n Two types of nucleic acids are present in living systems—ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
1. The Structure of Polynucleotide Chain
n A nucleotide is the basic unit of polynucleotide chain of DNA or RNA.
n Each nucleotide is composed of three components:
(i) a nitrogenous base,
(ii) pentose sugar (ribose in case of RNA and deoxyribose for DNA), and
(iii) a phosphate group.
n Nitrogenous base: It is of two types, purine (adenine and guanine) and pyrimidine (cytosine and thymine). Uracil is only present in RNA instead of thymine.
n A nitrogenous base is attached to the pentose sugar by an N-glycosidic linkage to form a nucleoside.
n When a phosphate group is attached to 5′–OH of a nucleoside through phosphodiester linkage, a nucleotide is formed.
n Two nucleotides are joined through 3′–5′ phosphodiester linkage and a dinucleotide is formed. Thus, when numerous nucleotides are joined, a polynucleotide chain is formed.
n One end of polynucleotide chain contains pentose sugar with free OH at 5′ end (it is called 5′–end) and the other end contains sugar with free OH at 3′ end (it is called 3′–end).
n Sugar and phosphate constitute the backbone of polynucleotide chain and nitrogenous bases are linked to sugar moiety which projects from the backbone.
2. Salient Features of Double Helical DNA
n James Watson and Francis Crick in 1953 proposed the double helix model of DNA based on the X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin and Erwin Chargaff’s rules of base pairing.
n Chargaff’s rules:
(i) The amount of adenine is always equal to the amount of thymine and the amount of guanine is always equal to the amount of cytosine, i.e., [A] = [T], [G] = [C]
(ii) Adenine is joined to thymine with two hydrogen bonds and guanine is joined to cytosine by three hydrogen bonds.
(iii) The ratio of adenine and guanine to that of thymine and cytosine is always equal to one, i.e.,
Following are some features of DNA:
(i) DNA is made up of two polynucleotide chains, where the backbone is made up of sugar and phosphate groups and the nitrogenous bases project towards the centre.
(ii) There is complementary base pairing between the two strands of DNA. ‘A’ pairs with ‘T’ with 2 hydrogen bonds and ‘C’ pairs with ‘G’ with 3 hydrogen bonds.
(iii) The two strands are coiled in right-handed fashion and are anti-parallel in orientation. One chain has a 5′→3′ polarity while the other has 3′→5′ polarity.
(iv) The diameter of the strand is always constant due to pairing of purine and pyrimidine, i.e., adenine is complementary to thymine while guanine is complementary to cytosine.
(v) The distance between two base pairs in a helix is 0.34 nm and a complete turn contains approximately ten base pairs. The pitch of the helix is 3.4 nm and the two strands are right-handed coiled.
(vi) Plane of one base pair stacks over the other in double helix. This in addition to hydrogen bonds confers stability to the helical structure.
(vii) Linkage between nitrogenous base and pentose sugar is N-glycosidic linkage.
3. Central Dogma of Molecular Biology
n Francis Crick proposed the central dogma of molecular biology which states that genetic information flows from DNA to mRNA (transcription) and then from mRNA to protein (translation) always unidirectionally (except bidirectionally in some viruses and the process is called reverse transcription).
4. Packaging of DNA
n Distance between two base pairs is 0.34 nm. If length of DNA double helix in a typical mammalian cell is calculated, i.e., total number of base pairs × by distance between two consecutive base pairs, i.e., 6.6 × 109 bp × 0.34 × 10–9 m/bp = 2.2 meters. This length is far greater than the dimension of a typical nucleus (= 10–6 m). Therefore, DNA needs to be packaged to fit in the nucleus.
(i) Packaging of DNA in prokaryotes
l In prokaryotes, well-defined nucleus is absent so DNA is present in a region called nucleoid. The negatively charged DNA is coiled with some positively charged non-histone basic proteins.
l DNA in nucleoid is organised in large loops held by proteins.
