分类： Molecular Biology

The blueprint of life [3] secondary and some special structures of DNA

Let’s pick up where we dropped, the secondary structure of DNA.

IN FACT, a number of different forms of nucleic acid double-helix have been observed and studied, all having the basic pettern of two helically-wound antiparallel strands.
Polymorphism （多样性）of DNA  Secondary Structure
―due to conformational changes of sugar-ring on the nucleotide chain.
1. B-form:
  • right-handed
  • the structure identified by Watson and Crick,
  • the most common form,
  • believed to be the idealized form of the structure adopted by virtually all DNAin vivo (in the living body of a plant or animal), or,  at physiological (characteristic of or appropriate to an organism’s healthy or normal functioning) pH and salt concentration.

characterized by:

  • a helical repeat of 10bp/turn (although now it is known that ‘real’ B-DNA has a repeat closer to 10.5bp/turn);
  • the presence of base pairs lying on the helix axis and almost perpendicular to it;
  • having well-defined, deep major and minor grooves.
2. A-form: 
  • right handed
  • adopted by DNA in vivo under unusual circumstances, (conversed from B-form in low moisture (<75%))
  • presents in certain DNA-protein complexes

characterized by:

  • a helical repeat of 11 bp/turn.
  • the presence of base pairs tilted with respect to the helix axis, and actually lying off the axis.
  • being the helix formed by RNA and DNA-RNA hybrids. (Similar to some RNA-DNA duplex or RNA-RNA duplex.)
3. Z-form:
  • left-handed
  • formed by stretches of alternating pyrimidine-purine sequence, e.g. GCGCGC, especially in negatively supercoiled DNA in high saline (盐) solution.
  • not easily form even in DNA regions of repeating GCGCGC, since the boundaries between the left-handed Z-form and the surrounding B-form would be very unstale

characterized by:

  • a zigzag (锯齿型) pattern where its name comes from
  • 12 bp/turn

(1)

A. B, Z-DNA
slide shown by prof.Dong,COMPARISON AMONG A-,B-,Z-DNA
(2)
A.B.Z-DNA instant notes
picture from Instant Notes in Molecular Biology (3rd Edtion)
====================================================
Some special structures of DNA 
1. Inverted repeats and direct repeats
Inverted Repeat is functionally important as recognition sites on the DNA for the binding of a variety of proteins (e.g. restriction and modification enzymes)
Inverted Repeat is either discontinuous or continuous,
Continuous → palindromic structure (回文结构:inverted repetitions of base sequence over the two strands form a symmetric structure )
Discontinuous→ hairpin & cruciform (发卡、十字结构:self complementary within each of the strands )
palindromic structure
palindromic structure
hairpin structure & cruciform structure
hairpin structure & cruciform structur
2. DNA triplex (The intramolecular triplex (H-DNA) as an example)
shown by prof.Dong, DNA TRIPLEX
shown by prof.Dong, DNA TRIPLEX
• AG-rich strand vs. CT-rich strand with Hoogsten hydrogen bond
(a quick glance at Hoodsten hydrogen bond:
image from wikipedia/hoogsten base pair
image from wikipedia/hoogsten base pair
• Mirror inverted repeat
• a barrier for different DNA and RNA polymerases, and so it is a negative regulator for gene expression.
3. G-quadruplex structure
G-rich sequence(s) to form 4-stranded structure by unusual G-G
H-bonds
shown by prof.Dong, G-AUADRUPLEX
shown by prof.Dong, G-AUADRUPLEX

the blueprint of life [2]: primary and secondary structure of DNA

Molecular structure of DNA:

professor Dong first showed us where DNA is:

molecular structure of DNA
slide shown by Prof.Dong,WHERE IS DNA?

Then he introduced the molecular structure of DNA with 4 parts:

1. Primary structure of DNA:

Arrangement of nucleotides along a DNA chain.

Annie: So…the primary structure of DNA is a line.

Conventionally, the repeating monomers of DNA are represented by their single letters A, T, G, C.

Professor: There’s a convention to write the DNA sequence with 5′ at the left, that is, in a 5′ to 3′ orientation from left to right.

====================================================

2. Secondary structure of DNA—stabilized partial structure formed by polymers of nucleotide

Professor Dong: First, please familiarize yourselves with Chargaff’s Rule:

A+G=T+C & G+T=A+C

↓↓

A=T & G=C

(Chargaff’s Rule)

(Based on analysis of the chemical composition of duplex DNA in the early 1950s, E. Chargaff deduced these rules about the amounts of different nucleotides in DNA.)

