Chapter 3 Nucleic Acids
Section 2 Structure of Nucleic Acids
Outline
12.1 Primary Structure of Nucleic Acids
12.2 ABZs of DNA Secondary Structure
12.3 Denaturation and Renaturation of DNA
12.4 Tertiary Structure of DNA
12.5 Chromosome Structure
12.7 Secondary and Tertiary Structure of
RNA
Primary Structure
Sequencing Nucleic Acids 核酸序列测定
Chain termination method (dideoxy method),
developed by F,Sanger
Base-specific chemical cleavage,developed by
Maxam and Gilbert
Both use autoradiography - X-ray film
develops in response to presence of radioactive
isotopes in nucleic acid molecules
Chain Termination Method
Based on DNA polymerase 聚合酶 reaction
Run four separate reactions
Each reaction mixture contains dATP,
dGTP,dCTP and dTTP,one of which is
P-32-labelled
Each reaction also contains a small
amount of one dideoxynucleotide,either
ddATP,ddGTP,ddCTP or ddTTP
5’
3’ |||||||||||||||
ddATP
DNA
dNTPs
ddTTP
DNA
dNTPs
ddGTP
DNA
dNTPs
ddCTP
DNA
dNTPs
A T C A T G T C A T C A A G T C T A G C A C
T A
T A G T A
T A G T A C A
T A G T A C A G T A
T A G T A C A G T A G T T C A
T A G T A C A G T A G T T C A G A
Each fragment is
terminated by a ddA
Chemical Cleavage Method
Not used as frequently as Sanger's
Start with ssDNA labelled with P-32 at one end
Strand is cleaved by chemical reagents
Assumption is that strands of all possible lengths,
each cleaved at just one of the occurrences of a
given base,will be produced,
Fragments are electrophoresed and sequence is
read
Chemical Cleavage Method
Four reactions are used
G-specific cleavage with dimethyl sulfate,
followed by strand scission with piperidine
G/A cleavage,depurination with mild acid,
followed by piperidine
C/T cleavage,ring hydrolysis by hydrazine,
followed by piperidine
C cleavage,same method (hydrazine and
piperidine),but high salt protects T residues
32pGCTACGTA
G G+A T+C C
specific chemical cleavage
The ABZs of DNA
Secondary Structure
See Figure 12.10 for details of DNA
secondary structure
Sugar-phosphate backbone outside
Bases (hydrogen-bonded) inside
Right-twist closes the gaps between base
pairs to 3.4 A (0.34 nm) in B-DNA
The,canonical规范的,base pairs
See Figure 12.10
The canonical A:T and G:C base pairs have
nearly identical overall dimensions 统一外形尺寸
A and T share two H-bonds
G and C share three H-bonds
G:C-rich regions of DNA are more stable
Polar atoms in the sugar-phosphate
backbone also form H-bonds
Comparison of A,B,Z DNA
See Table 12.1
A,right-handed,short and broad,2.3 A,11
bp per turn
B,right-handed,longer,thinner,3.32 A,10
bp per turn
Z,left-handed,longest,thinnest,3.8 A,12
bp per turn,Found in G:C-rich regions of DNA
See Figure 12.13
75% 92% d(CGCGCG)
溴化乙啶吖啶橙放线菌素 D
12.3 Denaturation of DNA
See Figure 12.17
When DNA is heated to 80℃ its UV
absorbance increases by 30-40%
This hyperchromic shift reflects the unwinding
of the DNA double helix
Stacked base pairs in native DNA absorb less
light
When T is lowered,the absorbance drops,
reflecting the re-establishment of stacking
Denaturation of DNA
Double-stranded DNA
A-T rich regions
denature first
Cooperative unwinding
of the DNA strands
Strand separation
and formation of
single-stranded
random coils
Extremes in pH or
high temperature
hyperchromicity (Hyperchromic effect)
hypochromic effect
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.
