1. Chapter 7 Structure of Nucleic Acids
"Scherzo in D & E" (detail) by David E. Rodale ( )

2. 7.1 · The Primary Structure of Nucleic Acids

3. Structure and Nomenclature of Nucleic Acids
Shorthand notation
of a nucleic acid 5’ P T A G C OH 5’ 3’
Two major classes of
nucleic acids:
DNA and RNA 3’

4. Sequencing Nucleic Acids
1 chain termination or dideoxy
method
base-specific chemical cleavage

5. DNA sequencing
The Sanger DNA sequencing method uses dideoxy nucleotidcs to terminate DNA synthesis, yielding a series of DNA fragments whose sizes can be measured by electrophoresis. The last base in each of these fragments is known, because we know which dideoxy nucleotide was used to terminate each reaction. Therefore, ordering these fragments by size--each fragment one (known) base longer than the next--tells us the base sequence of the DNA.

11. DNA sequencing can
be automated

13. Maxam-Gilbert Sequencing
The Maxam-Gilbert sequencing method follows a very different strategy. Instead of synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators, this method starts with full length, end-labeled DNA and cleaves it with base-specific reagents

15. TO G
We use dimethyl sulfate (DMS) to methylate guanines. we do the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand.
Next, we use a reagent (piperidine，哌啶) that does two things: It causes loss of the mcthylated base, then it breaks the DNA backbone at the site of the lost base (the apurinic site).

17. TO A+G
we can weaken the glycosidic bonds to both adenine and guanine with acid; then piperidine will cause depurination and strand breakage after both A's and G's.

18. TO C+T
Similarly, hydrazine(肼）opens both thymine and cytosine rings, and piperidine can then remove these bases and break the DNA strand at the resulting apyrlmidinic sites.
TO C
In the presence of 1 M NaC1, hydrazine is specific for cytosine only, so we can run this reaction next to the C + T reaction and obtain the T's by comparison.

20. Maxam-Gilbert sequencing is used only in special cases these days, but the methods of modifying and then breaking DNA used by Maxam and Gilbert are still useful for other purposes

21. 7.2 · The ABZs of DNA Secondary Structure
Structure Overview of Nucleic Acids
Unlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent on their sequences. There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries. The double helical structure is essential to the coding functional of DNA. Watson (biologist) and Crick (physicist) first discovered double helix structure in 1953 by X-ray crystallography.
RNA, on the other, can have as diverse structures as proteins, although they can also hand form double helix of type A. The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA World Hypothesis). In fact, some virus encode their genetic materials by RNA (retrovirus)

22. Structure of DNA
DNA is a double helix

25. Structural Equivalence of Watson–Crick Base Pairs ;
the A:T pair and G:C pair have virtually identical dimensions.

29. The DNA Double Helix Is a Stable Structure
Forces That Stabilize Nucleic Acid Double Helix
Hydrogen bonding in base-pairing
Hydrophobic interactions in base stacking
Electrostatical Interaction with cations in solution such as Mg2+. 5’ 3’
Same strand stacking
cross-strand stacking

30. Types of DNA Double Helix
Type A: major conformation of RNA, minor conformation of DNA;
Type B: major conformation of DNA;
Type Z: minor conformation of DNA 5’ 3’ 5’ 5’ 3’ 3’ Z A B 3’ 5’
Narrow
tight
Wide
Less tight
Left-handed
Least tight

31. Types of DNA Double Helix
A-DNA
A-RNA
Minor Groove
Major Groove

33. Comparison of the Structural Properties of A-, B-, and Z-DNA

34. Edges of Base-paired Nucleotides
Edges of basepairs have specific relationships to grooves;
Major groove edges are sequence specific thus provide sequence recognition sites; Many DNA-binding proteins bind to major grooves of DNA in gene transcription and regulation;

35. Intercalating Agents Distort the Double Helix
Several hydrophobic
molecules containing
flat aromatic and fused
heterocyclic rings can
insert between the
stacked base pairs
of DNA. These
molecules are called
intercalating agents.
Intercalating agents
are potential
Cancer-inducing
reagents.

36. 7.3 Denaturation and Renaturation of DNA
When duplex DNA molecules are subjected to conditions of pH, temperature or ionic strength that disrupt hydrogen bonds, the strands are no longer held together. The double helix is denatured.
If the temperature is the denaturing agent, the double helix is said to melt;
The phenomenon that the relative absorbance of the DNA solution at 260 nm increases as the bases unstack is called hyperchromic shift;
If one fellows the absorbance as a function of temperature, the midpoint temperature of the absorbance curve is termed melting temperature, Tm.

