Nucleic Acids

Nucleic acids are chain like macromolecules functioning in storage and transfer of genetic information. They are the major components of all cells, making up from 5 – 15% of their dry weight.

Although nucleic acids are so named because DNA was first isolated from cell nuclei, both DNA and RNA also occur in other parts of cells.

They are found in all living cells and viruses. They were first isolated in 1868, by a swiss scientist – Joseph Frederick Miescher from the nuclei of the pus cells on hospital bandages. He called them nuclein, because it came from nuclei and was something different from the protein. Altman (1889) coined the term nucleic acid, as it was found that nuclein was strongly acidic.

In 1930, A. Kossel demonstrated that nucleic acid on hydrolysis gave four nitrogen-containing compounds (adenine, guanine, cytosine and thymine; collectively called bases). P.A.T. Levene discovered for the first time that nucleic acids also contained a sugar molecule, which had a 5-carbon ring, and demonstrated that nucleic acids could be of two types, the deoxyribonucleic acid or DNA and ribonucleic acid or RNA

O.T. Avery, C.M. Macleod and M. McCarthy through transformation experiment indicated that DNA and not the protein is the genetic material. In 1952 A.D Hershey and M.J Chase using radioactive labeled phosphorous (³²PO₄) demonstrated that DNA is the sole genetic material.

Constituents of Nucleic Acids:

Chemical analysis have shown that nucleic acids are composed of the following 3 types of molecules:

1)      a pentose sugar

2)      a heterocyclic nitrogen base

3)      a phosphate group

Pentose Sugar:

Pentose sugar serve as building blocks of nucleic acids. The pentose sugar present is RNA is called D-ribose from which this nucleic acid gets its name. But DNA contains 2’-deoxy-D-ribose (simply deoxyribose). The oxygen atom present at the second carbon of ribose is missing in deoxyribose, giving its name 2’ – deoxyribose. The positions of carbon atoms of two pentose sugars are denoted as 1’,2’,3’,4’,5’ in order to differentiate them from the corresponding position in Nitrogen bases.

Nitrogen Bases:

The organic bases present in nucleic acids are heterocyclic compounds containing nitrogen in rings. Hence, they are also called as nitrogen bases. They are of two types: Purines and Pyramidines.

Pyrimidines:

            They have a six member ring containing two nitrogen atoms in place of carbon at position 1 & 3. The three pyrimidine bases : Thymine, Uracil, Cytosine contain a keto oxygen (= O) at position 2. In cytosine, an amino (-NH₂) group is present at position 4, in uracil another keto (=O) group is present at the fourth carbon, while thymine is essentially 5-methyl uracil i.e., a keto (=O) at position 4 and an CH₃ group at carbon 5.

All pyramidines, therefore, contain an –H atom at position 3, which is involved in their linkage with the 1C’ of pentose sugar.

Purines:

They have a six member pyrimidine ring, fused to a five member imidazole ring. Purines have 4 nitrogen atoms in place of carbon (C) at position 1,3ofpyrimidine ring and 7,9 of imidazole ring.

The nitrogen present at the 9^th position of purines participate in a covalent linkage with 1’C of the pentoses.

Adenine (A) and Guanine (G) are purines. In adenine, an amino (-NH₂) group is present at position 6. But in guanine, a keto (=O) group is found at position 6 and an additional –NH₂ group attached at the position 2.

Phosphate group:

Phosphoric acid (H₃Po₄) has 3 reactive hydroxyl groups (-OH) of which two are involved in forming the sugar – phosphate backbone of DNA. A phosphate moiety binds to the 5’C ov one and the 3’C of the other neighbouring pentose molecule of DNA to produce the phosphodiester (5’C – O – P –O – C 3’) linkage.

Nucleosides:

A nitrogen base combined with a sugar molecule is called as nucleoside. A base is linked with the pentose sugar molecule by a β – glycosidic bond.

The glycosidic linkage involves 1’C of the sugar and the nitrogen atom of N-9 (in purines) or N-3 (in pyrimidines) eliminating a molecule of water.

Nucleosides containing ribose sugar are called ribonucleosides, while those possessing deoxyribose sugar as deoxyribonucleosides.

