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Transcript
Chapter two
Structures and Functions
of Nucleic Acids
目录
Main contents
Introduction
1. The components of nucleic acids
2. The structures and functions of DNA
3. The structures, and functions of RNA
4. The physicochemical properties of
nucleic acids
5. Nucleases and Ribozymes
6. Genomics and Human Genome Project
Disease cases
Demands
Questions
目录
Nucleic acid
Polynucleotide or polymers of
nucleoties which can carry, transmit
and express the genetic information
目录
1.The discovery of nucleic acids and the
research advance
• 1868 Fridrich Miescher the isolation of nuclein from pus leukocytes
• 1944 Avery the confirmation of DNA as genetic molecules
• 1953 Watson and Crick the discovery of DNA double helix structure
• 1968 Nirenberg the discovery of genetic codons
• 1975 Temin and Baltimore
reverse transcriptase
• 1981 Gilbert and Sanger the method for DNA sequencing (dideoxy
chain terminator sequencing)
• 1985 Mullis the creation of PCR technology
• 1990 human genome project (HGP) start-up in America
• 1994 Chinese human genome project start-up
• 2001 HGP were nearly finished
目录
2. The sorts and distribution of nucleic
acids
Deoxyribonucleic
acid, DNA
more than 90% in nucleus , and other
found outside of nucleus such as
mitochondria, chloroplast, plasmid
Carry genetic information, determine
the genotype of individual or cell
Ribonucleic
acid, RNA
Distributed mainly in cytoplasm, minority
in nucleus
Transmit the genetic information and
gene expression , sometime work as the
carrier of genetic information such as
RNA virus
目录
Section One
The Monomeric Units of Nucleic Acids
Nucleotides
目录
Chemical composition of nucleic acids
1. Element component
C、H、O、N、P(9~10%)
2. Molecular component
—— base:purines,pyrimidines
—— ribose:ribose,deoxyribose
—— phosphate
3. Building units (monomeric units): nucleotides
目录
Chemical composition of nucleic acid
The composition of nucleotide
nucleic acid
base
ribonucleotide
Pentose
ribonucleoside
phosphate
phosphate
base
Pentose
Elementary unit of nucleic acid is nucleotide
DNA: dAMP, dGMP, dCMP, dTMP
RNA: AMP, GMP, CMP, UMP
目录
NH2
Bases
N
N
Purine
N
7
8
9
NH
NH
5
4
6
3
N
1N
2
N
adenine, A
O
N
NH
guanine, G
NH
N
NH2
目录
O
Pyrimidine
5
4
NH
N
3
2
6 1
NH
NH
O
uracil, U
O
NH2
H3 C
NH
N
NH
cytosine, C
O
NH
O
thymine, T
目录
Pentose
HO
CH 2
5´
O
OH
HO
CH 2
OH
O
1´
4´
3´
2´
OH
ribose
(RNA)
OH
OH
deoxyribose
(DNA)
目录
1. The structures of nucleotides
(1) The formation of ribonucleoside
It can be formed by glycoside
bond joined base to ribose
(ribonucleoside) or deoxyribose
HO CH2
(deoxyribonucleoside)
NH2
N
1
O
N
O
1´
ribonucleoside :AR, GR, UR, CR
OH OH
deoxyribonucleoside :dAR, dGR, dTR, dCR
目录
目录
(2) The naming of nucleoside or nucleotides
目录
目录
目录
Some important free nucleotide and
their ramifications in organisms

nucleotides:NMP,NDP,NTP

cyclic nucleotide:

cAMP,cGMP
Biological active substances containing
nucleotide:
NH2
NAD+、NADP+、CoA-SH、FAD
etc, containing
N AMP
NH
NH
NH22 2
N
NN
O
N
NN
O
OO
OO
NN
N
CH
HO PHOO PPHO
OO CH
CH
22O
OO PPP O
NN
2O N
O
OH
OH
OH OH
OH OH
cAMP
ADP
AMP
ATP
NAD+
O
OH
OH OH
OH
OH
OH
O
CH2
P
O
N
O
OHNADP+
OH
N
N
5´end
C
(3)The linkage of
nucleotides
The nucleotides
A
are linked together by
phosphodiester bonds
to form polynucleotides,
namely nucleic acids
G
3´end
目录
5′end
2. Primary Structure
of nucleic acids
C
Definition
The linear sequence of
(deoxy)nucleotides ( or base
sequences ) in DNA (RNA)
is termed primary
structure of DNA (RNA).