(ii) Packaging of DNA in eukaryotes
l Roger Kornberg (1974) reported that chromosome is made up of DNA and protein.
l Later, Beadle and Tatum reported that chromatin fibres look like beads on the string, where beads are repeated units of proteins.
l The proteins associated with DNA are of two types—basic proteins (histones) and acidic non-histone chromosomal (NHC) proteins.
l The negatively charged DNA molecule wraps around the positively charged histone proteins to form a structure called nucleosome.
l The nucleosome core is made up of four types of histone proteins—H2A, H2B, H3 and H4 occurring in pairs.
l 200 bp of DNA helix wrap around the nucleosome by 1¾ turns, plugged by H1 histone protein.
l Repeating units of nucleosomes form the chromatin in nucleus, which is a thread-like structure.
l The chromatin is packed to form a solenoid structure of 30 nm diametre.
l Further supercoiling forms a looped structure called the chromatin fibre.
l These chromatin fibres further coil and condense at metaphase stage of cell division to form chromosomes. Packaging of chromatin at higher level requires NHC proteins.
5. Differences between Euchromatin and Heterochromatin
S. No. | Euchromatin | Heterochromatin |
(i) | Regions of chromatin, which are loosely packed during interphase are called euchromatin. | Regions of chromatin, which are densely packed during cell division are called heterochromatin. |
(ii) | When chromosomes are stained with Feulgen stain (specific for DNA), these appear as lightly stained chromatin. | When chromosomes are stained with Feulgen stain, these appear as intensely stained chromatin. |
(iii) | Euchromatin contains active genes. | Heterochromatin contains inactive genes. |
(iv) | They do not contain repetitive DNA sequences. | They are enriched with highly repetitive tandemly arranged DNA sequences. |
(v) | It is transcriptionally active. | It is transcriptionally inactive. |
6. Transforming Principle
n Frederick Griffith (1928) conducted experiments with Streptococcus pneumoniae (bacterium causing pneumonia).
n He observed two strains of this bacterium—one forming smooth shiny colonies (S-type) with capsule, while other forming rough colonies (R-type) without capsule.
n When live S-type cells were injected into mice, they died due to pneumonia.
n When live R-type cells were injected into mice, they survived.
n When heat-killed S-type cells were injected into mice, they survived and there were no symptoms of pnuemonia.
n When heat-killed S-type cells were mixed with live R-type cells and injected into mice, they died due to unexpected symptoms of pneumonia and live S-type cells were obtained from mice.
n He concluded that heat-killed S-type bacteria caused a transformation of the R-type bacteria into S-type bacteria but he was not able to understand the cause of this bacterial transformation.
n He further stated that some ‘transforming principle’ transferred from heat killed S strain, enabled R strain to synthesize a smooth polysaccharide coat and become virulent. But biochemical nature of genetic material was not defined from his experiments.
7. Biochemical Characterisation of Transforming Principle
n Oswald Avery, Colin MacLeod and Maclyn McCarty repeated Griffith’s experiment in an in vitro system in order to determine biochemical nature of transforming principle.
n They purified biochemicals (proteins, DNA, RNA) from heat-killed S-type cells, and checked which of these could transform live R-type cells into S-type cell. They observed that DNA alone from S-type cells caused transformation of R-type cells into virulent S-type cells.
n They also discovered that proteases and RNases did not affect transformation while DNases inhibited the process.
n They concluded that DNA is the hereditary material.
8. Proof for DNA as the Genetic Material
n Hershey and Chase (1952) conducted experiments on bacteriophage to prove that DNA is the genetic material.
n Procedure:
(i) Some bacteriophage virus were grown on a medium that contained radioactive phosphorus (32P) and some in another medium with radioactive sulphur (35S).
(ii) Viruses grown in the presence of radioactive phosphorus (32P) contained radioactive DNA.
(iii) Similar viruses grown in presence of radioactive sulphur (35S) contained radioactive protein.
(iv) Both the radioactive virus types were allowed to infect E. coli separately.
(v) Soon after infection, the bacterial cells were gently agitated in blender to remove viral coats from the bacteria.
(vi) The culture was also centrifuged to separate the viral particle from the bacterial cell.
n Observations and Conclusions:
(i) Only radioactive 32P was found to be associated with the bacterial cell, whereas radioactive 35S was only found in surrounding medium and not in the bacterial cell.
