DNA AS GENETIC MATERIAL CHEMISTRY OF NUCLEIC ACIDS

DNA AS GENETIC MATERIAL, CHEMISTRY OF NUCLEIC ACIDS AND GENETIC CODE

DNA • The structure of DNA encodes all the information every cell needs to function and thrive. • DNA carries hereditary information in a form that can be copied and passed intact from generation to generation. GENE • A gene is a segment of DNA. • The biochemical instructions found within most genes, known as the genetic code, specify the chemical structure of a particular protein. • Proteins are composed of long chains of amino acids, and the specific sequence of these amino acids dictates the function of each protein.

Evidence that genes are made of DNA (or sometimes RNA) 1. Transformation in Bacteria • Frederick Griffith laid the foundation for the identification of DNA as the genetic material in 1928 with his experiments on transformation in the bacterium Pnuemococcus, now known as Streptococcus pneumoniae. 2. DNA: The transforming material. • Oswald Avery, Colin Mac Leod, and Maclyn Mc Carty showed the transforming substance to be DNA in 1944 in virulent cells of Streptococcus pneumoniae. • In 1952, A. D. Hershey and Martha Chase performed experiment in T 2 bacteriophage. The phage is composed of protein and DNA only. The experiment showed that the genes of phage are made of DNA.

The chemical nature of Polynucleotides • By the mid 1940 s, biochemists know the fundamental chemical structures of DNA and RNA. • When they broke DNA into its component parts, they found these constituents to be nitrogenous bases, phosphoric acid, and the sugar deoxyribose. • RNA yielded bases and phosphoric acid, plus a different sugar ribose. • The four bases found in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). • RNA contains the same bases, except that Uracil (U) replaces thymine.

• Adenine and Guanine are purines and are two ringed structures. • Thymine, Cytosine and Uracil are single ringed and are called pyrimidines. • These structures constitute the alphabet of genetics. Ribose contains a hydroxyl (OH) group in the 2 – position. • The subunits of DNA and RNA are nucleotides, which are nucleosides with a phosphate group attached through a phosphodiester bond.

• • • DNA Structure Linus Pauling elucidated that the - helix, an important feature of protein structure. Maurice Wilkins and Rosalind Franklin, another group tried to find out the structure of DNA at Kings College in London. They used X-ray diffraction to analyse threedimensional structure of DNA. Erwin Chargaff was another very important contributor. Chargaff studies (1950) of the base composition of DNAs from various sources revealed that the content of purines always equaled the content of pyrimidines. the amounts of adenine and thymine were always equal, as were the amounts of guanine and cytosine. These findings, known as Chargaff's rules, provided a valuable confirmation of Watson and Crick's model. Franklin's X-ray work strongly suggested that DNA was a helix.

The Double helix • DNA molecules form chains of building blocks called nucleotides. • Each nucleotide consists of a sugar molecule called deoxyribose that bonds to a phosphate molecule and to a nitrogen-containing compound, known as a base. • DNA uses four bases in its structure: adenine (A), cytosine (C), guanine (G), and thymine (T). • The order of the bases in a DNA molecule—the genetic code —determines the amino acid sequence of a protein. • In the cells of most organisms, two long strands of DNA join in a single molecule that resembles a spiraling ladder, commonly called a double helix. • Alternating phosphate and sugar molecules form each side of this ladder.

COMPLEMENTARY BASE PAIRING • Adenine always joins with Thymine Guanine always links to Cytosine. • Scientists use complementary base pairing to help identify the genes on a particular chromosome and to develop methods used in genetic engineering. • Watson and Crick found that the best model that satisfied all the X-ray data was a double helix • The two chains run in an anti parallel fashion with one chain having a 51 31 orientation and the other having a 31 51 orientation. • The width of the helix was found to be 2 nm. • The purine and pyrimidine bases were stacked 0. 34 nm apart in a ladder.

• The helix made one full turn every 3. 4 nm and, therefore, there should be 10 layers of bases stacked in one turn. . • In a given DNA, adenine is equal to thymine and guanine to cytosine. • There are two hydrogen bonds for A = T pairing and three bonds for C G pairing. • C G pairing is more stronger than A = T pairing. • Helical structure is right handed. • The fifth (5 - prime, of 5') carbon of the pentose ring is connected to the third (3 - prime, of 3 ') carbon of the next pentose ring via a phosphate group, and the nitrogenous bases stick out from this sugar-phosphate back bone. • By convention, DNA sequences are read from 5'→ 3' with respect to the polarity of the strand.

Genes made of RNA • A group of viruses, referred to as retroviruses, has RNA as the genetic material. • These tumour viruses can integrate with the host genome DNA, only after the RNA makes a DNA copy. • The central dogma says that the flow of information is unidirectional. i. e. , DNA → RNA → Protein. • With the discovery of the enzyme reverse transcriptase, RNA can also go back to DNA and the central dogma is now represented as: RNA → DNA → Protein.

A variety of DNA structures • B form is present in most DNA in the cell. The plane of a base pair is no longer perpendicular to the helical axis, but tilts 20 degrees away from horizontal. • The A helix packs in 11 base pairs per helical turn instead of 10 found in the B form, and turn occurs in 31 angstroms instead of 34. • The distance between base pairs, is only 2. 8 nm instead of 3. 4 nm, as in B-DNA. • Both the A and B form DNA structures are right handed; the helix turns clockwise.