37. Denaturation and Renaturation of DNA
DNA of different sequences
have different Tm.
Tm is higher for DNA
that contain more GC
pairs; Tm is also directly
proportional to the
ionic strength of the
solution.
(salts can shield repulsions
of negatively charged
phosphate groups) Tm

38. Structural Changes in DNA Melting

39. pH Extremes or Strong H-Bonding Solutes Also Denature DNA Duplexes At pH values greater than 10, extensive deprotonation of the bases occurs, destroying their hydrogen bonding potential and denaturing the DNA duplex. Similarly, extensive protonation of the bases below pH 2.3 disrupts base pairing. Alkali is the preferred denaturant because, unlike acid, it does not hydrolyze the glycosidic linkages in the sugar–phosphate backbone. Small solutes that readily form H bonds are also DNA denaturants at temperatures below Tm if present in sufficiently high concentrations to compete effectively with the H-bonding between the base pairs. Examples include formamide and urea.

40. Renaturation (or Annealing)
Renaturation refers to the process of DNA strands associate into a double helix;
Renaturation can be analyzed quantitatively: C is the amount of single stranded DNA remaining, C0 is the initial single stranded DNA.
C0t Plot
t1/2

41. Nucleic Acid Hybridization
If DNA from two different species are mixed, denatured, and allowed to cool slowly so that reannealing can occur, artificial hybrid duplexes may form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other.

43. Nucleic acid hybridization is a commonly employed procedure in molecular biology. First, it can reveal evolutionary relationships. Second, it gives researchers the power to identify specific genes selectively against a vast background of irrelevant genetic material. An appropriately labeled oligo- or polynucleotide, referred to as a probe, is constructed so that its sequence is complementary to a target gene. The probe specifically base pairs with the target gene, allowing identification and subsequent isolation of the gene. Also, the quantitative expression of genes (in terms of the amount of mRNA synthesized) can be assayed by hybridization experiments.

44. Buoyant Density of DNA
Not only the melting temperature of DNA but also its density in solution is dependent on relative G:C content.
G:C-rich DNA has a significantly higher density than A:T-rich DNA. Single-stranded DNA is denser than double-helical DNA

46. Cesium chloride centrifugation is an excellent means of removing RNA and proteins in the purification of DNA.
Because of its relatively high density, DNA can be purified from cellular material by a form of density gradient centrifugation known as isopycnic centrifugation.

47. 7.4 · Supercoils and Cruciforms: Tertiary Structure in DNA
Many DNA molecules are circular (e.g., bacterial chromosomes, all plasmid DNA). Circular DNA can form supercoils.
Human chromosome contains 3x109 basepairs and are wrapped around proteins to form nucleosomes. Nucleosomes are packed tightly to form helical filament, a structure called chromotin.

48. Supercoiling means the coiling of coiling
DNA Supercoiling
Supercoiling means the coiling of coiling

49. Supercoiling
of DNA
Relaxed
state

50. Supercoiling induced by separating the strands of a helical structure(during replication or transcription)

51. Supercoils: Tertial Structure in DNA
Supercoils refer to the DNA structure in which double-stranded circular DNA twists around each other. Supercoiled DNA contrasts relaxed DNA;
In DNA replication, the two strands of DNA have to be separated, which leads either to overwinding of surrounding regions of DNA or to supercoiling;
A specialized set of enzymes (gyrase, topoisomerases) is present to introduce supercoils that favor strand separation;
The degree of supercoils can be quantitatively described.

52. DNA from a lysed E..coli cell

53. Relaxed and supercoiled plasmid DNAs
Relaxed and supercoiled plasmid DNAs. The degree of supercoiling increases from left to right.

54. Varieties of Supercoiled DNA
Note that all black
lines represents double
stranded DNA

55. DNA underwinding is defined by topological linking number(Lk, or L).

56. The Linking Number (L) of DNA
The linking number of DNA, a topological property, determines the degree of supercoiling;
The linking number defines the number of times a strand of DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie in a plane;
If both strands are covalently intact, the linking number cannot change;
For instance, in a circular DNA of 5400 basepairs, the linking number is 5400/10=540, where 10 is the basepair per turn for type B DNA.

57. The Twist (T) and The Writhe of DNA
Twist is a measure of the helical winding of the DNA strands around each other. Given that DNA prefers to form B-type helix, the preferred twist = number of basepair/10;
Writhe is a measure of the coiling of the axis of the double helix. A right-handed coil is assigned a negative number (negative supercoiling) and a left-handed coil is assigned a positive number (positive supercoiling).
Topology theory tells us that the sum of T (or Tw)and W (or Wr)equals to linking number:
L=T+W
For example, in the circular DNA of 5400 basepairs, the linking number is 5400/10=540
If no supercoiling, then W=0, T=L=540;
If positive supercoiling, W=+20, T=L-W=520;

60. An Example of L, T, and W Calculation
A relaxed circular, double stranded DNA (1600 bps) is in a solution where conditions favor 10 bps per turn. What are the L, T, and W?
During replication, part of this DNA unwinds (200 bps) while the rest of the DNA still favor 10 bps per turn. What are the new L, T, and W?
1400 bps
1600 bps
200 bps
L=160
T=( )/10=140
W=L-T=+20
L=1600/10=160
W=0 (relaxed)
T=L-W =160

61. Wr=-2
Wr=2
Tw=200

62. DNA Gyrase The bacterial enzyme DNA gyrase is a topoisomerase that introduces negative supercoils into DNA

64. Mechanism of topoisomerase II (DNA gyrase)

65. Topoisomerases catalyze changes in the linking number of DNA
Topoisomerase I treats in different length of time

66. Superhelix Density
The superhelix density or specific linking difference is defined as L/L0 and is sometimes termed sigma, s . For our example, s = -4/40, or As a ratio, s is a measure of supercoiling that is independent of length. Its sign reflects whether the supercoiling tends to unwind (negative s ) or overwind (positive s ) the helix.