(ii) This indicates that only DNA and not the protein coat entered the bacterial cell.
(iii) This proves that DNA is the genetic material which is passed from virus to bacteria and not protein.
9. Characteristics of Genetic Material
n In some viruses, RNA is the genetic material (e.g., Tobacco Mosaic virus). RNA also performs functions of messenger and adapter.
(i) DNA and RNA have the ability to direct their duplications because of rule of base pairing and complementarity but proteins fail to fulfill first criteria itself.
(ii) Genetic material should be stable so as not to change with different stages of life cycle, age or change in physiology of organism.
(iii) RNA being unstable mutates at a faster rate. Thus, viruses having RNA genome and having shorter life span mutate and evolve faster.
(iv) RNA can code directly for protein synthesis and hence can easily express characters. But DNA is dependent on RNA for protein synthesis. Protein synthesizing machinery has evolved around RNA.
Conclusion: Both RNA and DNA can function as genetic material, but DNA being chemically less reactive and structurally being more stable is a better genetic material. DNA is more stable than RNA because of:
(a) being double standard.
(b) two strands being complementary; even if separated by heating they come together.
(c) DNA is less reactive than RNA as has
(d) Presence of thymine in place of uracil provides additional stability to DNA.
10. Ribonucleic Acid (RNA)
n RNA was the first genetic material.
n Essential life processes (such as metabolism, translation, splicing, etc.) evolved around RNA.
n RNA acts as genetic material as well as catalyst.
n RNA being a catalyst was reactive and hence unstable. Therefore, DNA has evolved from RNA with chemical modifications that make it more stable.
n DNA being double stranded and having complementary strand resist changes by evolving a process of repair.
11. Differences between DNA and RNA
S. No. | DNA | RNA |
(i) | The sugar present is deoxyribose. | The sugar present is ribose. |
(ii) | Nitrogenous bases present are adenine, guanine, thymine and cytosine. | Nitrogenous bases present are adenine, guanine, cytosine and uracil. |
(iii) | It is always double stranded. | It can be single stranded or double stranded. |
(iv) | It is the genetic material of almost all living organisms. | It is the genetic material of only some viruses. |
(v) | It is chemically less reactive and structurally more stable. | It is chemically more reactive and structurally less stable. |
12. DNA Replication
n Watson and Crick in 1953 proposed a scheme that DNA replication was semi-conservative.
n According to the scheme, the two parental strands separate and each strand acts as a template for synthesising a complementary strand over it.
n After completion of replication, each DNA had one parental strand and one newly synthesised strand.
(i) Experimental proof for semi-conservative mode of DNA replication
l Matthew Meselson and Franklin Stahl in 1958 performed experiments on E. coli to prove that DNA replication is semi-conservative.
l They grew E. coli in a medium containing 15NH4Cl (in which 15N is the heavy isotope of nitrogen) for many generations.
l As a result, 15N got incorporated into newly synthesised DNA.
l This heavy DNA can be differentiated from normal DNA by centrifugation in caesium chloride (CsCl) density gradient.
l Then they transferred the cells into a medium with normal 14NH4Cl and took the samples at various definite time intervals as the cells multiplied.
l The extracted DNAs were centrifuged and measured to get their densities.
l The DNA extracted from the culture after one generation of transfer from the 15N medium to 14N medium (i.e., after 20 minutes; E. coli divides every 20 minutes) showed an intermediate hybrid density.
l The DNA extracted from culture after two generations (i.e., after 40 minutes) showed equal amounts of light DNA and hybrid DNA.
l When allowed to grow for 80 minutes, it showed more amounts of light DNA but the hybrid DNA still maintained itself.
l Similar experiment was performed by Taylor and colleagues in 1958, on Vicia faba using radioactive thymidine to detect distribution of newly synthesized DNA in chromosomes to prove that the DNA in chromosome also replicate semi-conservatively.