Linkage bond
phosphodiester linkage
A
G
3′end
目录
writing
A
5 P
G
P
T
P
G
P
C
P
T
P
OH 3
5 pApCpTpGpCpT-OH 3
5 A C T G C T 3
目录
1.2 Properties of nucleosides and nucleotides
Tautomerism between the lactam and lactim
Absorbance of ultraviolet light at 260 nm
目录
Section Two
Dimensional Structure
and Function of DNA
目录
目录
• The secondary structure of DNA----double helix
structure
– The research background and historic significance
of DNA helix structure
– The key points of DNA double helix structure
• The superhelix structure of DNA and the its
composition in chromatin
– The DNA superhelix structure
– The spatial structure of DNA in prokaryote
– DNA constitution in the nucleus of eukaryote cells
• DNA functions
目录
5′end
1. Primary Structure
of nucleic acids
C
Definition
The linear sequence of
deoxynucleotides ( or base
sequences ) in DNA is
termed primary structure
of DNA.
Linkage bond
phosphodiester linkage
Backbone: ---Pi-dR-Pi-dR---
A
G
3′end
目录
2. Secondary Structure of DNA---Double Helix Model
目录
2.1 Background
The analysis of base composition of DNA
Chargaff rules
The analysis of chemical and physical
data on DNA building model
Primarily x-ray diffraction data
collected by Rosalind franklin and
Maurice Wilkins
目录
Chargaff rules
a. A = T, G = C;
b. The base composition
is different in different
organism species.
c. The base composition
of different organs in
same individual is same.
目录
2.2 The key points of DNA
Double Helix Model
(1) The DNA double helix is
oriented to right-handed
running.
(2) The two DNA strands minor
groove
are organized in an
antiparallel arrangement (i. e.
The two strands run in
major
groove
opposite directions, one
strand is oriented 5’3’ and
the other is oriented 3’ 5’ ).
目录
3’
5’
5’
3’
目录
2.2 The key points of
DNA Double Helix Model
(3) The bases of the
two strands form
hydrogen bonds to
each other; A pairs
with T and G pairs
with C. For each round
of the helix, there are 10
pairs of bases
目录
3’
5’
minor
groove
major
groove
5’
3’
Base pairing rules
A
T
Hydrogen bond
A=T
G≡C
C
G
Hydrogen bond
目录
2.2 The key points of DNA
Double Helix Model
(4) The stable forces are
hydrogen bonds
between base pairs and
base stack force.
(5) There are major
grooves and minor
grooves in DNA double
helix.
3’
5’
minor
groove
major
groove
5’
目录
3’
The summary on DNA double helix
(Watson, Crick, 1953)
(1) right-handed oriented double helix, antiparallel
(2) Backbone outside, bases inside
(3) base-pairing, A=T, G≡C
(4) A running of the helix containing 10 pairs of
bases
hydrogen
bonds,
base
force
(5) Structure
stable
depends
on stack
the vice-bonds
(6) There are minor grooves and major grooves
目录
.
Polymorphism of
secondary structure of DNA
2
4
目录
Forms of DNA
Form
Pitch(nm)
Residues
per turn
inclination of base
pair from horizontal
A
2.8
11
20°
B
3.4
10
0°
Z
4.5
12
7°
11
20°
RNA-DNA
hybrid
2.8
Source: From Davidson, The biochemistry of the Nucleic Acids, 8thed., revised
by Adams, et al. Copyright ©Chapman & Hall, London.
