DNA Strucutre

Chargaff's rules
Erwin Chargaff and his colleagues carefully measured the amounts of the four bases in DNA from a variety of organisms and found that DNA from different organisms varies greatly in base composition. This finding disproved the tetranucleotide theory. They discovered that, within each species, there is some regularity in the ratios of the bases:
the total amount of adenine is always equal to the amount of thymine (A = T), and the amount of guanine is always equal to the amount of cytosine (G = C; Table 10.1). These findings became known as Chargaff s rules.

The discovery of the three-dimensional structure of DNA by James Watson and Francis Crick in 1953.
In 1947, William Ashbury began studying the three dimensional structure of DNA by using a technique called X-ray diffraction. Maurice Wilkins and Rosalind Franklin, also was studying the structure of DNA by using X-ray diffraction and obtained strikingly better pictures of the molecule. Watson and Crick investigated the structure of DNA, not by collecting new data but by using all available information about the chemistry of DNA to construct molecular models

For their discovery, Watson and Crick, along with Maurice Wilkins, were awarded a Nobel Prize in 1962.

The Structure of DNA

The primary structure of DNA refers to its nucleotide structure and how the nucleotides are joined together. The secondary structure refers to DNAs stable three-dimensional configuration, the helical structure worked out by Watson and Crick.

The Primary Structure of DNA

A DNA strand is a polymera large molecule built from repeating units. The repeating units of DNA are nucleotides, each comprising three parts:
a a five-carbon sugar, a phosphate , and a nitrogen-containing base denoted A, T, G, and C.

Polynucleotide Chains
In DNA, each base is chemically linked to one molecule of the sugar deoxyribose, forming a compound called a nucleoside. When a phosphate group is also attached to the sugar, the nucleoside becomes a nucleotide. Thus a nucleotide is a nucleoside plus a phosphate. A "nucleotide" is a 5'-phosphate ester of a nucleoside.

Pentose Sugar
The sugars of nucleic acidscalled pentose sugars have five carbon atoms, numbered 1, 2, 3, and so forth ( FIGURE 10.9). Four of the carbon atoms are joined by an oxygen atom to form a five-sided ring; the fifth (5) carbon atom projects upward from the ring. Hydrogen atoms or hydroxyl groups (OH) are attached to each carbon atom. The sugars of DNA and RNA are slightly different in structure. RNAs ribose sugar has a hydroxyl group attached to the 2carbon atom, whereas DNAs sugar, called deoxyribose, has a hydrogen atom at this position and contains one oxygen atom fewer overall. This difference gives rise to the names ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)

Pentose Sugar
This difference gives rise to the names ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). This minor chemical difference is recognized by all the cellular enzymes that interact with DNA or RNA, thus yielding specific functions for each nucleic acid. Further, the additional oxygen atom in the RNA nucleotide makes it more reactive and less chemically stable than DNA. For this reason, DNA is better suited to serve as the long-term repository of genetic information.

Nitrogenous Base
The second component of a nucleotide is its nitrogenous base, which may be of two typesa purine or a pyrimidine ( FIGURE 10.10). Each purine consists of a sixsided ring attached to a five-sided ring, Whereas each pyrimidine consists of a six-sided ring only.

Nitrogenous Base
In a nucleotide, the nitrogenous base always forms a covalent bond with the 1-carbon atom of the sugar. A deoxyribose (or ribose) sugar and a base together are referred to as a nucleoside. The sugar is linked to the heterocyclic base via a N- glycosidic bond, almost always to N-1 of pyrimidine or to N9 of a purine The N- -glycosyl bond is formed by removal of the elements of water (a hydroxyl group from the pentose and hydrogen from the base).

Fig. Minor bases of DNA. 5-Methylcytidine occurs in the DNA of animals and higher plants, N6-methyladenosine in bacterial DNA, and 5hydroxymethylcytidine in the DNA of bacteria infected with certain bacteriophages.

Some minor bases of tRNAs. Inosine contains the base hypoxanthine. Note that pseudouridine, like uridine, contains uracil; they are distinct in the point of attachment to the ribosein uridine, uracil is attached through N-1, the usual attachment point for pyrimidines; in pseudouridine, through C-5.

Phosphate group
Phosphorus atom bonded to four oxygen atoms Phosphate groups are found in every nucleotide and frequently carry a negative charge, which makes DNA acidic. The phosphate is always bonded to the 5-carbon atom of the sugar in a nucleotide

Nucleotide
In a ribonucleotide or a deoxyribonucleotide, one or more phosphate groups is bonded to atom C3 or atom C5 of the pentose to form a 3nucleotide or a 5-nucleotide, respectively.

A 5-nucleotide can therefore be called a nucleoside-5-phosphate.