(ii) Enzymes for DNA replication
l Various enzymes are required as catalysts during DNA replication in living cells.
l DNA-dependent DNA polymerase: It catalyses the polymerisation of deoxynucleotides on DNA template at a fast rate. Its average rate of polymerisation is 2000 bp per second. [It completes process of replication for E.coli within 38 minutes which has only 4.6 × 106 bp whereas for human genome, diploid content is 6.6 × 109 bp]. It also has to catalyse reaction with high degree of accuracy as any mistake during replication would result into mutations.
l Helicase: It unwinds the DNA strand to form the replication fork.
l DNA ligase: It joins the Okazaki fragments which are formed on the lagging strand.
l Dual Purpose of Deoxyribonucleoside triphosphates:
(i) Act as substrate because Deoxyribonucleotides are joined to form DNA.
(ii) Provide energy for polymerisation reaction as polymeristion is energetically very expensive (The two terminal phosphates in a deoxyribonucleoside triphosphates are high energy phosphates as in ATP).
(iii) Process of DNA Replication
l DNA replication begins at a unique and fixed point called origin of replication or ‘ori’.
Initiation
n The complementary strands of DNA double helix are separated by enzyme, DNA helicase. This is called unwinding of double-stranded DNA.
n The separated strands tend to rewind, therefore these are stabilised by proteins called single strand binding proteins (ssBPs), which bind to the separated strands.
n Unwinding of double-stranded DNA forms a Y-shaped configuration in the DNA duplex, which is called replication fork.
Elongation
n An enzyme called primase initiates replication of the strand oriented in the 3′ (towards origin)→5′ (towards fork) direction. This generates 10–60 nucleotides long primer RNA (replicated in 5′→3′ direction).
n The free 3′–OH of this RNA primer provides the initiation point for DNA polymerase for sequential addition of deoxyribonucleotides.
n DNA polymerase progressively adds deoxyribonucleotides to the free 3′-end of the growing polynucleotide chain so that replication of the 3′→5′ strand of the DNA molecule is continuous (growth of the new strand in 5′→3′ direction).
n The replication of 3′→5′ strand is continuous and it is called leading strand, while the replication of second strand (5′→3′ strand) of the DNA molecules is discontinuous and it is known as the lagging strand.
n The replication of lagging strand generates small polynucleotide fragments called ‘Okazaki fragments’ (after R. Okazaki, who first identified them).
n These Okazaki fragments are then joined together by enzyme called DNA ligase.
13. Transcription
n The process of copying genetic information from one strand of the DNA into RNA is termed as transcription.
n The principle of complementarity governs the process, except that adenosine now base pairs with uracil instead of thymine, as in replication.
n Unlike replication, only a single-stranded fragment of DNA gets copied into RNA.
Transcription unit
n The transcription unit of DNA contains three regions in the DNA:
(i) The promoter: It is the binding site for RNA polymerase for initiation of transcription.
(ii) The structural gene: It codes for enzyme or protein for structural functions.
(iii) The terminator: It is the region where transcription ends.
n The DNA-dependent RNA polymerase helps in DNA replication by catalysing the polymerisation in only one direction, i.e., 5′→3′.
n The DNA strand that has the polarity 3′→5′ acts as a template and is also referred to as template strand.
n The strand which does not get transcripted is called coding strand and has the polarity 5′→3′. Its sequence is same as RNA formed.
n The promoter is located towards 5′-end (upstream) of the structural gene of coding strands and provides the binding site for RNA polymerase.
n The sequence of DNA located towards the 3′-end (downstream) of the coding strand where the process of transcription would stop is called terminator.
14. Transcription Unit and the Gene
n The segment of DNA coding for a polypeptide is called cistron.
n In eukaryotes, the transcription unit possess a structural gene specific only for a single polypeptide. Thus it is called monocistronic.
n In prokaryotes, the transcription unit possessing the structural genes for many polypeptides which are part of single metabolic pathway are called polycistronic.
n The gene in eukaryotes are split into the coding or expressed sequence of DNA called exon, and non-expressable sequence of DNA called intron or intervening sequence.
n mRNA contains only exon but no intron.
n Regulatory sequences are loosely defined as regulatory genes, though they do not code for any RNA or protein.