目录
2.5 The Superhelix Structure of DNA
(1)Superhelix structure of DNA in
prokaryote ------Circular double stranded
superhelical DNA
目录
(2)Superhelix structure of DNA in
eukaryote ------Nucleosomes
The nucleosome in eukarytic cells
DNA double helix  superhelix 
nucleosome  chromatin 
chromosome
The nucleosomes in eukarytic cells
consist of DNA and proteins
目录
The elementary unit of chromosome is
nucleosome which consists of DNA
and histone. Histone includes H1, H2A,
H2B, H3, H4. Two molecules of each
H2A, H2B, H3, H4 constitute the core
particle of nucleosome.
目录
Core particles
Linkage DNA
H1, H2A, H2B, H3, H4 histones
目录
目录
10 nm fiber
lengthwise
section
Transv
erse
section
30 nm fiber
300nm solenoid
chromosome
chromatin
目录
The folding of nucleosomes, the chromatin condensation, the formation of chromosome
2.6 Functions of DNA
Genes consist of regulatory region and large
protein-coding segments.
Genome is a whole sequence of DNA in an
organism.
Genetic code: The letters A,G,T and C
correspond to the nucleotides found in DNA.
They are organized into three-letter code
words called codons, and the collection of
these makes up the genetic codes.
目录
DNA functions:
1. Template of replication
2. Template of transcription
3. To accept some mutations
目录
Section Three
Structures and
Functions of RNA
目录
Structure and Function of RNA
目录
1. Structure of RNA
The primary structures of RNA belong to
single stranded linear polynucleotide, but
contain part self-complementary pairing,
namely hairpin structures
Elementary unit of nucleic acid is
nucleotide
RNA: AMP, GMP, CMP, UMP
目录
2. Types and Functions of RNAs
2.1 Messenger RNA
Structure Characters of mRNA:
(1) 5’-cap sequence: m’GpppNm
The cap functions are to boost the
binding of ribosome with mRNA, and
to increase the stability of mRNA.
目录
Cap structure of mRNA 5’-end
Guanine
目录
Structure Characters of mRNA:
(2) 3’-end sequence: poly A
The 3’-end functions are related to
increase the stability of mRNA and the
lifetime worked as template for
translation.
3’ poly A
5’ cap structure
5’ uncoding region
coding region
3’ uncoding region
目录
(3) Mature process of mRNA *
(intron)
(exon)
hnRNA
mRNA
目录
(4) mRNA functions:
As a template of protein synthesis,
it contains triplet codes.
So, mRNA carries the information for the
primary structure of proteins, serves as
template of protein synthesis during
translation.
目录
intron
Eukaryotic
真核细胞cells
Prokaryotic
cells
细胞质
extron
外显子
细胞核
内含子
DNA
转录
Transcript
DNA
Transcript
转录
hnRNA
转录后剪接
Prost-transcript
转运
modification
mRNA
翻译
translation
蛋白
protein
mRNA
protein
蛋白
翻译
translation
目录
2.2 Transfer RNA (tRNA)
(1) Composition of transfer RNA, tRNA
It’s the smallest RNA among RNAs,
only consists of 70~90 nucleotides.
tRNAs contain some unusual (modified)
bases, such as 7-methylguanine,
pseudouridine, dehydrouridine,
目录
O
N
NH
H
H
NH
N
N
CH3
H
H
CH3
N,Ndimethylguanine
HN
N
NH
O
unusual bases
CH2
CH
C
CH3
CH3
NH
NH
O
Dihydrouridine
S
NH
N
N
N6-isopreneadenine
NH
O
4-thiouracil
目录
(2) Functions of transfer RNA, tRNA
Two roles:
Activating amino acids
Recognizing codons in mRNA
The tRNA molecules serve as adapters
for the translation of the information
in the sequence of nucleotides of the
mRNA into specific amino acids.
目录
(3) The structure characters of tRNA
◆Contain
rare bases, such as DHU,
pseudouridine (  ), mG, mC
◆ Stem-loop structure ( local double
strands)
Secondary structure: cloverleaf pattern
◆ Anticodon in the anticodon loop
◆ The base sequence of an anticodon
can reversely complement with codon
on mRNA.
目录
Amino acid arm
TΨC loop
DHU loop
variable loop
The cloverleaf pattern of
tRNA ( secondary structure)
Anticodon
loop
目录
The anticodon at the end of a basepaired stem recognizes the triplet
nucleotide or codon of the template mRNA.
