Chapter at A Glance
- Genetic material is that substance which not only controls the formation and expression of traits in an organism but can replicate & pass from a cell to its daughter cell or from one generation to another.
- DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are two types of nucleic acids, found in living systems.
DEOXYRIBONUCLEIC ACID (DNA)
- DNA is the hereditary material in humans and almost all other organisms. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA).
- DNA consists of a nitrogenous base, pentose sugar and a phosphate group. A nucleotide is a basic building block of DNA.
- A nitrogenous base is attached to the pentose sugar by an N-glycosidic linkage to form a nucleoside, i.e., Nucleoside =Nitrogen base+ Pentose sugar. In 1953, James Watson and Francis revealed the ‘double helix’ model of the structure of DNA, based on X-ray diffraction data from Maurice Wilkins and Rosalind Franklin.
- It consists of two polynucleotide chains where the sugar and phosphate group form the backbone and the nitrogenous bases project inside the helix. The two polynucleotide chains have anti-parallel polarity i.e. if one strand has 5′ 3′ polarity, the other strand has 3′ 5′ polarity.
- Nitrogenous base are of two types – purines (9 membered double rings with nitrogen at 1, 3, 7 & 9th positions) and pyrimidines (6 membered rings with nitrogen at 1 and 3rd position).
- Purines are of two types – adenine (A) and guanine (G) and pyrimidines are of three types – thymine (T), cytosine (C) and uracil (U).
- A characteristics that differentiate DNA from RNA is that DNA contains all of the nitrogen bases except uracil and RNA contains all of the nitrogen bases except thymine.
- Pentose sugar is deoxyribose in DNA and ribose in RNA.
- DNA is composed of 2 strands wound around each other to form a double helix, with base inside and sugar phosphate backbone on outside.
- 2 DNA strands are organised in antiparallel and complementary arrangement [i.e., 2 strands run in opposite orientation (one in 5′ – 3′ and allies in 3′ – 5′)]. A= T, G = C is called complementary base pairing that makes sequencing possible if template strand sequence is known.
- Erwin Chargaff (1950) found that purine and pyrimidine contents of DNA are equal.
- DNA of bacteria such as E.coli is a circular double stranded molecule which contain 4.6 million base pairs.
- Eukaryotic DNA is organised into bead structure called nucleosome. Negatively charged DNA is wrapped around this positively charged histone octamer to form nucleosome.
- On the basis of packaging, chromatin is classified into 2 types-euchromatin and heterochromatin.
- Euchromatin is transcriptionally active, loosely packed, rich in gene concentration, lightly stained.
- Heterochromatin is transcriptionally inactive, densely packed, darkly stained.
- Establishment of DNA as genetic material by Griffith who carried out experiment with Streptococcus pneumoniae (bacterium causing pneumonia) which is used to infect mice.
- Hershey & Chase (1962) discovered that DNA is the genetic material of bacteriophage.
- DNA replication or DNA synthesis is the process of copying a double stranded DNA prior to cell division.
- Replication of DNA is found to be semiconservative and its experimental proof has been provided by Meselson and Stahl in 1958.
- DNA replication occurs during S phase of cell cycle and require template and DNA polymerase along with primer.
- DNA replication begins at a specific site called ori (origin) which has recognition site for DNA polymerase, which also provides site for attachment of RNA primer.
- The enzyme which form RNA from DNA are called RNA polymerase. The synthesis of RNA primer is brought about by enzyme primase.
- In bacterial chromosome, replication starts at a single origin and both strands of a double helix serve as template for DNA synthesis which proceeds outwards in both directions from single origin (i.e., it is bidirectional).
- dsDNA is antiparallel, one strand runs in 5′-3′ direction and its complementary runs in 3′-5′. New DNA strand is made against each template strand. DNA polymerase makes DNA only in 5′-3′ direction. So are template strand with 3′- 5′ orientation, new DNA strand is made in continuous piece in correct 5′-3′ direction. This DNA is called leading strand.
- On template strand which has 5′ 3′ orientation, DNA polymerase synthesizes
- short pairs of new DNA (about 1000 nucleotides long) in 5′ 3′ direction and then joins these piece together. These small fragments are called okazaki fragments and new DNA strand made in this discontinuous manner is called lagging strand. Okazaki fragments are joined by means of DNA ligase.
RIBONUCLEI ACID(RNA)
- RNA is a vital molecule with a long chain of nucleotides. It is a first genetic material. A nucleotide chain comprises a phosphate, a ribose sugar, and nitrogenous base.
- RNA acts as a catalyst and as a genetic material.
- RNA is a non hereditary nucleic acid except in some viruses (retro viruses). On the basis of molecular size & function, three forms of non genetic RNA are-
- mRNA (messenger RNA)– provides template for translation.