15. Transcription in Prokaryotes
n In prokaryotes, the structural gene is polycistronic and continuous.
n In bacteria, the transcription of all the three types of RNA (mRNA, tRNA and rRNA) is catalysed by single DNA-dependent enzyme, called the RNA polymerase.
n All three RNA’s are needed to synthesize a protein in cell. mRNA provides the template, tRNA brings amino acids and reads the genetic code, and rRNA plays structural and catalytic role durinng translation.
n The transcription is completed in three steps: initiation, elongation and termination.
n Initiation: s (sigma) factor recognises the start signal and promotor region on DNA which then along with RNA polymerase binds to the promoter to initiate transcription. It uses nucleoside triphosphates as substrate and polymerises in a template-dependent fashion following the rule of complementarity.
n Elongation: The RNA polymerase after initiation of RNA transcription loses the s factor but continues the polymerisation of ribonucleotides to form RNA. It facilitates opening of helix and continues elongation with only a short stretch of RNA being bound to enzyme at a time.
n Termination: Once the RNA polymerase reaches the termination region of DNA, the RNA polymerase is separated from DNA–RNA hybrid, as a result nascent RNA separates. This process is called termination which is facilitated by a termination factor r (rho).
n In prokaryotes, mRNA does not require any processing, so both transcription and translation occur in the cytosol. It can be said that transcription and translation are coupled together as many times translation can begin much before mRNA is fully transcribed.
16. Transcription in Eukaryotes
n The structural genes are monocistronic in eukaryotes.
n The process of transcription is similar to that in prokaryotes.
n It takes place in the nucleus.
n Coding gene sequences called exons form the part of mRNA and non-coding sequence called introns are removed during RNA splicing and exons are joined in a defined order.
n In eukaryotes, three types of RNA polymerases are found in the nucleus:
(i) RNA polymerase I transcribes rRNAs (28S, 18S, and 5.8S).
(ii) RNA polymerase II transcribes the precursor of mRNA (called heterogeneous nuclear RNA or hnRNA).
(iii) RNA polymerase III transcribes tRNA, 5S rRNA and snRNAs (small nuclear RNAs).
Post-transcriptional modifications
n The primary transcripts are non-functional, containing both the coding region, exon, and non-coding region, intron, in RNA and are called heterogenous RNA or hnRNA.
n The hnRNA undergoes splicing and two additional processes called capping and tailing.
n In capping, an unusual nucleotide, methyl guanosine triphosphate, is added to the 5′-end of hnRNA.
n In tailing, adenylate residues (about 200–300) are added at 3′-end in a template independent manner.
n Now the hnRNA undergoes a process where the introns are removed and exons are joined to form mRNA by the process called splicing.
17. Genetic Code
n The relationship between the sequence of nucleotides on mRNA and sequence of amino acids in the polypeptide is called genetic code.
n George Gamow suggested that the code must be of 3 bases in order to code for 20 amino acids because there are only 4 bases (i.e., 43 or 4 × 4 × 4 = 64) which code for 20 amino acids. So, codon is a triplet.
n Har Gobind Khorana developed chemical method for synthesising RNA molecules with defined base combinations (homopolymers and copolymers) to develop the genetic code.
n Marshall Nirenberg developed cell-free system for protein synthesis and thus artificially synthesised proteins to understand the nature of codons.
n Severo Ochoa demonstrated that polynucleotide phosphorylase also helped in polymerising RNA with defined sequences in a template-independent manner (enzymatic RNA synthesis).
Table 6.1: The Codons for the Various Amino Acids
First position
| Second position | Third position
| |||
U | C | A | G | ||
U | UUU Phe UUC Phe UUA Leu UUG Leu | UCU Ser UCC Ser UCA Ser UCG Ser | UAU Tyr UAC Tyr UAA Stop UAG Stop | UGU Cys UGC Cys UGA Stop UGG Trp | U C A G |
C | CUU Leu CUC Leu CUA Leu CUG Leu | CCU Pro CCC Pro CCA Pro CCG Pro | CAU His CAC His CAA Gln CAG Gln | CGU Arg CGC Arg CGA Arg CGG Arg | U C A G |
A | AUU Ile AUC Ile AUA Ile AUG Met/Start | ACU Thr ACC Thr ACA Thr ACG Thr | AAU Asn AAC Asn AAA Lys AAG Lys | AGU Ser AGC Ser AGA Arg AGG Arg | U C A G |
G | GUU Val GUC Val GUA Val GUG Val | GCU Ala GCC Ala GCA Ala GCG Ala | GAU Asp GAC Asp GAA Glu GAG Glu | GGU Gly GGC Gly GGA Gly GGG Gly | U C A G |
Salient features of genetic code
(i) The codons are triplet. Out of 64 codons, 61 code for 20 amino acids and 3 codons (UAA, UGA, UAG) do not code for any amino acid hence, function as stop or terminating codons.