5’
AUG UAC GCA CCA UCG
Met AUG
mRNA
3’
5’
5’
Ala
5’
3’
Tyr
Pro
目录
The cloverleaf
pattern of tRNA
( secondary structure)
目录
The tertiary structure of tRNA
目录
2.3 Ribosomal RNA (rRNA)
(1) rRNA structure
(2) rRNA functions
a. A component of
ribosomes
b. Ribosomes work
as the apparatus of
protein synthesis
目录
(3) rRNA sorts ( dependent on the
sedimentation coefficient, S)
Eukaryotes
Prokaryotes
5S rRNA
28S rRNA
5S rRNA
Large subunit
5.8S rRNA
18S rRNA
23S rRNA
16S rRNA
Large
subunit
small
subunit
small subunit
目录
The components of ribosome
Prokaryote ( E coli.)
Eukaryote (mouse liver)
30S
40S
Small
subunit
rRNA
16S
1542 nucleotides
18S
1874 nucleotides
proteins
21
Occupy 40% of
total weight
33
Occupy 50% of
total weight
Large
subunit
50S
60S
rRNA
23S
5S
2940 nucleotides
120 nucleotides
28S
5.85S
5S
4718nucleotides
160nucleotides
120nucleotides
proteins
31
Occupy 30% of
total weight
49
Occupy 35% of
total weight
目录
2.4 Other Small Stable RNA
A large number of discrete, highly
conserved, and small stable RNA
species are found in eukaryotic cells.
The majority of these molecules exist
as ribonucleoproteins and are
distributed in the nucleus, in the
cytoplasm, or in both.
目录
The major sorts of RNA and their functions
In the nucleus or
in the cytoplasm
Ribosomal RNA
Messenger RNA
Transfer RNA
rRNA
mRNA
tRNA
mitochondrion
mt rRNA
mt mRNA
mt tRNA
heterogeneous
nuclear RNA
hnRNA
Small nuclear RNA snRNA
Small nucleolus RNA SnoRNA
Small cytoplast RNA scRNA/7SL-RNA
functions
the components of ribosomes
the template for translation
activating AA and recognizing
codons on mRNA
the precursor of mRNA
related to the splicing and
transfering of hnRNA
related to the processing and
modifying of rRNA
the components of signal
discriminator for proteins
located in endoplasmic reticulum
目录
Small nuclear RNAs are significantly
involved in mRNA processing and
gene regulation. Of the several
snRNAs, U1, U2, U44, U5, and U6 are
involved in intron removal and the
processing of hnRNA into mRNA.
hnRNA ( heterogeneous nuclear RNA )
snRNA ( small nuclear RNA)
目录
Section Four
The Properties of Nucleic Acid
目录
1. The general properties of nucleic
acids
1.1
1.2
1.3
1.4
Acidic molecules
Macromolecules
High viscosity (DNA)
Ultraviolet absorption ( 260 nm)
目录
Adenine
Extinction coefficient
Uracil
Cytosine
Guanine
Thymine
Wavelength
Ultraviolet absorbent spectrum of various bases (pH 7.0)
目录
2. DNA denaturation
Definition
Double helix of DNA
Single
strand
The denaturation factors
heating, chemical reagents, ultraviolet
light
目录
The essence of DNA denaturation is the
breaking of hydrogen bonds between the
double strands of DNA
heating
Cooling slowly
Natural DNA
Denatural DNA
目录
The changes of DNA properties after
denaturation
* Hydrogen linkage broken
* A260  ( hyperchromic effect )
* Double helix  single strand
* Tm ( melting temperature )
* Tm and G + C
Tm = 69.3 + 0.41 ( % G+ C )
< 20 bp, Tm = 4 ( G + C ) + 2 ( A + T )
目录
The application of OD260
(1) The determination of the amount of DNA or RNA
OD260=1.0
is corresponded to
50μg/ml double stranded DNA
40μg/ml single stranded DNA(or RNA)
20μg/ml oligonucleotides
(2) To estimate the purification of nucleic acid
samples
Purified DNA : OD260/OD280 = 1.8
Purified RNA: OD260/OD280 = 2.0
目录
For example: the change of ultraviolet absorption
spectrum of DNA induced by denaturation
Absorbent value
Denatured state
Natural state
Wave length
The ultraviolet absorption spectrum of DNA

Hyperchromic effect : the phenomenon of OD260 increase
companied by DNA denaturation in solution
目录
Heated denaturation of DNA
Melting curve: a
graph in which
absorbance versus
temerature
is
plotted
Temperature-optical density profile for DNA
目录
 Tm:
Tm is the
temperature at which
50% of DNA in
solution are denatured
by heated , or called
melting temperature,
Tm. The value of Tm
is related to the
contents of G and C.