Professor: The secondary structure of DNA is a partial structure formed by polymers of nucleotide. The structure is referred to as the double-helix structure.

Two separate chains of DNA are wound around each other, each following a helical (coiling) path, resulting in a right-handed double helix structure.

In 1953, Watson and Click proposed the DNA double-helix structure based on Chargaff’s Rule and DNA Crystallography and X-ray diffraction images of DNA structure by Wilkins and Franklin.  (Rosalind Franklin, who was not that well-known as Watson, Click and Wilkins but apparently played a equally significant role in the discovery of the structure.The whole was later nominated Nobel Prize but her, is that even fair?)

—————————————————————————

The backbone of duplex DNA is a serious of phosphodiester group (the covalent linkage of a phosphate group between the 5′-hydroxyl of one sugar and the 3′-hydroxyl of the next, that is , repeats of P-sugar unit) linked by phosphodiester bond.

—————————————————————————
The strands are joined noncovalently by hydrogen bonding between the bases on opposite strands, to form base pairs.
There are around 10 base pairs per turn in the DNA double-helix. The two strands are oriented in opposite directions in terms of their 5’to3′ direction(the nucleotides in one strand is opposite to their direction in the other strand).
stand direction
More crucially, the two strands are complementary in terms of sequence. The bases hydrogen-bond to each other as purine-pyrimidine pairs which have very similar geometry and dimensions.
         A–T:  2 H-bonds ;    C–G:  3 H-bonds
   5’- A   T   G    T   C -3’
   ¦¦    ¦¦  ¦¦¦   ¦¦   ¦¦¦
   3’- T   A   C   A   G -5’

 Thus, the sequence of one strand uniquely specifies the sequence of the other, with all that which implies for the mechanism of replication of DNA and its transcription to RNA.

——————————————————————————-
Professor Dong added:
Between the backbone stands run the major and minor grooves.

In a detailed analysis of DNA structure, there are two types of grooves that can be seen; the major groove has the nitrogen and oxygen atoms of the base pairs pointing inward toward the helical axis, while in the minor groove,the nitrogen and oxygen atoms point outwards;

major groove A_T
Shown by prof.Dong, MAJOR GROOVE A-T
major groove GC
Shown by prof.Dong, MAJOR GROOVE G-C
—Major Groove                        —Minor Groove
—Depth: 8.5 Å                             —Depth: 7.5 Å
—Width: 11.7 Å                          —Width: 5.7 Å
Å
Definition: Symbol for Ångström, a unit equal to 0.1 nanometer, mainly used in expressing sizes of atoms, lengths of chemical bonds, and wavelengths of electromagnetic radiation.
Supplement: The unit is named after the Swedish physicist, Ångström, Anders Jonas.
instant notes

picture from Instant Notes in Molecular Biology

Professor: The major groove is more dependent on base composition. and major grooves and minor grooves are also recognition and binding sites for certain  protein factors, and are involved in the regulation of gene expression.

——————————————————————————
grooves
slide shown by Prof.Dong

Professor: Summary of “Double Helix” Model (B-DNA):

  • —Right Handed Double Helix
  • —Outside: P-Sugar backbone
  • Inside: Base pairing linked by H-bonds
  • —Minor and Major grooves
 （note: bp=base pair(s)）

the blueprint of life[1]

Of course the blueprint of life is DNA.

DNA is important because it is the genetic material ←contain all the info for the synthesis and functioning of a living form duplicate and passes through to the next generation.

Hearing this Annie questioned herself: DNA is not the genetic for ALL viruses. RNA “blueprints” for the rest of the viruses. Virus, though not even having a cell structure, is a form of life. So why not be fair, and say the blueprint of life are DNA and RNA?

Professor Dong continued,

Proofs:

  1. Bacterial Transformation Experiment

—Griffith, 1928

Professor: So what is the transforming principle? 

Annie: It could be a cool type of enzyme that moved the toxic part of S strain onto R strain…

Professor: Well, Enzyme did help a lot in the experiment we’ll talk about later, but it is not the hero of the story.

Annie: So what’s the story?