260
Ab
so
rba
nc
e
Single-stranded
Double-stranded
220 300
melting curves and Tm
完整 helix
Helix解旋转换中点
midpoint of
the transition
Melting temperature,Tm
Electron micrograph of partially melted DNA
A-T rich regions melt first,followed by G-C rich regions
Double-stranded,G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
Renaturation
Reassociation of DNA
DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
Slower,rate-limiting,
second-order process of finding
complementary sequences
to nucleate base-pairing
Faster,
zippering
reaction to
form long
molecules
of double-
stranded
DNA
Complexity of chromosomal DNA
>>>>>>>>>>>>>>
110,000
Hybridization
Nucleic Acids from Different Species
Can Form Hybrids
12.4 Supercoils and Cruciforms十字形
In duplex DNA,10 bp per turn of helix
Circular DNA sometimes has more or less than
10 bp per turn - a supercoiled state
Enzymes called topoisomerases 拓扑异构酶 or
gyrases 旋转酶 can introduce or remove supercoils
Cruciforms occur in palindromic 回文 regions of
DNA
Negative supercoiling may promote cruciforms
Palindrome 回文
Chromosome Structure染色体结构
Human DNA’s total length is ~2 meters!
This must be packaged into a nucleus that is
about 5 μmeters in diameter
This represents a compression of more than
100,000!
It is made possible by wrapping the DNA
around protein spools called nucleosomes核小体
and then packing these in helical filaments
Nucleosome Structure
Chromatin染色质,the nucleoprotein complex,
consists of histones组蛋白 and nonhistone
chromosomal proteins
12.7 Sec/Tert Structure of RNA
Transfer RNA
Extensive H-bonding creates four
double helical domains,three loops,
four stems
Phenylalanine tRNA is "L-shaped"
Many non-canonical（ 稀有） base
pairs found in tRNA
Tertiary structureSecondary structure
Ribosomal RNA
Ribosomes synthesize proteins
All ribosomes contain large and small
subunits
rRNA molecules make up about 2/3 of
ribosome
High intrastrand sequence complementarity
leads to extensive base-pairing
Ribosomal RNA
Secondary structure features seem to be
conserved（ 保守，恒定）,whereas sequence is
not
There must be common designs and
functions that must be conserved
Section 2 Structure of Nucleic Acids
Outline
12.1 Primary Structure of Nucleic Acids
12.2 ABZs of DNA Secondary Structure
12.3 Denaturation and Renaturation of DNA
12.4 Tertiary Structure of DNA
12.5 Chromosome Structure
12.7 Secondary and Tertiary Structure of
RNA
Primary Structure
Sequencing Nucleic Acids 核酸序列测定
Chain termination method (dideoxy method),
developed by F,Sanger
Base-specific chemical cleavage,developed by
Maxam and Gilbert
Both use autoradiography - X-ray film
develops in response to presence of radioactive
isotopes in nucleic acid molecules
Chain Termination Method
Based on DNA polymerase 聚合酶 reaction
Run four separate reactions
Each reaction mixture contains dATP,
dGTP,dCTP and dTTP,one of which is
P-32-labelled
Each reaction also contains a small
amount of one dideoxynucleotide,either
ddATP,ddGTP,ddCTP or ddTTP
5’
3’ |||||||||||||||
ddATP
DNA
dNTPs
ddTTP
DNA
dNTPs
ddGTP
DNA
dNTPs
ddCTP
DNA
dNTPs
A T C A T G T C A T C A A G T C T A G C A C
T A
T A G T A
T A G T A C A
T A G T A C A G T A
T A G T A C A G T A G T T C A
T A G T A C A G T A G T T C A G A
Each fragment is
terminated by a ddA
Chemical Cleavage Method
Not used as frequently as Sanger's
Start with ssDNA labelled with P-32 at one end
Strand is cleaved by chemical reagents
Assumption is that strands of all possible lengths,
each cleaved at just one of the occurrences of a
given base,will be produced,
Fragments are electrophoresed and sequence is
read
Chemical Cleavage Method
Four reactions are used
G-specific cleavage with dimethyl sulfate,
followed by strand scission with piperidine
G/A cleavage,depurination with mild acid,
followed by piperidine