目录
3. The renaturation of DNA and
nucleic acid hybridization
(1) Renaturation
Single strand of DNA  double helix
( annealing )
The best annealing temperature:
25℃ < Tm
目录
Renaturation process
denaturation
Various source
DNA
renaturation
Hybridization
double stranded
molecules of
DNA-DNA
目录
(2) Nucleic Acid Hybridization
Definition
The process of forming a double stranded
structure from two polynucleotide strands
( either DNA or RNA) from different source is
termed hybridization
Hybridization Principle
( To see the above slice or next slice)
目录
Double stranded DNA 1
Heterogeneous double
stranded DNA
single stranded DNA
heating
cooling
Double stranded DNA 2
slowly
Double stranded DNA
single stranded DNA
heating
Adding marked
single stranded
DNA probes
Forming the marked
heterogeneous
stranded DNA
Cooling slowly
The denaturation and renaturation of DNA, nucleic acid hybridization
目录
(3) The application of Hybridization technology
★ Determining whether a certain sequence occurs
more than once in the DNA of a particular organism
★ Demonstrating a genetic or evolutionary relationship
between different organisms
★ Determining the number of genes transcribed in a
particular mRNA
★ Determining the location of any given DNA
sequence by annealing with a complementary
polynucleotide probe
目录
Section 5
Nucleases
catalytic RNA and DNA
目录
1. Nucleases
(1) Definition
Nucleases are the enzymes capable of degrading
nucleic acids.
(2) Catalogue
DNase Those which exhibit specificity for
deoxyribonucleic acid are referred to as
deoxyribonucleases
RNase Those which specifically hydrolyze
ribonucleic acids are ribonucleaes.
Endonuclease
Exonuclease
目录
Classification according to the substrates
•deoxyribonuclease, DNase
Specially degrade DNA
•ribonuclease, RNase
Specially degrade RNA。
–Classification according to the cut sites
Endonuclease
restriction endonuclease (RE) and nonRE
Exonuclease
5’-3’ exonuclease or 3’-5’ exonuclease
目录
2. Ribozymes
 Ribozymes are RNA molecules with
catalytic activity
 There are five classes of ribozymes
★ Three classes carry out self-processing
reactions.
★ Two classes ( RNase P and rRNA )are
true catalysts that act on separate
substrates (containing proteins)
目录
3. Deoxyribozymes
 Some single-stranded DNA molecules are
capable of adopting intricate tertiary
structures and performing efficient catalysis.
 These special DNA sequences with catalytic
capability are called deoxyribozymes.
 At present, deoxyribozymes are only found in
laboratories.
目录
Section 6
Genomics and
the Human Genome Project
目录
1. Characteristics of Eukaryotic Genome
DNA
 Genomes in eukaryotes are much larger
than in prokaryotes
 Only a few percent ( 2-4%) of DNA in a
mammalian cell may suffice for all genes.
 Eukaryotes genes do not overlap but are
spaced, on the average, 40 000 bp apart.
 There are split genes that consist of exons
and introns in eukaryote genomes.
