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Topics 1. 2. 3. 4. 5. 6. 7. 8. Basic Composition of Nucleic Acids Structural Individuality of DNA DNA, Replication, Repair and Recombination RNA: Structure and Classification Genetic Code: An Overview Process of Transcription Translation: Basis of Protein synthesis Human Genome Project and DNA Fingerprinting
Basic Composition of Nucleic Acids:
Nucleic acids are the molecules which carry the genetic information from one generation to the next. The genetic carriers are of two type, Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). These macromolecules are long chain polymers formed by a larger number of nucleotides linked together. Each of the nucleotide has its own specific sugar, a phosphate and a nitrogenous base. The back bone of the structure is formed by the sugars linked by phosphates but the nitrogenous bases differ. The primary structures of DNA and RNA are similar to each other. They form a long chain of nucleotides composed of single nucleosides (a sugar and a phosphate moiety)
A nucleotide is a complex structure formed by the combination of many biomolecules. Chemically a nucleotide is composed of: (i) A nitrogenous base (ii) A sugar [pentose-deoxyribose (DNA) or ribose (RNA)] (iii) A phosphate group
Nitrogenous bases are of two types: a) Purines comprising of Adenine (A) and Guanine (G) and b) Pyrimidines comprising of Cytosine (C) and Thymine (T).
In general, the composition of a nucleotide is a phosphate group linked with the help of a phosphodiester bond to a sugar molecule that consecutively associated with an organic base molecule. The sugar molecule is a pentose consisting of five carbon atoms. In RNA, the pentose is a ribose and in case of DNA, it is deoxyribose.
Among the four special inorganic nitrogenous bases, one of the four differs between these two similar long chain polymers. Adenine, Cytosine and Guanine are found in both RNA and DNA, whereas Uracil is present only in RNA and Thymine is present only in DNA.
The nitrogenous bases have a peculiar property of forming specific pairs with one another popularly known as the ‘base pairing mechanism’. Out of the four kinds of bases in a DNA molecule, Adenine (A), Guanine (G), Cytosine (C) and Thymine (T), Adenine of one DNA strand pairs up only with Thymine of the opposite DNA strand. Cytosine of one DNA strand pairs up only with Guanine of the opposite DNA strand. Thus Purine and Pyrimidine bases complement each other in a DNA molecule.
The nitrogenous bases present on one polynucleotide strand form weak hydrogen bonds with the nitrogenous bases of the opposite polynucleotide strand. These hydrogen bonds between the nitrogenous bases of the two polynucleotide strands hold them together. This bonding gives stability to the structure with the formation of a double helix. The double helix is a helical structure consists of two single strands running anti parallel to each other.
DNA is the predominant genetic material in most of the cellular forms. In case of some viruses such as TMV (Tobacco Mosaic Virus) and some bacteriophages, RNA acts as the genetic material. Both DNA and RNA are able to generate an exact copy of itself through the process of replication. This individual characteristic of these macromolecules suits them to act as a genetic material.
Both the forms are structurally and chemically stable. Evolutionary changes are possible in these molecules due to the scope of ‘mutations’ occurring in the sequence of bases in the linear polymeric chain.
Structural Individuality of DNA:
In 1869, Friedrich Mischer identified the DNA as an acidic substance present inside the nucleus. ‘Nuclein’ was the first name given by Mischer to the DNA. The structural explanation was not able to produce by them due to the technical difficulties in isolating the long chain polymers. Later, the double helical structure of the DNA molecule was first described by James Watson and Francis Crick in 1953 based on the X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin.
The DNA structure is illustrated by a right handed double helix, with about 10 nucleotide pairs per helical turn. It contains two polynucleotide chains twisted each other to form a double helix. Each DNA molecule contains a pentose sugar, a phosphate backbone and nitrogenous base. The two polynucleotide chains are attached to each other through nitrogenous bases. A nitrogenous base is linked to the pentose sugar through the N –Glycosidic linkage to form a nucleoside, such as guanosine or deoxyguanosine, adenosine or deoxyadenosinse, cytidine or deoxycitidine and uridine or deoxythymidine. When the 5’ – OH of any such nucleoside is linked with a phosphate groups through a phosphoester linkage, the respective nucleotide or deoxyribonucleotide (depending on the type of sugar present) is formed. A dinucleotide unit is formed by linking two such nucleotides through 3' – 5' phosphodiester linkage. The polynucleotide strand thus formed has a free phosphate moiety attached at the 5'-OH end of the ribose sugar which is known as the 5’ end whereas the other end is referred as 3' end since it contains a free 3'-OH group. The two polynucleotide strands has antiparallel polarity, if one of the chain possess the polarity 3'→ 5', then 5'→ 3'. The pitch of the right handed helix is 3.4 nm. Each turn consists of around 10bp. Hence the distance between successive base pairs in the helix is approximately 0.34 nm.
