Molecular Basis Of Inheritance
– A good summary makes the whole chapter easy.
DNA, RNA and Search for Genetic Material The DNA DNA (Deoxyribonucleic acid) is a double-stranded, helical molecule. Its structure was cracked by Watson and Crick based on the X-ray crystallography results provided by Maurice Wilkins and Rosalind Franklin. Each strand of the DNA molecule is composed of repeating units of nucleotides. A nucleotide consists of 3 components, namely, a pentose sugar(deoxyribose sugar),nitrogenous base (purines or pyrimidines) and a phosphate group.
There are two types of purines, namely, adenine and guanine. Pyrimidines are of three types, i.e. thymine, cytosine and uracil. The nitrogenous bases are common in DNA and RNA, except uracil is found in RNA and thymine is present only in DNA. The negative charge on DNA is due to the presence of the negatively charged phosphate groups. A nitrogenous base is linked to the pentose sugar through the N-glycosidic linkage. Two nucleotides are linked through 3′-5′ phosphodiester linkage. A polymer formed in such a manner has a free phosphate group at 5′-end of ribose sugar, which is referred to as 5′-end of polynucleotide chain. The other end of the polymer has a free 3′-OH (hydroxyl) group of the deoxyribose sugar. This is the 3′-end of the polynucleotide chain. The bonding between sugar and phosphate groups forms the backbone of a polynucleotide chain. The nitrogenous bases are linked to sugar moieties and project from the sugar-phosphate backbone. Structure of DNA The characteristic features of the double helical structure of DNA are as follows:
- Two polynucleotide chains wrap around each other, where the backbone is constituted by pentose sugar and phosphate, and the bases project inside.
- The two DNA chains run antiparallel to each other. It implies that if one chain has the polarity 5′-3′, then the other has 3′-5′.
- The bases in the two strands are paired through hydrogen bonds between the base pairs. Adenine forms two hydrogen bonds with thymine whereas cytosine forms three hydrogen bonds with guanine.
- The two strands are coiled in a right-handed pattern.
- The plane of one base pair lies over the other in a double helix. This, in addition to H-bonds, gives stability to the helical structure.
Packaging of DNA Helix Positively charged basic proteins that surround the DNA are known as histones. Histones are rich in amino acids like lysine and arginine; these are basic in nature. Histone proteins are organized to form a unit of eight molecules called the histone octamer. The DNA is negatively charged and is packaged by wrapping around the positively charged histone octamer. This forms a structure called nucleosome. A nucleosome contains 200 base pairs. Nucleosomes form the repeating unit of a structure called chromatin in the nucleus. Chromatin are thread-like stained bodies seen in nucleus. The nucleosomes in chromatin appear as ‘beads-on-string’structures when viewed under the electron microscope.
RNA RNA (Ribonucleic acid) was the first genetic material. There was evidence to prove that life processes, like metabolism, translation, splicing, etc., have evolved around RNA. (i) There are some important biochemical reactions in living systems that are catalysed by RNA catalysts and not by protein enzymes. (ii) DNA has evolved from RNA with chemical modifications that makes it more stable because RNA being single stranded and a catalyst, is reactive and hence, unstable. Types of RNA (i)mRNA (messenger RNA) acts as the template for transcription. (ii) tRNA (transfer RNA) carries amino acids and reads the genetic code. (iii) rRNA (ribosomal RNA) plays a structural and catalytic role during translation. All the three RNAs are essential to synthesise a protein in a cell. DNA as a Genetic Material Griffith performed an experiment known as transformation experiment. He used two strains of Streptococcus pneumoniae. These were the two different strains which were used to infect the mice. The two strains used were type III-S (smooth), that contained outer capsule made up of polysaccharide and type II-R (rough) strain, that did not contain capsule. The capsule protects the bacteria from the host’s immune system. The S strain was disease-causing whereas the R strain was non-infective.
- Rough strain of Streptococcus was injected into the mouse. The mouse lived.
- Smooth strain of Streptococcus was injected into the mouse. The mouse died.
- When heat-killed smooth strain of Streptococcus was injected into the mouse, the mouse lived.
- In the last set of experiments, rough strain and heat-killed smooth strain were injected into the mouse. The mouse died.
This proved that there was some substance present in heat-killed S strain that was converting or transforming the rough strain into virulent strain, leading to the death of the mouse. This transforming substance was later found to be DNA.
Biochemical Nature of Transforming Material Oswald Avery, Colin MacLeod and Maclyn McCarty, worked to determine the biochemical nature of transforming material in Griffith’s experiment.