Four common ribosides are adenosine, gauanosine, uridine and cytidine. Similarly, the four common deoxyribosides are deoxy-adenosine, deoxyguanosine, deoxycytidine and thymidine. (the name of pyrimidine nucleoside end with the suffix –dine, and those of purine end with suffix –sine)

Nucleotides:

The nucleotide is derived from a nucleoside by addition of one molecule of phosphate group. The phosphate molecule is linked with sugar molecule at carbon no. 5 or at carbon no.3. Correspondingly nucleotides will be called 5’p3’OH nucleotide and 3’p5’OH nucleotide. The four nucleotides found in DNA are deoxycytidylic acid, deoxythymidylic acid, deoxyadenylic acid and deoxyguanylic acid. Similarly, the four nucleotides found in RNA are cytidylic acid, uridylic acid, adenylic acid and guanylic acid.

Structure of DNA:

•      In 1953 using critical information from Rosalind Franklin and Linus Pauling, Watson and Crick determined the double helical structure of DNA  in 1953. Their double-helix model of DNA structure was based on two major kinds of evidence.

•      1. When the composition of DNA from many different organisms was analyzed by E. Chargaff and colleagues, it was observed that the concentration of thymine was always equal to the concentration of adenine (A =T) and the concentration of cytosine was always equal to the concentration of guanine (C = G).

This strongly suggested that thymine and adenine as well as cytosine and guanine were present in DNA with some fixed interrelationship. It also necessitated that the total concentration of pyrimidines (C + T) always equal to total concentration of purines ( A + G)

There is an equivalence between the bases carrying amino groups at the 6 or 4 positions (A + C) and those carrying keto groups at these positions ( G + T).This is popularly known as Chargaff’s rule.

Chargaff’s rule suggests that A is always paired with T. G always paired with C.

2. When X rays are focused through isolated macromolecules or crystals of purified molecules, the X rays are deflected by the atoms of the molecules in specific patterns, called diffraction patterns, which provide information about the organization of the components of the molecules. These X-ray diffraction patterns can be recorded on X-ray sensitive film.

Watson and Crick had available X-ray crystallographic data on DNA structure from the studies of M.H.F Wilkins, R. Franklin. 

On the basis of Chargaff’s chemical data, Wilkins and Franklin’s X – ray diffraction data, and inferences drawn from model building, Watson and Crick proposed that DNA exists as a double helix.

The main features of Watson – Crick model of DNA are:

1.      A DNA molecule is made up of two polynucleotide chains or strands which are coiled about one another in a spiral.

2.      Each polynucleotide chain consists of a sequence of nucleotides linked together by phosphodiester bonds between their sugar and phosphate residues.

3.      The two strands of a DNA molecule are oriented antiparallel to each other i.e., one strand runs in

5’      3’ direction, while the other strand runs in 3’      5’direction. This opposite polarity of the strands is very important in considering the mechanism of replication of DNA.

4.      The anitparallel orientation is essential as the two polynucleotide strands are held together in their helical configuration by hydrogen bonding between bases in opposing strands, the resulting base-pairs being stacked between the two chains perpendicular to the axis of the molecule like the steps of a spiral staircase.

5.      The base – pairing is specific, adenine present in one strand of DNA is always paired with thymine located opposite to it in the other strand. Similarly guanine located in one strands is always paired with the cytosine located opposite to it in the other strand.

6.      Thus, all base-pairs consists of one purine and one pyrimidine. This specificity of base-pairing results from the hydrogen – bonding capacities of the bases. Adenine and thymine form two hydrogen bonds, and guanine and cytosine form three hydrogen bonds.

7.      Once the sequence of bases in one strand of a DNA double helix is known, the sequence of bases in the other strand is also known because of the specific base-pairing. The two strands of a DNA double helix are thus said to be complementary (not identical). The formation of hydrogen bonds between A and T, and between G and C is thus known as Complementary base pairing.

8.      This property, complementarity of the two strands that makes DNA uniquely suited to store and transmit genetic information.

9.      The two strands of a DNA molecule are coiled together in a right-handed helix forming the DNA double helix. The diameter of this helix is 20 A^o.