Base Pairing- A unique property:
The nitrogenous base present on one polynucleotide stands forms weak hydrogen bonds with the specific nitrogenous base of the opposite polynucleotide strand. Such specific pairing of bases is called complementary base pairing i.e. A pairing with T and G pairing with C. Cytosine (C) is common for both DNA and RNA. Thymine is present in DNA and Uracil (U) is present in RNA in the place of Thymine. Adenine and thymine are connected by two hydrogen bonds (non-covalent) while guanine and cytosine are connected by three. In a DNA molecule, the nitrogenous bases of the two strands always align with the same pair i.e. cytosine always pairs with guanine and adenine always pairs with thymine. Since both nucleotides are essential for the pair to be complete, Cytosine and Guanine or Adenine and Thymine, they will always be in exactly the same proportion.
This observation is at par with the results of Erwin Chargaff which states that ‘For a double stranded DNA, the ratios between Adenine and Thymine and Guanine and Cytosine are always constant and equals one’. In addition to this, the arrangement of base pairs is in such a manner that the plane of one base pair stacking over the other, awards the structural stability of DNA.
DNA, Repair and Recombination
Mutations mean ‘sudden changes’ which occur in the sequence of bases which subsequently alters the genotype, the genetic make up of an individual. The changes in the genotype certainly affect the phenotype, the external appearance of the individual too. Mutations are of many types, the most common are insertions, deletions and substitutions.
Insertions are the types of mutation occur due to the insertion of one or more base pairs in between the normal sequence of bases. JIM HAD BIG CAR If an unknown letters ST is inserted in between HAD and BIG, the statement can be rearranged as JIM HAD STB IGC AR This changes the reading frame and hence the information contained is also wrongly interpreted.
Deletion of one or more base pairs from the regular sequence of bases. JIM HAD BIG CAR Similarly, if the letter B is deleted in between HAD and BIG, the statement can be rearranged as JIM HAD IG CAR which is meaningless.
Substitutions refers to the phenomenon in which one or more base pair(s) is (are) substituted instead of another. JIM HAD BIG CAR
Similarly, if the letters RED are substituted in place of BIG, the rearranged statement gives different information. JIM HAD RED CAR Apart from these normal mutations, point mutations also occur where a single base pair in the DNA sequence is changed. Sickle cell anemia is one such harmful hereditary disease occurs as a result of point mutations. Genetic information is carried to the next generation without any discontinuity is the basis of life existence. This is made possible in nature only by the repair of damaged DNA molecules.
Every cell possesses its individual mechanism to carry out DNA repair. This mechanism can be broadly classified into three categories:
1. Base Excision Repair
The base excision repair involves the removal of the damaged regions of DNA by specialized enzyme systems and filling the gaps with newly synthesized DNA.
2. Mismatch Nucleotide Repair
This mechanism of DNA repair followed by the replication of DNA is just like a proof reading process using the parental strand as template in order to rectify the incorrect nucleotide incorporated into the daughter strand.
3. Direct repair of double stranded breaks in DNA
Sometimes pyrimidine dimers are formed due to the action of Ultra Violet rays from the sun. The photocleavage of such pyrimidine dimers along with an enzyme like DNA photolyase is called as direct repair.
Recombination is the process in which the genome of an individual can change in order to create a new combination of genes. Recombination simply occurs between the DNA molecules with similar sequences, often designated as homologous chromosomes. During meiotic stage of cell division, the two homologous chromosomes exchange block of genes between them to form a cross like structure which are known as ‘Holliday junctions’.
The maternal and the paternal chromosomes have their specific pairs of alleles. During this recombination process, the alleles are exchanged together so that a new set of alleles are formed with a part of both the maternal and the paternal chromosomes. A number of enzymes catalyze these events of recombination as this mechanism involves the breakage of two homologous duplex molecules of DNA and exchange happens at the break, then both are perfectly fastened together without leaving any knots. Hence recombination serves as the basis for diversity in genes happening in almost all plants, animals, even in smaller organisms like viruses, prokaryotes, plasmids and in the smaller DNA of the cell organelles like mitochondria also.