Nucleotides most commonly contain one to three phosphate groups at the C5 position and are called nucleoside monophosphates, diphosphates, and triphosphates.

dTTP thymidine triphosphate:

Polynucleotide strands
DNA is made up of many nucleotides connected by covalent bonds, which join the 5-phosphate group of one nucleotide to the 3 carbon atom of the next nucleotide. These bonds, called phosphodiester linkages, are relatively strong covalent bonds; a series of nucleotides linked in this way constitutes a polynucleotide strand.

 Thus the covalent backbones of nucleic acids consist of alternating phosphate and pentose residues, and the nitrogenous bases may be regarded as side groups joined to the backbone at regular intervals. The backbones of both DNA and RNA are hydrophilic. Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone allowing them to easily dissolve in water.

The role of the salt

The phosphate groups, with a pKa near 0, are completely ionized and negatively charged at pH 7, and the negative charges are generally neutralized by ionic interactions with positive charges on proteins, metal ions, and polyamines. The positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water.

 Water dielectric constant = 80.1

Ethanol on the other hand has a much lower dielectric constant, making it much easier for Na+ to interact with the PO3-, shield its charge and make the nucleic acid less hydrophilic, causing it to drop out of solution.

 A short nucleic acid is referred to as an oligonucleotide. A longer nucleic acid is called a polynucleotide. An important characteristic of the polynucleotide strand is its direction, or polarity. At one end of the strand a phosphate group is attached only to the carbon atom of the sugar in the nucleotide. This end of the strand is therefore referred to as the 5end. The other end of the strand, referred to as the 3end, has an OH group attached to the 3-carbon atom of the sugar.

Fig. DNA consists of two polynucleotide chains that are antiparallel

Tautomerism

Tautomers are isomers of a compound which differ only in the position of the protons and electrons. The carbon skeleton of the compound is unchanged. A reaction which involves simple proton transfer in an intramolecular fashion is called a tautomerism. The oxo and amino groups of purines and pyrimidines exhibit keto-enol and amine-imine tautomerism, but physiologic conditions strongly favor the amino and oxo forms.

Purine and pyrimidine bases exist in different forms called tautomers

The Base Composition of DNA

Erwin Chargaff and his colleagues in the late 1940s. They found that the four nucleotide bases of DNA occur in different ratios in the DNAs of different organisms and that the amounts of certain bases are closely related. These data, collected from DNAs of a great many different species, led Chargaff to the following conclusions:

1. The base composition of DNA generally varies from one species to another. 2. DNA specimens isolated from different tissues of the same species have the same base composition. 3. The base composition of DNA in a given species does not change with an organisms age, nutritional state, or changing environment. 4. In all cellular DNAs, regardless of the species, the number of adenosine residues is equal to the number of thymidine residues (that is, A = T), and the number of guanosine residues is equal to the number of cytidine residues (G =C). From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues; that is, (A+G=T+C).

Secondary Structures of DNA

The secondary structure of DNA refers to its 3D configurationits fundamental helical structure. DNAs secondary structure can assume a variety of configurations, depending on its base sequence and the conditions in which it is placed.

The Double Helix

In 1953 Watson and Crick postulated a three dimensional model of DNA structure that accounted for all the available data. It consists of two helical DNA chains wound around the same axis to form a right handed double helix. Two polynucleotide chains wind around a common axis to form a double helix. The two strands of DNA are antiparallel (run in opposite directions),

 In the doublestranded DNA molecules, the genetic information resides in the sequence of nucleotides on one strand, the template strand. This is the strand of DNA that is copied during nucleic acid synthesis. It is sometimes referred to as the noncoding strand. The opposite strand is considered the coding strand because it matches the RNA transcript that encodes the protein.

 The hydrophilic backbones of alternating deoxyribose and phosphate groups are on the outside of the double helix, facing the surrounding water. The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis. The offset pairing of the two strands creates a major groove and minor groove on the surface of the duplex.

 The strand backbones are closer together on one side of the helix than on the other. The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together. The grooves twist around the molecule on opposite sides. Certain proteins bind to DNA to alter its structure or to regulate transcription (copying DNA to RNA) or replication (copying DNA to DNA). It is easier for these DNA binding proteins to interact with the bases (the internal parts of the DNA molecule) on the major groove side because the backbones are not in the way.

Base-stacking
Two antiparallel polynucleotide chains of double-helical DNA are not identical in either base sequence or composition. Instead they are complementary to each other. The complementarity between the DNA strands is attributable to the hydrogen bonding between base pairs. The DNA double helix, or duplex, is held together by two forces, : hydrogen bonding between complementary base pairs and base-stacking interactions. The base-stacking interactions, which are largely nonspecific with respect to the identity of the stacked bases, make the major contribution to the stability of the double helix.