• Alexander Rich and his colleagues discovered in 1979, DNA can exist in an extended left-handed helical form • The zigzag look of this DNA's backbone when viewed from the side, it is often called Z DNA. • The distance between base pair is 4. 5 nm and number of bases per turn is 12. • RNA-DNA hybrid strand assumes the A form. • Normal DNA has 2 groove (major and minor). • Z- DNA has single groove.

Separating the two strands of a DNA double helix • While the ratios of G to C and A to T in an organisms DNA are fixed, • the GC content (percentage of G + C) can vary considerably from one DNA to another. • The values of GC content range from 22% to 73% and these differences are reflected in differences in the properties of DNA.

PROPERTIES OF DNA 1. DNA denaturation, or DNA melting The temperature at which the DNA strands are half denatured is called the melting temperature, or Tm. This is known as DNA denaturation. • The amount of strand separation or melting is measured by the absorbance of the DNA solution at 260 nm. • The higher a DNA's GC content, the higher its Tm. C G pairing form 3 hydrogen bonds, whereas A = T pairs have only 2. • In addition to heating, DMSO and formamide also disrupt the hydrogen bonding between DNA.

2. Annealing or Renaturation • • • Once the two strands of DNA separate, they can under the proper conditions, come back together again. This is called annealing or renaturation. Factors that contribute to renaturation are 1. Temperature : Best temperature for renaturation of a DNA is about 25 o. C below its Tm 2. DNA concentrations: The higher the concentration, the faster the annealing 3. Renaturation time: If longer time allowed for annealing, the more will occur.

Activities of genes • A gene is a unit of information which is held as a code in a discrete segment of DNA. • This code specifies the amino acid sequence of a protein • The coding parts of a gene sequence are exons, and the non- coding parts are introns. • Before a gene can be expressed, the DNA that encodes has to be transcribed into RNA.

A gene participates in 3 major activities 1. A gene can be replicated • genetic information can be passed from generation to generation unchanged. 2. The sequences of bases in the RNA depends directly on the sequences of bases in the gene. • Most of these RNAs, in turn, serve as templates for making protein molecules. • Thus, most genes are essentially blueprints for making proteins. • The production of protein from a DNA blueprint is called gene expression. 3. A gene can accept occasional changes, or mutations.

GENETIC CODE • Most genes encode proteins and although only a small part of the total DNA , the coding regions of genes act as a template for the protein. • Proteins are made up of amino acids. • It is the sequence of amino acids which give the protein its specific properties. • DNA template is first transcribed into m. RNA. • The m. RNA template is then translated into a chain of amino acids. • There are 20 different amino acids which are used to build up proteins.

• Marshall Nirenberg and his associates in the early 1960 sdevised an elegant technique, called the triplet binding test, and discovered the first word of code dictionary. • A system was developed for synthesizing proteins in vitro. • The system included a cell extract containing ribosomes, t. RNAs and other cellular components. • When synthetic m. RNAs consisting entirely of a single type of nucleotide were added, polypeptides composed of only a single type of amino acid were formed. • Thus phenylalanine was formed when polyuridylic acid (poly U) was added.

• Marshall Nirenberg, Severo Ochoa, Hargobind Khorana, Francis Crick and many others contributed significantly to decipher the genetic code. • They figured out that the order in which amino acids are arranged in proteins. • On the basis of a variety of experiments, it was found out that a particular sequence of 3 bases (triplet) would code for a particular amino acid and this triplet is referred to as codon.

• Many amino acids have more than one codon and codons specifying the same amino acid are said to be degenerate and differ in only the third base. • The complex process by which the information in RNA is decoded into a polypeptide is an exciting story and its understanding represents a great achievement of the twentieth century in Biology.

Properties of the Genetic code • The code is highly degenerate, i. e. , most of the amino acids are coded for more than one amino acids. • Leucine, serine and arginine have 6 different codons. • Proline, threonine and alanine, have four. • Isoleucine has three. • Methionine and tryptophan have only one codon. • The code is not overlapping. There is no punctuation or spacing between different codons. • The starting signal for protein synthesis is the codon AUG (for methionine). •

• The code appears to be highly universal, i. e. , it is the same for various different kind of organisms. • Coding regions can be transferred from one organism to another and the correct protein produced. • However, a few exceptions to this are known. For example, in yeast mitochondria, UGA codes for tryptophan instead of stop. • In Paramecium, UAA and UAG code for glutamine instead of stop codon

Point Mutation will cause change in the amino acid sequence 1 2 3 4 5 Normal gene frame BIG FAT CAT ATE RAT Delete 1 base (F) BIG ATC ATA TER AT Add 1 base (X) BIG FAT CXA TAT ERA + The universality provides strong evidence that life on earth started only once. • When the first living forms appeared some 3 billion years ago, the genetic code was established and it has not changed since then through out the evolution of living organisms. • The selective pressure has been less strict in mitochondrial DNA. Mitochondria code only for few proteins and have their own protein synthetic machinery. The overall code has been maintained

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