RNA: Structure and Classification:
Ribo nucleic acid (RNA) is structurally similar to DNA. Both nucleic acids are sugarphosphate polymers and both have nitrogen bases attached to the sugars of the backbone. Even then some of the important differences make these macromolecules distinct from one another. RNA is single stranded, not double stranded like DNA RNA molecules are smaller (shorter) than DNA molecules. Both DNA and RNA differ in composition, size and structure. RNA contains ribose sugar, whereas the deoxyribose sugar is present in DNA The nitrogenous base Uracil is present in RNA instead of thymine in DNA.
The major difference between RNA and DNA lies in their function. DNA has only one exclusive function of storing the genetic information of the individual in its sequence of nucleotide bases. But there are three main kinds of ribonucleic acid, each of which is characterized according to the specific jobs carried out by them.
1. Ribosomal RNAs:
Ribosomal RNAs (rRNAs) exist outside the nucleus in the cytoplasm of a cell in structures called ribosomes. Ribosomes are small, granular structures which serve as the site of protein synthesis. Each ribosome is a complex consisting of about 60% ribosomal RNA (rRNA) and 40% proteins.
2. Messenger RNAs:
Messenger RNAs (mRNAs) are the nucleic acids that "record" information from DNA in the nucleus of the cell. They carry this copied information to the ribosomes and are known as messenger RNAs (mRNA).
3. Transfer RNAs (tRNAs):
The function of transfer RNAs (tRNA) is to select amino acids from the amino acid pool of the cell and deliver one by one to protein chains which are being constructed.
The 2' – OH group present in every nucleotide of RNA makes RNA as more reactive and easily degradable also. RNA can also be addressed as catalytic at this stage. Hence comparing the structure of DNA and RNA, DNA is found to be more stable. Regarding the chemical nature of reactivity, RNA is more reactive than DNA. In case of expressing the characters, RNA is more powerful then DNA since they directly code for the proteins to be synthesized. DNA has to be always dependent on RNA for its transmission of genetic characters.
5. Genetic Code: An Overview
Chemically, gene is a segment of a large polynucleotide molecule called deoxyribonucleic acid (or DNA) which forms most important constituent of a chromosome. Actually, a gene is a part of the DNA molecule which has a code for the making of a particular protein in the cells. Genes have fixed locations on a chromosome i.e. their locations do not vary. Genes are around 30,000 to 40,000 in number which far exceed the number of chromosomes, which are only 23 pairs in total. A retrovirus uses RNA as its genetic material whereas all other organisms, including frogs, use DNA as their genetic material. Genetic material refers to the means or carrier by which genetic information is passed on through the generations.
George Camow, a physicist made a bold proposition that the code is made up of a combination of three bases (triplets), since there are only 20 amino acids. The genetic code consists of 64 triplets of nucleotides. These triplets are called codons. With three exceptions, each codon encodes for one of the 20 amino acids used in the synthesis of proteins. This phenomenon produces some redundancy in the code and hence most of the amino acids being encoded by more than one codon.
The genetic code can be expressed as either RNA codons or DNA codons. RNA codons occur in messenger RNA (mRNA) and are the codons that are actually "read" during the process of translation. But each mRNA molecule acquires its sequence of nucleotides through the process of transcription from the corresponding gene.
For each table, the left-hand column gives the first nucleotide of the codon, the 4 middle columns give the second nucleotide, and the last column gives the third nucleotide.
Exceptions to the Code:
The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes from the lowest microorganisms like bacteria, plants to humans. However, some exceptions have been found in mitochondrial codons and protozoans. Most of these involve assigning one or two of the three STOP codons to an amino acid instead.
6. Process of Transcription
Transcription is the process of converting the information contained in a DNA segment into RNA. It begins with the synthesis of mRNA molecules containing nearly several hundred to several thousand ribonucleotides. The number of ribonucleotides depends upon the size of the protein to be made. In case, if the DNA were to project out into the cytoplasm where the ribosomes are located, in order to give the instructions for which proteins were to be made, it would be more susceptible to get mutations from the mutagens. Hence DNA is secured inside. It uses the mRNA as the messenger and sends the information it wants to encode out to the ribosomes which in turn carry out the instructions in the cytoplasm. A large number of mRNA has been transcribed according to the specific genetic sequence inside the DNA. Each of the 100,000 or so proteins in the human body is synthesized from such different mRNAs.