- tRNA (transfer RNA)– brings aminoacids and reads genetic code.
- rRNA (ribosomal RNA)- structural and catalytic role during translation.
- RNA processing : In prokaryotes, mRNA transcribed from protein coding genes requires no modification prior to translation. In fact, many mRNA molecules begin to translate even before RNA synthesis has finished.
- Ribosomal RNA (rRNA) and transfer RNA (tRNA) are synthesised as precursor molecule that do not require post-transcriptional processing.
- RNA transcription requires enzyme RNA polymerase, DNA template, all four types of ribonucleoside triphosphates (ATP, GTP, CTP & UTP), divalent metal ions Mg2+ or Mn2+ as cofactor, rho (p) factor etc.
- RNA polymerase can initiate transcription at specific DNA sequences known as promoters. It then produces an RNA chain which is complementary to the DNA strand used as template.
GENETICCODEANDPROTEIN SYNTHESIS
- Genetic code is defined as a relationship between the sequence of amino acids in a nucleotide chain of mRNA or DNA and amino acid in a polypeptide chain.
- Triplet nature of genetic code was proposed by F.H.C.Crick. Nirenberg gave the first experimental proof for triplet code.
- UGA, UAG and UAA are nonsense codon which do not code for any amino acid.
- Wobble hypothesis explains why multiple codons can code for a single amino acid. This hypothesis states that the third position (3′) of the codon on mRNA and the first position (5′) of the anticodon on tRNA are bound less tightly than the other pair and therefore unusual base combination
- The characteristics of the genetic code are degeneracy of genetic code, non overlapping, universality, triplet in nature, comma-less, non-ambiguous.
- Protein synthesis is a process of creating protein molecules. It involves amino acid synthesis, transcription, and translation.
- Process of copying genetic information from one strand of DNA to RNA is called transcription.
- The strand of DNA with polarity 3′ —> 5′ act as template strand and the DNA strand with polarity 5′ —> 3′ act as coding strand.
- Transcription is carried out by DNA dependent RNA polymerase. Transcription unit is the segment of deoxyribonucleic acid between the sites ofinitiation & termination of transcription.
- Transcription and translation takes place in same compartment (there is no separate cytosol and nucleus in bacteria). So transcription and translation are coupled in bacteria where as in eukaryotes transcription and translation occur in nucleus and cytosol respectively and thus compartmentalised.
- Transfer of genetic information from a polymer of nucleotides to a polymer of amino acids is called translation.. This is accomplished with the help of genetic code which is row of three consecutive nucleotides – coding for 20 amino acids.
- Codon exists on mRNA, corresponding anticodon on tRNA and protein synthesis and elongation occurs in rRNA.
- tRNA is intermediate between reading of codons on mRNA and formation of amino acid chain for protein.
- The main step in protein synthesis are activation, initiation, elongation & termination of polypeptide chain.
REGULATION OF GENE EXPRESSION
- Gene expression is the mechanism at the molecular level by which a gene is able to express itself in the phenotype of an organisms.
- In eukaryotes, the regulation could be exerted at-
- Transcriptional level (formation of primary transcript).
- Processing level (RNA splicing) level.
- Transport of mRNA from nucleus to cytoplasm.
- Translational level.
- One of the best studied example of gene expression is lac operon.
- Model of lac operon was suggested by Jacob and Monod in 1961.
- In lac operon, a polycistronic structural gene is regulated by a common promoter & regulatory genes.
- Structural genes produces 3 enzymes for the degradation of lactose to glucose & galactose.
- Operator gene: DNA sequence that regulates transcription of the structural genes.
- Regulator gene : Encodes a protein that recognizes operon sequence. Regulation of the operon by repressor is called as negative regulation as it decreases expression of structural gene.
- Positive control of gene expression is when the regulatory protein binds to DNA and increases rate of transcription. In this case the regulating protein is called an activator.
HUMAN GENOME PROJECT AND DNA FINGERPRINTING
- Human Genome Project established whole genome of human with the help of genetic engineering techniques, cloning techniques and bioinformatics.
- DNA fingerprinting is a technique for identifying individuals, generally using repeated sequences (repetitive DNA) in the human genome that produces a pattern of band that is unique for every individual.
- DNA technique has following steps:
- Isolation of DNA and digestion of DNA by restriction endonucleases. Separation of DNA fragments by (gel) electrophoresis and transferring (blotting) of separated DNA fragments to synthetic membrane or nitrocellulose or nylon.
- Hybridisation using VNTR probe and detection of hybridised DNA fragments by autoradiography.
- Matching the banding pattern so obtained with that of relative.
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