Base-stacking
The upper and lower faces of each nitrogenous base are relatively flat and non polar (uncharged). These surfaces are said to be hydrophobic because they bind poorly to water molecules, which are very polar. Owing to their repulsion of water molecules, the paired nitrogenous bases tend to stack on top of one another in such a way as to exclude the maximum amount of water from the interior of the double helix. Hence a double stranded DNA molecule has a hydrophobic core composed of stacked bases, and it is the energy of base stacking that provides double-stranded DNA with much of its chemical stability.

Forces that help to form the DNA double helix

Rigid phosphate backbone Stacking interactions - electronic interactions between planar bases Hydrophobic interactions - highly negative phosphate backbone vs. nonpolar bases Hydrogen bonding is not the most energtically signicant component Ionic interactions - salt stabilizes the duplex form of DNA shielding of phosphate backbone

Different secondary structures

Structural variation in DNA reflects three things:
the different possible conformations of the deoxyribose, rotation about the contiguous bonds that make up the phosphodeoxyribose backbone (Fig. 818a), and free rotation about the C-1N-glycosyl bond (Fig. 818b).

Because of steric constraints, purines in purine nucleotides are restricted to two stable conformations with respect to deoxyribose, called syn and anti (Fig. 818b). Pyrimidines are generally restricted to the anti conformation because of steric interference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine.

ring pucker: endo vs. exo and C-3'

ENDO: OUT-OF-PLANE ATOM ON SAME SIDE OF RING AS C5 EXO; DISPLACED TO OPPOSITE SIDE

Double helical parameters

helical sense: right vs left major vs minor groove base pairs per helical turn helix rise per base pair or helical pitch:distance from one step to the next helical twist: angle between two adjacent base pairs =360 deg/base pairs per turn base tilt: slant of the step, not completely planar glycosidic conformation: anti vs syn sugar ring pucker: 4 out of 5 ring atoms are nearly planar the 5th atom is usually the C-2 or C-3 atom endo vs exo

A-DNA Conditions required to produce structure Overall shape Helical sense Base pairs per helical turn Diameter Helix rise per base pair (Distance between adjacent bases) Helix pitch (rise per turn) Base tilt normal to the helix axis 75% H2O Short and wide Right handed 11 ~26

B-DNA 92% H2O Long and narrow Right handed 10.5 ~20

Z-DNA Alternating purine and pyrimidine bases Elongated and narrow Left handed 12 ~18

2.6

3.4

3.7

28

34

44

20

7 9 for purinepyrimidine; 51 for pyrimidinepurine C2-endo for pyrimidine; C3-endo forpurines) Anti for pyrimidines; syn for purines Flat Narrow and deep

Helix twist per base pair

32

36

Sugar pucker

C3-endo

C2-endo

Glycosyl bond conformation Major groove Minor groove

Anti Narrow and deep Wide and shallow

Anti Wide and deep Narrow and deep

B-DNA
The Watson-Crick structure is also referred to as B form DNA, or B-DNA. Conditions required to produce strucutre: 92% H2O The B form is the most stable structure for a random-sequence DNA molecule under physiological conditions and is therefore the standard point of reference in any study of the properties of DNA B-DNA is an alpha helix, meaning that it has a right handed, or clockwise, spiral. Each chain makes one complete turn every 34 . The bases are spaced at 3.4 , so there are ten bases per helical turn in each strand and ten base pairs per turn of the double helix.

B-DNA
It possesses approximately 10 base pairs (bp) per 360-degree rotation of the helix; so each base pair is twisted 36 degrees relative to the adjacent bases. The diameter of the helix is 2 nm, and the bases are perpendicular to the long axis of the DNA molecule Overall shape is Long and narrow.

A-DNA
A-DNA structure, which exists when less water is present. (75% H2O) The DNA is still arranged in a right-handed double helix, but the helix is wider and shorter than BDNA . The number of base pairs per helical turn is 11, rather than 10 as in B-DNA.

A-DNA
The plane of the base pairs in A-DNA is tilted about 20 with respect to the helix axis. These structural changes deepen the major groove while making the minor groove shallower. The reagents used to promote crystallization of DNA tend to dehydrate it, and thus most short DNA molecules tend to crystallize in the A form.

Z-form DNA
Z-DNA forms a left handed helix. In this form, the sugar-phosphate backbone zigzag back and forth, giving rise to the name Z-DNA (for zigzag). There are 12 base pairs per helical turn, and the structure appears more slender and elongated.

Z-form DNA
Certain nucleotide sequences fold into left handed Z helices much more readily than others. Prominent examples are sequences in which pyrimidines alternate with purines, especially alternating C and G or 5-methyl-C and G residues. To form the left-handed helix in Z-DNA, the purine residues flip to the syn conformation, alternating with pyrimidines in the anti conformation. The major groove is barely apparent in Z-DNA, and the minor groove is narrow and deep.

Z-form DNA
There is evidence for some short stretches (tracts) of Z DNA in both prokaryotes and eukaryotes. These Z-DNA tracts may play a role (as yet undefined) in regulating the expression of some genes or in genetic recombination.