C/T cleavage,ring hydrolysis by hydrazine,
followed by piperidine
C cleavage,same method (hydrazine and
piperidine),but high salt protects T residues
32pGCTACGTA
G G+A T+C C
specific chemical cleavage
The ABZs of DNA
Secondary Structure
See Figure 12.10 for details of DNA
secondary structure
Sugar-phosphate backbone outside
Bases (hydrogen-bonded) inside
Right-twist closes the gaps between base
pairs to 3.4 A (0.34 nm) in B-DNA
The,canonical规范的,base pairs
See Figure 12.10
The canonical A:T and G:C base pairs have
nearly identical overall dimensions 统一外形尺寸
A and T share two H-bonds
G and C share three H-bonds
G:C-rich regions of DNA are more stable
Polar atoms in the sugar-phosphate
backbone also form H-bonds
Comparison of A,B,Z DNA
See Table 12.1
A,right-handed,short and broad,2.3 A,11
bp per turn
B,right-handed,longer,thinner,3.32 A,10
bp per turn
Z,left-handed,longest,thinnest,3.8 A,12
bp per turn,Found in G:C-rich regions of DNA
See Figure 12.13
75% 92% d(CGCGCG)
溴化乙啶吖啶橙放线菌素 D
12.3 Denaturation of DNA
See Figure 12.17
When DNA is heated to 80℃ its UV
absorbance increases by 30-40%
This hyperchromic shift reflects the unwinding
of the DNA double helix
Stacked base pairs in native DNA absorb less
light
When T is lowered,the absorbance drops,
reflecting the re-establishment of stacking
Denaturation of DNA
Double-stranded DNA
A-T rich regions
denature first
Cooperative unwinding
of the DNA strands
Strand separation
and formation of
single-stranded
random coils
Extremes in pH or
high temperature
hyperchromicity (Hyperchromic effect)
hypochromic effect
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.
260
Ab
so
rba
nc
e
Single-stranded
Double-stranded
220 300
melting curves and Tm
完整 helix
Helix解旋转换中点
midpoint of
the transition
Melting temperature,Tm
Electron micrograph of partially melted DNA
A-T rich regions melt first,followed by G-C rich regions
Double-stranded,G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
Renaturation
Reassociation of DNA
DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
Slower,rate-limiting,
second-order process of finding
complementary sequences
to nucleate base-pairing
Faster,
zippering
reaction to
form long
molecules
of double-
stranded
DNA
Complexity of chromosomal DNA
>>>>>>>>>>>>>>
110,000
Hybridization
Nucleic Acids from Different Species
Can Form Hybrids
12.4 Supercoils and Cruciforms十字形
In duplex DNA,10 bp per turn of helix
Circular DNA sometimes has more or less than
10 bp per turn - a supercoiled state
Enzymes called topoisomerases 拓扑异构酶 or
gyrases 旋转酶 can introduce or remove supercoils
Cruciforms occur in palindromic 回文 regions of
DNA
Negative supercoiling may promote cruciforms
Palindrome 回文
Chromosome Structure染色体结构
Human DNA’s total length is ~2 meters!
This must be packaged into a nucleus that is
about 5 μmeters in diameter
This represents a compression of more than
100,000!
It is made possible by wrapping the DNA
around protein spools called nucleosomes核小体
and then packing these in helical filaments
Nucleosome Structure
Chromatin染色质,the nucleoprotein complex,
consists of histones组蛋白 and nonhistone
chromosomal proteins
12.7 Sec/Tert Structure of RNA
Transfer RNA
Extensive H-bonding creates four
double helical domains,three loops,
four stems
Phenylalanine tRNA is "L-shaped"
Many non-canonical（ 稀有） base
pairs found in tRNA
Tertiary structureSecondary structure
Ribosomal RNA
Ribosomes synthesize proteins
All ribosomes contain large and small
subunits
rRNA molecules make up about 2/3 of
ribosome
High intrastrand sequence complementarity
leads to extensive base-pairing
Ribosomal RNA
Secondary structure features seem to be
conserved（ 保守，恒定）,whereas sequence is
not
There must be common designs and
functions that must be conserved