10.  The pitch i.e., the length of helix required to complete one turn, is 34 A^o.  Thus, each turn contains 10 equally spaced base pairs. The distance between successive base pairs is 3.4 A^o and the angle between them is 36^o

11.  Each turn of DNA molecule include one major (wider) groove and one minor (narrow) groove along the phosphodiester backbone. Proteins interact with DNA at these grooves.

12.  The high degree of stability of DNA double helices results in part from the large number of hydrogen bonds between the base- pairs(even though each hydrogen bond by itself is quiet weak), and in part from the hydrophobic bonding between the stacked bas e-pairs. The planar sides of the base-pairs are relatively non polar and thus tend to be water insoulube or hydrophobic. This hydrophobic core of stacked base-pairs contributes considerable stability to DNA molecules present in the aqueous protoplasms of living cells.

Why DNA is helix

      The tendency towards a helix comes from the stacking of the individual bases on the top of one another. Both sugar and phosphate which constitute the backbone are quite soluble in water.   

       However, the DNA bases which are in the middle of the helix are relatively hydrophobic and insoluble.

    

      Since, the bases are flat, they stack on top of each other in order to form a more hydrophobic environment. The bases twist slightly in order to maximize their hydrophobic interactions with each other and it is this twisting of the stacked bases that gives rise to a helix. Thus, the reason for a helix in DNA is primarily due to hydrophobic stacking interactions of the bases.

Types of DNA

B-DNA:

The vast majority of the DNA molecules present in the aqueous protoplasms of living cells almost certainly exists in the Watson-Crick double-helix form. This is the B-form of DNA. The B-form is the conformation that DNA takes under physiological conditions i.e., in aqueous solutions containing low concentrations of salts.

B-DNA shows clockwise (right-handed) helix structure with 10 base-pairs per turn and has a pitch of 34A A^o.

However, DNA is not a static, invariant molecule. DNA molecules exhibit a considerable amount of conformational flexibility. The structures of DNA molecule change as a function of their environment.

A-DNA:

A-DNA for any sequence is favoured under dehydrating conditions ( 75% relative humidity). It appears that at least four purines (ex. GAGGGA) or pyrimidines in a row are enough to set up a local A-DNA helix. A- DNA helix is bit wider than B-DNA, and this is mainly due to the fact that the base pairs stack nearly on top of each other in B-DNA but stack a little off-centre in the A-conformation. There are about 11 base pairs per turn and with a pitch of 28 A^o.

Z-DNA:

It is an left handed helix. The backbone is not smooth helix, but is irregular and zig-zag in shape, hence its name. Alternating purine-pyrimidine can form, left handed Z-DNA. Z-helix is narrower than A and B –Conformation. It has 12 base-pairs per turn and with a pitch of 45 A^o.

Whether a DNA sequence will be in the A-, B- or Z-DNA conformation depends on 1. Ionic or hydration environment, A-DNA is favoured by low hydration, where as Z-DNA can be fovoured by high salt.

The second condition is the DNA sequence – A-DNA is favoured by certain stretches of purines or pyrimidines, where as Z-DNA can be most readily formed by alternating Purine-Pyrimidine steps.

The second condition is the presence of proteins that can bind to DNA in one helical conformation and force the DNA to adopt a different conformation.

In living cells, most of the DNA is in a mixture of A- or B-DNA conformation, with a few small regions capbable of forming Z-DNA.

Character	A-DNA	B-DNA	Z-DNA	C-DNA
Coiling	Right handed	Right handed	Left handed	Right handed
Pitch	28 Ao	34 Ao	45 Ao	31 Ao
Base pairs per turn	11	10	12	9.33
Diameter	23 Ao	20 Ao	18 Ao	19 Ao

Functions of DNA:

DNA performs two functions, they are:

I.                   Autocatalysis: the process of duplication of a single DNA molecule into two daughter DNA molecules is called autocatalysis or replication. DNA replicates by semi-conservative mechanism i.e., the daughter DNA molecules show one old or parent strand and one newly synthesized strand.

II.                Heterocatalysis: DNA promotes the synthesis of proteins and regulates bio-chemical reactions of cell. In this process the DNA templates transfer genetic message to mRNA by a process called transcription. mRNA helps in the synthesis of proteins.

DNA     Transcription         mRNA       Translation           Protein