(ii) One codon codes for only one particular amino acid, hence the code is unambiguous and specific.
(iii) Some amino acids are coded by more than one codon, hence the code is degenerate.
(iv) The codon is read on mRNA in a contiguous fashion, i.e., without punctuations and thus the code is commaless.
(v) The genetic code is nearly universal, i.e., a particular codon codes for the same amino acid in all organisms from bacteria to human except in mitochondria and few protozoans.
(vi) AUG is a dual function codon, it codes for methionine (met) and it also acts as initiator codon.
18. Mutations
Mutation is defined as the sudden inheritable change in the genetic material. It can be of the following two major types:
(i) Point mutation: It is the mutation in a single base pair, which is replaced by another base pair. For example, in sickle-cell anaemia, point mutation in β-globin chain results in change of glutamate to valine.
(ii) Frameshift mutation: It is the change in the reading frame because of insertion or deletion of base pairs.
(a) Insertion: It is the addition of one or more nucleotides in the DNA segment. Insertion of three or its multiple bases do not change the reading frame but add a new amino acid.
(b) Deletion: It is the removal of one or more nucleotides from the DNA segment. Deletion of three or its multiple bases do not change the reading frame but remove one or more amino acids.
Normal DNA: ATC GAT CGA
Insertion: ATC C
A TCG
Deletion: ATC ATC GA
Example:
RAM HAS RED CAP
If we insert a letter B in between HAS and RED and rearrange the statement, it would read as follows:
RAM HAS BRE DCA P
Similarly, if we now insert two letters at the same place, say BI. Now it would read:
RAM HAS BIR EDC AP
Now we insert three letters together, say BIG, the statement would read:
RAM HAS BIG RED CAP
The same exercise can be repeated, by deleting the letters R, E and D, one by one and rearranging the statement to make a triplet word.
RAM HAS EDC AP
RAM HAS DCA P
RAM HAS CAP
19. tRNA—the Adapter Molecule
n Francis Crick proposed the presence of an adapter molecule which could read the code on one end and on the other end would bind to the specific amino acids.
n However, tRNA was known before the genetic code was postulated and was then called sRNA (soluble RNA). Its role as an adapter molecule was reported later.
Structure
n The secondary structure of tRNA is clover-leaf like but the three-dimensional tertiary structure depicts it as a compact inverted L-shaped molecule.
n tRNA has five arms or loops:
(i) Anticodon loop, which has bases complementary to the code.
(ii) Amino acid acceptor end to which amino acids bind.
(iii) T loop, which helps in binding to ribosome.
(iv) D loop, which helps in binding aminoacyl synthetase.
(v) Variable arm.
n Each tRNA is specific for a particular amino acid.
n A specific tRNA for initiation is called initiator tRNA.
n There is no tRNA for stop codons.
20. Translation
n Translation is the process of synthesis of protein from amino acids, sequence and order of amino acids being defined by sequence of bases in mRNA. Amino acids are joined by peptide bonds.
n A translational unit in mRNA from 5’ → 3’ comprises of a start codon, region coding for a polypeptide, a stop codon and untranslated regions (UTRs). UTRs are additional sequences of mRNA that are not translated. They are present at both 5’ end (before start codon) and at 3’ end (after stop codon) for efficient translation process.
(i) Initiation
l In prokaryotes, initiation requires the large and small ribosome subunits, the mRNA, initiation tRNA and three initiation factors (IFs).
l Activation of amino acid: Amino acids become activated by binding with aminoacyl tRNA synthetase enzyme in the presence of ATP.
Amino Acid (AA) + ATP
l Transfer of amino acid to tRNA: The AA–AMP–Enzyme complex formed reacts with specific tRNA to form aminoacyl-tRNA complex.