- They purified biochemicals (RNA, Proteins and DNA, etc) from heat-killed S-cells and discovered that DNA alone from S-strain caused R-strain to be transformed.
- They also discovered that proteases (protein-digesting enzymes) and RNAases (RNA-digesting enzymes) did not affect the transformation of the non-virulent strains into the virulent strains.
- But it was observed that DNAase did inhibit transformation. This indicated that DNA caused the transformation.
- It was thus concluded that DNA is the hereditary material. But, still all the biologists were not convinced.
- Alfred Hershey and Martha Chase (1952) gave proof that DNA is the genetic material.
- In the experiment, bacteriophages (viruses that infect bacteria) were used.
- They grew some bacteriophages on a medium that contained radioactive phosphorus and some others on a sulphur-containing radioactive medium.
- The bacteriophages grown in the presence of radioactive phosphorus contained radioactive DNA. Radioactivity was not observed in the protein part. This is because DNA contains phosphorus but protein is devoid of phosphorus. In the same way, bacteriophages grown on radioactive sulphur contained radioactive protein, but not radioactive DNA. This is because DNA does not contain sulphur.
- Radioactive phages were allowed to attach to E. coli bacteria. As the infection proceeded, phage coats were removed from the bacteria by centrifuging. The phage particles were separated from the bacteria by rotating them in a centrifuge.
- Bacteria which were infected with phages that had radioactive DNA were radioactive, indicating that DNA was the material that passed from the phage to the bacteria.
- Bacteria that were infected with phages that had radioactive proteins were not radioactive. This indicated that the proteins did not enter the bacteria from phages. It proved that DNA is the genetic material that is passed from the bacteriophages to the bacteria.
Central Dogma, Replication, Transcription and Translation, Genetic Code Central Dogma of Molecular Biology
- It was given by Crick. It explains how the genetic information flows in a biological system.
- Central dogma explains how DNA replicates and then how it gets converted into messenger RNA (mRNA) by the process of transcription. Then this mRNA is translated to form proteins. Now we have a latest advancement called Reverse transcription in which DNA is formed from RNA. It is found in some viruses e.g. HIV.
- DNA replication is a process where two identical copies of DNA are produced from a single DNA molecule.
- It involves the separation of the two strands of a DNA helix to form two new DNA molecules. Out of the two new strands of DNA formed, one is identical to one of the parent strands and the one is complementary to the parent strand.
- This is the semi-conservative mode of replication. Before the cell enters the mitotic phase, the DNA is replicated in the S phase of interphase.
Enzyme Machinery and Process of DNA Replication
- The most important enzyme involved in DNA replication is the DNA polymerase. DNA replication is an energy dependent process.
- Replication begins on the specific site on the DNA, known as the origin of replication (ori).
- During the process of replication, the two DNA strands do not separate completely, the replication occurs within the small opening of the DNA helix known as a replication fork.
- At the origin, the Helicase enzyme starts unzipping and unwinds the DNA. The strands are thus separated. The Single-stranded binding proteins (SSBs) help in keeping the strands separated.
- Topoisomerase prevents the DNA from supercoiling and primase plays a role in making RNA primers on both the strands. It helps the DNA polymerase to identify where to start from.
- The DNA polymerase adds the nucleotides in 5′ to 3′ direction. Consequently, on the strand with polarity 3′-5′, the replication is continuous, while on the other strand with polarity 5′-3′, it is discontinuous.
- The strand with continuous replication is known as the leading strand whereas the one with discontinuous replication is known as the lagging strand.
- The enzyme DNA ligase later joins the discontinuously synthesized fragments, called Okazaki fragments.
- Transcription is the process where genetic information from a strand of DNA is copied to form the RNA. The RNA is formed as a complementary strand to the DNA strand. Here during the addition of bases in RNA, adenine forms a base pair with uracil instead of thymine.
- During transcription, one of the strands of DNA acts as a template for mRNA formation. RNA polymerase enzyme synthesises the mRNA. Transcription is carried out for a particular DNA segment which is required further for gene expression.
- A transcription unit in DNA comprises three regions, namely, a promoter, structural gene and a terminator. DNA dependent RNA polymerase brings about the addition of nucleotides in 5′-3′ direction.
- Promoter is the region where RNA polymerase binds. Terminator defines the end of transcription.
- RNA polymerase binds to the promoter and initiates the process of transcription This is the initiation stage.
- It adds nucleotides following the rule of complementarity. It also facilitates opening of the helix which then ensures the elongation of the strand.
