfer RNA
tRNA serves as an amino acid carrier to
transport AA for protein synthesis.
• tRNA is about 15% of total RNA.
• tRNA is 65-100 nucleotides long.
• There are at least 20 types of tRNA in
one cell.
Structure of tRNA
• The overall structure is a cloveleaf,
reversed L-shape structure.
• There are three loops (DHU loop,
anticodon loop, TψC loop), and four
stems.
• The 3-D structure is stabilized by
hydrogen bonds of local intrachain base
pairs on these stems.
Reversed L-shape structure
Two key sites of tRNA
• A tRNA molecule has an amino acid
attachment site and a template-recognition
site, bridging DNA and protein.
• The template-recognition site is a
sequence of three bases called the
anticodon complementary to the mRNA
codon.
Codon and anticodon
The anticodon
on tRNA pairs
with the codon
on mRNA.
Amino acid attachment
• The OH group at the
3' end of tRNA links
covalently to an
amino acid.
• Only the attached
AA becomes
activated and
capable of being
transported.
Rare Bases
tRNA contains a high portion of unusual
bases.
§ 3.3 Ribosomal RNA
rRNA provides a proper place for protein
synthesis.
• rRNA is the most abundant RNA in cells
(>80%).
• rRNA assembles with numerous
ribosomal proteins to form ribosomes.
Ribosomes
• Ribosomes associate with mRNA to form a
place for protein synthesis.
• Ribosomes of eukaryotes and prokaryotes
are similar in shapes and functions.
Components of ribosomes
Prokaryote
(E.coli)
Eukaryote
(Liver of mouse)
Smaller subunit
rRNA
proteins
30s
16s
21
40s
1542 nucleotides
18s
40% of total weight 33
1874 nucleotides
50% of total weight
Larger subunit
rRNA
50s
23s
5s
proteins
31
60s
2940 nucleotides
28s
120 nucleotides
5.85s
5s
30% of total weight 49
4718 nucleotides
160nucleotides
120nucleotides
35% of total weight
Ribosome of E. coli
23S rRNA
50S large subunit
31 proteins
16S rRNA
70S ribosome
30S small subunit
21 proteins
5S rRNA
Secondary structure of 18S rRNA
The secondary
structure of rRNA
has many loops
and stems, which
can bind ribosomal
proteins to form an
assembly for
protein synthesis.
Ribosomal complex
氨基酸
肽链
N末端
退位
进位
E位
A位
核糖体大亚基
核糖体移动方向
m7GpppG
AAA...AAA
mRNA
核糖体小亚基
P位
Polysomes
核糖体
mRNA
5'末端
3'末端
合成中的多肽
EM of polysomes
Section 4
Physical and Chemical
Properties of Nucleic Acids
General properties
• Acidity
– Negative backbone
• Viscosity
– Concentration and aggregation effects
• 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
Application of OD260
Quantify DNAs or RNAs
OD260=1.0 equals to
50μg/ml dsDNA
40μg/ml ssDNA (or RNA）
20μg/ml oligonucleotide
Determine the purity of nucleic acid samples
pure DNA: OD260/OD280 = 1.8
pure RNA: OD260/OD280 = 2.0
Transition of dsDNA to ssDNA
The absorbance at
260nm of a DNA
solution increases
when a dsDNA is
melted into two
single strands. The
change is called
hyperchromicity.
Melting curve of dsDNA
DNA melting
• Melting curve: a graphic presentation of
the absorbance of dsDNA at 260nm
versus the temperature.
• Melting temperature (Tm): the
temperature at which the UV adsorption
reaches the half of the maximum value,
also means that about 50% of the dsDNA
is disassociated into the single-stranded
DNA.
Melting curve shift
Tm of dsDNA depends on its average G+C
content. The higher the G+C content, the
higher the Tm.
§ 4.2 Thermal stability
• Dissociation of dsDNA into two ssDNAs
is referred to as denaturation.
• Denaturation can be partially and
completely.
• The nature of the denaturation is the
breakage of H-bonds.
• Denaturation is a common and
important process in nature.
Denaturation of DNA
Extremes in pH or
high temperature
Cooperative unwinding
of DNA strands
EM image of denatured DNA
Renaturation of DNA
Two separated complementary DNA strands
can rejoin together to form a double helical
form spontaneously when the temperature
or pH returns to the biological range. This
process is called renaturation or annealing.
§ 4.3 Hybridization
• The ability of DNA to melt and anneal
reversibly is extremely important.
• An association between two different
polynucleotide chains whose base
sequences are complementary is referred
to as hybridization.
• The stability of the hybridized strand
depends on the complementary degree.
Two dsDNA molecules
from different species
are completely
denutured by heating.
When mixed and slowly
cooled, complementary
DNA strands of each
species will associate
and anneal to form
normal duplexes.
• Two ssDNAs, two ssRNAs, as well as one
ssDNA and one ssRNA can also be
hybridized.
• Ionic strength, degree of complementary,
temperature, as well as base composition,
fragment length of nucleic acids will affect
the hybridization.
• It is a common phenomenon in biology,
and has been used as a convenient
techniques in medicine and biology.
Target DNA detection
• complementary hybridization
probe:
target:
…. TAGCTGAG …
…. ATCGACTC …
• mismatched hybridization
probe:
…. TAGCTGAG …
non-target: …. ATCAGCTC …
Applications
• Gene structure and expression
• Microarray or gene chip
• mRNA separation
• Gene diagnosis and therapy
• PCR technique
Section 5
Nuclease
Definition and classification
Nucleases are enzymes that are able to
hydrolyze phosphoester bonds and cleave
DNA or RNA into fragments.
• Deoxyribonuclease (DNase)
- specially cleave DNA
Ribonuclease (RNase)
- specially cleave RNA
Classification
Exonucleases
They can cleave terminal nucleotides either
from 5’-end or from 3’-end, such as
enzymes used in the DNA replication.
Endonucleases
They can cleave internally at either 3’ or 5’
side of a phosphate group, such as the
restriction endonucleases used to construct
the recombinant DNA.
Exonuclease
5’
3’
Endonuclease
Endonuclease
Exonuclease
3’
5’
Applications
• Participate in DNA synthesis and repair,
as well as RNA post-translational
modification
• Digest nucleic acids of food for better
absorption
• Degrade the invaded nucleic acids
• Construct the recombinant DNA