Synthesis of mRNA:
A messenger RNA is synthesized in the cell nucleus by transcription of a particular segment of DNA. A small section of the DNA double helix unwinds, and the bases on the two strands are exposed.
By forming hydrogen bonds with their complementary bases on DNA, RNA nucleotides (ribonucleotides) line up in the proper order. Then the nucleotides are joined together by a DNA dependent RNA polymerase enzyme, and mRNA forms.
When DNA gets replicated, both the strands are copied. Here in this case, only one of the two DNA strands is transcribed into mRNA. This proves again that the RNA is a singlestranded molecule.
If both the DNA strands are converted into respective mRNAs, the resulting m RNAs would be complementary to each other and there is a possibility of forming a double stranded RNA. Hence protein synthesis will not take place and the purpose of transcription will not be solved.
The DNA strand that is transcribed is called the template strand or the antisense strand while its complement is called the informational strand or the coding or sense strand.
The template strand and the coding strand are complementary and also the template strand and the mRNA molecule are also complementary. This fact follows that the messenger RNA molecule produced during transcription is a copy of the DNA coding strand. The only difference is Uracil in RNA instead of Thymine in DNA.
7. Translation: Basis of Protein Synthesis
Translation is the process of polymerization of amino acids to form a polypeptide chain. The mRNA determines the sequence of amino acids with the sequence of bases contained in it. The formation of peptide bond in between the amino acids requires energy and hence this energy is sustained inside the amino acids by the process of amino acylation of tRNA. Peptide bond formation is much easier between two such charged tRNAs. Ribosomes which are attached to the rough Endoplasmic reticulum (ER) are the sites of protein synthesis. The ribosome is made up of two units, a smaller subunit and a larger subunit. The translation process begins when the small subunit encounters an mRNA. Inside the larger subunit, there are two sites where the succeeding amino acids bind to and form a peptide bond. Ribosome also plays its role as a catalyst in peptide bond formation.
The initiator tRNA locates the start codon region in the mRNA and directs the ribosome to bind at that site. The main phase of elongation continues where the amino acid taken by tRNA will get linked to the respective codon in mRNA by the formation of complementary base pairs with the anticodons of tRNA. The ribosome moves to codon by codon and the amino acid are added like beads in a chain. When the ribosome comes across the stop codon, the elongation terminates and the completely synthesized polypeptide chain of amino acids is released out.
Human Genome Project and DNA Fingerprinting:
Human Genome Project: Genetics is the branch of biology that deals with the study of heredity and variation.
The most advancement in the field of genetics is the Human Genome Project (HGP). It was a 13-year mega project coordinated by the U.S. Department of Energy and the National Institutes of Health. During the early years of the HGP, the Wellcome Trust (U.K.) became a major partner. Apart from that, additional contributions came from Japan, France, Germany, China, and others. It was accomplished in the year 2003. The goals of the HGP were
• • • • • •
Determining the sequences of the 3 billion chemical base pairs that constitute human DNA. identifying all the approximately 20,000-25,000 genes in human DNA, storing all this information in databases, improving tools for data analysis, transferring related technologies to the private sector, and Addressing the ethical, legal, and social issues (ELSI) that may arise from the project.
Though the HGP is finished, analyses of the data are still continuing for many years.
Importance of genome:
Genome is the entire DNA in an organism, including its genes. Genes carry information for making all the proteins required by all organisms. These proteins determine, among other things, how the organism looks, how well its body metabolizes food or fights infection, and sometimes even how it behaves. DNA is made up of four similar chemicals (called bases and abbreviated A, T, C, and G) that are repeated millions or billions of times throughout a genome. The human genome, for example, has 3 billion pairs of bases. Assigning the genetic and physical maps on the genome is the next challenging task where researches are under progress. Many non – human experimental organisms like yeast, bacteria, Drosophila (fruit fly), Caenorhabditis elegans (a free living nematode) have been sequenced for their genomes.