- Once the polymerase reaches the terminator region, the newly made mRNA falls off. The RNA polymerase also detaches. This results in termination of transcription.
- It is the process of protein synthesis by reading the mRNA codons. It occurs in cytoplasm. Ribosomes are the sites for protein synthesis in a cell.
Process of Translation
- Charging of tRNA: Binding of specific amino acid to the tRNA molecule.
- Initiation of polypeptide synthesis: Identifying the initiation codon on mRNA and attachment of the specific charged tRNA with the complementary anticodon.
- Elongation of polypeptide synthesis: It involves the bonding of amino acids to form the polypeptide chain.
- Termination of polypeptide synthesis: It involves the end of the process of protein synthesis by identification of stop codon on mRNA.
- Genetic code is the sequence of nucleotides in DNA or RNA that determines the sequence of amino acids in a polypeptide chain.
- George Gamow suggested that genetic code should be a combination of 3 nucleotides to code 20 amino acids.
- H.G. Khorana developed a chemical method to synthesise RNA molecules with a defined combination of bases.
- Marshall Nirenberg’s system for protein synthesis finally helped in deducing the code.
Salient Features of Genetic Code
- The code is triplet. 61 codons code for amino acids and 3 codons do not code for any amino acids; these are called stop codons (UAG, UGA and UAA).
- Codon is unambiguous and specific. It codes for just one amino acid.
- The code is degenerate; this means that some amino acids are coded by more than one codon.
- The codon is read in mRNA in a continuous fashion; without any punctuation.
- The codon is nearly universal. AUG has a dual function. It codes for methionine amino acid and also acts as an initiator codon.
Regulation of Gene Expression, HGP and DNA Fingerprinting Regulation of Gene Expression All the genes are not activated constantly. The genes are needed only when proteins are needed. These are thus called regulatory genes and are made to function only when required and remain non-functional at other times. Such regulated genes, therefore, are required to be switched ‘on’or ‘off’ when a particular function is to begin or stop. These genes form an ‘operon’. Lac Operon
- An operon consists of structural genes, operator genes, promoter genes, regulator genes, and repressor.
- Lac operon consists of Lac Z, Lac Y, and Lac A genes as structural genes. These genes code for specific enzymes. Lac Z codes for galactosidase, Lac Y codes for permease and Lac A codes for transacetylase. When repressor molecules bind the operator, the transcription process is inhibited.
- When the repressor does not bind the operator and instead inducer binds, transcription is switched on. In the case of lac operon, lactose is an inducer. So, binding of the lactose to the repressor, switches on the transcription.
- In Transcription, with the help of RNA polymerase enzymes, the messenger RNA is produced. The main function of mRNA is to facilitate the synthesis of a protein.
Human Genome Project (HGP) The project was coordinated by the United States Department of Energy and the National Institute of Health. The method involved the two major approaches- first identifying all the genes that express as RNA called Express sequence tags (EST). The second is the sequencing of the all set of genomes that contained the all the coding and non-coding sequence called sequence Annotation. Goals of HGP
- To identify all the genes (20,000 to 25,000) in human DNA.
- To determine the sequence of the 3 billion chemical base pairs that make up human DNA.
- To store this information in the database.
- To improve tools for data analysis.
- To transfer related information to other sectors.
- To address the legal, ethical and social issues that may arise due to the project.
Salient Features of the Human Genome
- The human genome is made up of 3164.7 million nucleotide bases.
- On an average, a gene consists of 3000 bases, but size may vary.
- Human beings have about 30,000 genes.
- For over 50 percent of the discovered genes, the functions are still unclear.
- Less than 2 percent of the genome contributes for protein synthesis.
- Human genome consists of a large portion of repeated sequences.
- Chromosome 1 with 2968 genes has the most number of genes. The Y chromosome with 231 genes has the least number of genes.
DNA Fingerprinting Alec Jeffreys et al (1985) developed the procedure of genetic analysis and forensic medicine, called DNA fingerprinting. Of the total base sequence present in humans, 99.9% in all human beings are identical. The remaining 0.1% differs from person to person and makes every individual unique. DNA fingerprinting involves identifying the difference between two DNA molecules at the specific regions where the sequence is repeated many times called repetitive DNA. Steps in DNA Fingerprinting
Applications of DNA Fingerprinting This technique is used to:
- Identify criminals in forensic laboratories.
- Settle paternity disputes.
- Verify whether a hopeful immigrant is really a close relative of an already established resident.
- Identify racial groups to rewrite biological evolution.