67. Toroidal Supercoiled DNA
Negatively supercoiled DNA can arrange into a toroidal state. The toroidal state of negatively supercoiled DNA is stabilized by wrapping around proteins which serve as spools for the DNA “ribbon.” This toroidal conformation of DNA is found in protein: DNA interactions that are the basis of phenomena as diverse as chromosome structure and gene expression.

68. The toroidal state of negatively supercoiled DNA is stabilized by wrapping around proteins which serve as spools for the DNA “ribbon.”

69. palindrome is a word, phrase,or sentence that is spelled identically reading forward or backward; two examples are ROTATOR and NURSES RUN..

70. Such sequence are self-complementary within each strand and therefore have the potential to form hairpin or cruciform (cross-shaped) structures

71. Cruciforms

72. DNA compaction requires a special form of supercoiling There are two types of supercoiling: plectonemic (from the Greek plektoe, “twisted”, and nema, “thread”) and solenoidal.

73. Plectonemic Supercoiling An electron micrograph of
plectonemically supercoiled
plasmid DNA

74. (b) An interpretation of the observed structure
(b) An interpretation of the observed structure. The purple lines show the axis of the supercoil.

75. (c) An indealized representation of this structure

76. plectonemic and solenoidal supercoiling.

77. 7.5 · Chromosome Structure
Human
chromosomes

78. Chromatin and nucleoid structure
nucleosomes

79. Nucleosomes
Nucleosomes look like “beads on a string” under microscope. The beads contain a pair of four histone proteins, H2A, H2B, H3, and H4 (octamer). The string is double stranded DNA;
The surface of the octamer contain features that guide the course of DNA such that DNA can wrap 1.65 turns around in a left-handed conformation. H1 proteins serves to seal the ends of the DNA and connects consecutive nucleosomes.
nucleosomes

82. Histones are small, basic proteins

83. Each of histones has variant forms because certain amino acid side chains are enzymatically modified by methylation, ADP-ribosylation, phosphorylation, or acetylation. Such modifications affact the net electric charge, shape, and other properties of histones, as well as the structural and functional properties of chromatin, and they play a role in the regulation of transcription.

84. Nucleosome are the fundamental organizational units of chromatin

90. Chromatin assembly

92. The 30 nm fiber, a higher-order organization of nucleosomes

95. A partially unraveled human chromosome, revealing numerous loops of DNA attached to a scaffoldlike structure.

96. Loops of chromosomal DNA attached to a nuclear scaffold

97. Organization
of Chromotin
and Chromosomes:

98. 7.6 · Chemical Synthesis of Nucleic Acids

99. DNA synthesis
DMT::二甲氧三苯甲基

100. DNA synthesis
DMT::二甲氧三苯甲基

103. Commercially available automated instruments, called DNA synthesizers or “gene machines,” are capable of carrying out the synthesis of oligonucleotides of 150 bases or more.

104. 7.7 · Secondary and Tertiary Structure of RNA
Types of RNAs:
Transfer RNA (adaptor molecule)
Messenger RNA (template for protein synthesis)
Ribosomal RNA (protein synthesis)
Small nuclear RNA (splicesomal RNA)
Small nucleolar RNA (ribosomal RNA processing)
Interference RNA (gene silencing)
microRNA (translation regulation)
Virus RNA (code virus genome)
In comparison with DNA structures, much less is known about RNA structures. Most RNA are associated with proteins which facilitate their structural folding.

105. Transfer RNA Structures
Secondary Structure
Of large ribosomal RNA
Tertiary Structure
Of large ribosome subunit
TyC Loop
Anticodon
Stem
V ariable
loop
D Loop
Anticodon Loop

106. Ribosomal RNA Secondary Structure Of large ribosomal RNA
Tertiary Structure
Of large ribosome subunit
Ban et al., Science 289 ( ), 2000

107. Catalytic RNA Secondary Structure Of large ribosomal RNA
Tertiary Structure
Of large ribosome subunit

108. Objectives of Chapter 7 Describe DNA helix structure properties;
Recognize supercoiling in DNA and its handness;
Calculate L, or T, or W when the other two parameters are known;
Describe nucleosome and chromotin structure;
Describe the structural elements in tRNA;