Professor:

—Avery et al., 1944

slide shown by Prof. Dong ,AVERY REPEATED GRIFFITH’S EXPERIMENT WITH MODIFICATION

Only DNA is responsible for the transformation.

Annie thought: Well, there still existed possibilities that DNA and some other things that were not sugar, lipid or protein cooperated to complete the transformation… The “other things” also contributed to the transformation but could not complete it without DNA?  In this case DNA is not the only one that is responsible,  

  1. T2 Bacteriophage Infection (Blender Experiment)

—Hershey &Chase, 1952

Experiment 1

Experiment 2

Radioactive labeling of proteins and DNA

The professor continued. So now let’s go deeper and see the chemical composition of DNA.

A 5-carbon sugar , hand in hand with base group and phosphorous group, thought Annie.

It’s not that simple as you learned in high school, said the professor. We need to know the chemical composition of the base group, deoxyribon and phosphorous group as well.

sugar group
slide shown by Prof. Dong,SUGAR GROUP

 Professor:

Base
slide shown by Prof. Dong,BASE GROUPS

Annie thought: Pyrimid-ine, Could it have anything to do with the Pyramid in Egypt?

The professor was explaining the structure and Annie didn’t interrupt him with her question.

After class, Annie searched the Internet for an answer, and this is what she found.

 The professor continued.

Bonds

P group——phosphester bond–—Sugar—–glycosidic bond—–B group

What is molecular biology

Generally speaking, molecular biology is the study of structure and function of macromolecules such as nucleic acids, proteins and other polymers. In a narrow sense, molecular biology focuses on nucleic acids and their activities, such as transcription, translation, DNA replication, recombination, translocation and so on.

Writing in Nature in 1961, William Astbury described molecular biology as:

“…not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned particularly with the forms of biological molecules and […] is predominantly three-dimensional and structural—which does not mean, however, that it is merely a refinement of morphology. It must at the same time inquire into genesis and function.”

Foundation of Molecular Biology:
  • —1869: Discovery of nucleic acids (nuclein) 
  •  —F. Miescher
  • —1944: Proofing of nucleic acids are genetic materials
  • —1953: Proposition of DNA structure – “double helix model”  (Watson and Crick, 1962 Nobel)
  • 1954: Establishment of “central dogma” (—Crick)
  • 1958: Mechanism of DNA replication (Meselson and Stahl)
  • —1961: “Operon” model of gene regulation (Jacob and Monod)
  • —1964: Identification of genetic codes (Nirenberg, 1968 Nobel)

Era of Genetic Engineering

  • —1970: Discovery of reverse transcriptase  (Temin and Baltimore, 1975 Nobel)
  • —1972: First recombinant DNA in vitro (—Berg, 1980 Nobel)
  • —1973: First transformation of hybrid plasmid into E. coli (—Cohen and Boyer)
  • 1977: First genetically modified product (Boyer, somatostatin )
  • 1977: DNA sequencing methods (Sanger and Gilbert, 1980 Nobel)
  • 1985: DNA in vitro amplification technology-PCR）
  • —1970: Discovery of reverse transcriptase (Temin and Baltimore, 1975 Nobel)
  • 1972: First recombinant DNA in vitro (—Berg, 1980 Nobel)
  • —1973: First transformation of hybrid plasmid into E. coli (—Cohen and Boyer)
  • —1977: First genetically modified product (Boyer, somatostatin)
  • —1977: DNA sequencing methods (—Sanger and Gilbert, 1980 Nobel）
  • —1985: DNA in vitro amplification technology-PCR（—Mullis, 1993 Nobel)
Era of Genomics and post-Genomics
  • —1986: Establishment of “Genomics” concept (Dulbecco, Roderick and McKusick)
  • —1990: Human genome project starts (—USA department of energy)
  • —2003: Completion of sequence mapping of human genome (—International HGP organization)
  • —2003-now: functional analysis of genome (post-genomics)

Genome Projects-Current Researches:

  • —Genetic engineering
  1. GMO (Genetically modified organism 基因修饰生物)
  2. —Cancer and gene therapy
  • —Regulation analysis of gene expression
          Signal transduction, TFs, RNA splicing, etc
  • —Structural and functional analysis of biological macromolecules
  1. —X-ray crystallography, NMR, EM etc
  2. —Yeast two-hybrid, immunoprecipitation etc
  • —Genomics, proteomics and bioinformatics
  • —…..

Hotspots