目录
2. The human Genome Project
 1990, the project started
 An international program
 Make a sequence map for the total 3 billion
bp of human genome
 Resource for the investigation of hereditary
diseases as well as normal gene structure
and expression
目录
Disease Cases
1. Hemoglobin diseases (血红蛋白病)
2. Horsebean disease (蚕豆病)
3. X-Fragile Syndrome (X-脆性综合征)
目录
Demands
♣Types of nucleic acids and their
functions
♣ Basic units for nucleic acid composition
♣ The differences between DNA and RNA
♣ Secondary structure characters of DNA
♣ Structure characters of mRNA, tRNA,
rRNA
♣ Physicochemical properties of DNA, RNA
目录
Questions:
1. Describe the sorts, functions of nucleic
acids in nature. What are the
characteristics of their chemical
components or structures respectively?
2. What are the double helix of DNA?
How to prove the structural characters
of DNA double helix?
3. Why is it said that DNA are the
carriers of genetic substances ? How to
demonstrate it ?
目录
4. Which important physical and
chemical properties of nucleic acids are
there that can be utilized ?
5. Illustrate those important free
mononucleotides existing in organisms
6. Do you know how to compare
DNA with RNA?
7. What are the differences between
nucleases and ribozymes?
目录
Friedrich Miescher (1844-1895)
Friedrich Miescher worked at the Physiological Laboratory of the
University of Basel and in Tübingen and is most well known for his
discovery of the nucleic acids.
目录
Friedrich Miescher(1844-1895)
From an early age Friedrich was recognized as being highly
intelligent, but he was shy and introspective - perhaps in part as
the result of a serious hearing impairment he had suffered from
since boyhood. Despite this handicap he took great interest in
music.
Miescher had originally intented to study lymphocytes, he
collected laudable pus from used bandages in the nearby
hospital. He obtained a precipitate from the cell nuclei solution.
Obviously this material must have come from the nucleus, and
he therefore named it nuclein. Using elementary analysis, one of
the few methods available to characterize an unknown
compound. He extracted high-molecular-weight DNA from
damaged cells eventually.
目录
Oswald T. Avery (1877-1955). Courtesy of The
Rockefeller University Archives.
Reichard, P. J. Biol. Chem.
2002;277:13355-13362
目录
O.T. Oswald Theodore Avery
(1877~1955)
Oswald Theodore Avery (1877-1955) was a distinguished
bacteriologist and research physician and one of the founders of
immunochemistry. He is best known, however, as a discoverer that
deoxyribonucleic acid (DNA) serves as genetic material. The Oswald
T. Avery Collection is a part of the Joshua Lederberg Papers, which
are at the National Library of Medicine and available digitally. The
collection was assembled by Nobel laureate Dr. Lederberg because of
the strong connection between Dr. Avery's work and his own. The
work of Avery and his lab, observes Dr. Lederberg, was "the
historical platform of modern DNA research" and "betokened the
molecular revolution in genetics and biomedical science generally.
目录
Rosalind Franklin
-
(
19
20
19
58
)
目录
Rosalind Franklin(1920-1958)
Born in July of 1920, Rosalind Franklin graduated from Cambridge
University and in 1951 went to work as a research associate for John Randall
at King's College. A chemist by training, Franklin had made original and
essential contributions to the understanding of the structure of graphite and
other carbon compounds. It was Franklin alone whom Randall had given the
task of elucidating DNA's structure.
The technique with which Rosalind Franklin set out to do this is called Xray crystallography. With this technique, the locations of atoms in any crystal
can be precisely mapped by looking at the image of the crystal under an X-ray
beam. By the early 1950s, scientists were just learning how to use this
technique to study biological molecules. Rosalind Franklin applied her
chemist's expertise to the unwieldy DNA molecule. After complicated analysis,
she discovered (and was the first to state) that the sugar-phosphate backbone
of DNA lies on the outside of the molecule. She also elucidated the basic
helical structure of the molecule.
目录
More advanced reading:
• Nelson, D. L., and Cox, M. M. (2000)
Lehninger Principles of Biochemistry,
third edition, Worth Publishers.
• Berg, J. M., Tymoczko, J. L., and Stryer,
L. (2002) Biochemistry, Fifth edition, W.
H. Freeman and Company, New York.
• Mathews, C. K., van Holde, K. E., and
Ahern, K. G. (2000) Biochemistry, third
edition, Benjamin/Cummings
(www.aw-bc.com/mathews/).
目录