DNA finger printing:
The differences in the sequences of DNA give every individual unique in their phenotypic appearance. DNA finger printing is the technique which involves the identification of differences in some specific regions called ‘repetitive DNA’ in the sequence of DNA in the genome. Since this repetitive DNA contains a small stretch of DNA repeated many a times, it is easy to identify the variations between individuals without overwhelming task of comparing the entire genome. This works on the basis of polymorphism in DNA sequences. It has its wider applications in field of medical biotechnology, forensic science, genetic biodiversity and evolutionary biology. Having a thorough knowledge about the effects of DNA variations among individuals can lead to revolutionary new ways to diagnose, treat, and someday prevent the thousands of disorders that affect us. Besides providing clues to understand the human biology, the study about nonhuman organisms' DNA sequences can lead to an understanding of their natural capabilities. We can apply those potential characteristics toward solving challenges in health care, agriculture, energy production and also in environmental remediation.
Points to Remember:
• • • Nucleic acids are the genetic carrier molecules which carry the genetic information from one generation to the next. Nucleic acids are of two type, Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). Primary structures of DNA and RNA are similar to each other forming a long chain of nucleotides composed of single nucleosides (a sugar and a phosphate moiety). • A nucleotide is a complex structure formed by the combination of a nitrogenous base, a sugar [pentose-deoxyribose (DNA) or ribose (RNA)], and a phosphate group. • Nitrogenous bases are of two types: Purines comprising of Adenine (A) and Guanine (G) and Pyrimidines comprising of Cytosine (C) and Thymine (T). • Adenine, Cytosine and Guanine are found in both RNA and DNA, whereas Uracil is present only in RNA and Thymine is present only in DNA. • • In 1869, Friedrich Mischer identified the DNA as an acidic substance present inside the nucleus and named it first as ‘Nuclein’. Double helical structure of the DNA molecule was first described by James Watson and Francis Crick in 1953 based on the X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin. • • • • • A dinucleotide unit is formed by linking two such nucleotides through 3' – 5' phosphodiester linkage. The pitch of the right handed helix is 3.4 nm. Each turn consists of around 10bp. Hence the distance between successive base pairs in the helix is approximately 0.34 nm. Adenine and thymine are connected by two hydrogen bonds (noncovalent) while guanine and cytosine are connected by three. Chargaff’s rule states that ‘For a double stranded DNA, the ratios between Adenine and Thymine and Guanine and Cytosine are always constant and equals one’.
Mutations mean ‘sudden changes’ which occur in the sequence of bases which subsequently alters the genotype, the genetic make up of an individual.
Mutations are of many types, the most common are insertions, deletions and substitutions. DNA repair mechanism can be broadly classified into three categories as Base excision repair, Mismatch nucleotide repair and direct repair of double stranded breaks in DNA.
• • • • •
Recombination is the process in which the genome of an individual can change in order to create a new combination of genes. Recombination occurs between two homologous chromosomes, during meiotic stage of cell division. Homologous chromosomes exchange block of genes during recombination forming a cross like structure known as ‘Holliday junctions’. RNA is single stranded, not double stranded like DNA. Three main kinds of ribonucleic acid are Ribosomal RNAs, Messenger RNAs, ransfer RNAs (tRNAs), each of which is characterized according to the specific jobs carried out by them.
• • • • • •
Comparing the structure of DNA and RNA, DNA is found to be more stable. A gene is a part of the DNA molecule which has a code for the making of a particular protein in the cells. Genetic code consists of 64 triplets of nucleotides. These triplets are called codons. Transcription is the process of converting the information contained in a DNA segment into RNA. A messenger RNA is synthesized in the cell nucleus by transcription of a particular segment of DNA. The DNA strand that is transcribed is called the template strand or the antisense strand while its complement is called the informational strand or the coding or sense strand.
Translation is the process of polymerization of amino acids to form a polypeptide chain. The mRNA determines the sequence of amino acids with the sequence of bases contained in it.
Genetics is the branch of biology that deals with the study of heredity and variation.
The chief goal of the Human Genome Project [HGP] is to determine the sequence of the 3 billion chemical base pairs that constitute human DNA. Many non – human experimental organisms like yeast, bacteria, Drosophila (fruit fly), Caenorhabditis elegans (a free living nematode) have been sequenced for their genomes.
DNA finger printing is the technique which involves the identification of differences in some specific regions called ‘repetitive DNA’ in the sequence of DNA in the genome.
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