AA–AMP–Enzyme complex + tRNA → AA–tRNA + AMP + Enzyme
l The cap region of mRNA binds to the smaller subunit of ribosome.
l The ribosome has two sites, A-site and P-site.
l The smaller subunit first binds to the initiator mRNA and then binds to the larger subunit so that initiation codon (AUG) lies on the P-site.
l The initiation tRNA, i.e., methionyl tRNA then binds to the P-site.
(ii) Elongation of polypeptide chain
l Another charged aminoacyl tRNA complex binds to the A-site of the ribosome at the second codon.
l A peptide bond is formed between carboxyl group (—COOH) of amino acid at P-site and amino group (—NH) of amino acid at A-site by the enzyme peptidyl transferase.
l The ribosome slides over mRNA from codon to codon in the 5′→3′ direction i.e. called translocation.
l According to the sequence of codons, amino acids are attached to one another by peptide bonds and a polypeptide chain is formed.
(iii) Termination of polypeptide
l When the A-site of ribosome reaches a termination codon, which does not code for any amino acid, no charged tRNA binds to the A-site.
l Dissociation of polypeptide from ribosome takes place, which is catalysed by a ‘release factor’.
l There are three termination codons namely UGA, UAG and UAA.
21. Regulation of Gene Expression
n Regulation of gene expression means controlling the amount and time of formation of gene products according to the requirements of the cell.
n In eukaryotes, gene regulation can take place at four levels:
(i) Transcription level (regulation of primary transcript formation),
(ii) Processing level (regulation of splicing),
(iii) Transport of mRNA from nucleus to the cytoplasm,
(iv) Translation level.
n In prokaryotes, control of rate of transcriptional initiation is the predominant site for control of gene expression. It can be seen in lac operon and trp operon.
22. The lac Operon
n Operon: The concept of operon was first proposed in 1961, by Jacob and Monod. An operon is a unit of prokaryotic gene expression which includes coordinately regulated (structural) genes and control elements which are recognised by regulatory gene product.
n Components of an operon:
(i) Structural gene: The fragment of DNA which transcribe mRNA for polypeptide synthesis.
(ii) Promoter: The sequence of DNA where RNA polymerase binds and initiates transcription of structural genes is called promoter.
(iii) Operator: The sequence of DNA adjacent to promoter where specific repressor protein binds is called operator.
(iv) Regulator gene: The gene that codes for the repressor protein that binds to the operator and suppresses its activity as a result of which transcription will be switched off.
(v) Inducer: The substrate that prevents the repressor from binding to the operator, is called an inducer. As a result transcription is switched on. It is a chemical of diverse nature like metabolite, hormone substrate, etc.
n The lactose operon: The lac z, lac y, lac a genes are transcribed from a lac transcription unit under the control of a single promoter. They encode enzyme required for the use of lactose as a carbon source. The lac i gene product, the lac repressor, is expressed from a separate transcription unit upstream from the operator.
n Regulation of lac operon by repressor is referred to as negative regulation.
n lac operon consists of three structural genes (z, y, a), operator (o), promoter (p) and a separate regulatory gene (i). Lactose is the inducer in lac operon.
n The three structural genes (z, y, a) transcribe a polycistronic mRNA.
n Gene z codes for β-galactosidase (β-gal) enzyme which breaks lactose into galactose and glucose.
n Gene y codes for permease, which increases the permeability of the cell to lactose (β-galactosides).
n Gene a codes for enzyme transacetylase, which catalyses the transacetylation of lactose in its active form.
n When Lactose is Absent
(i) When lactose is absent, i gene regulates and produces repressor mRNA which translates into repressor protein.
(ii) The repressor protein binds to the operator region of the operon and as a result prevents RNA polymerase to bind to the operon.
(iii) The operon is switched off.
n When Lactose is Present
(i) Lactose acts as an inducer which binds to the repressor and forms an inactive repressor.
(ii) The repressor fails to bind to the operator region.
(iii) The RNA polymerase binds to the operator and transcribes lac mRNA.
(iv) lac mRNA is polycistronic, i.e., produces all three enzymes, β-galactosidase, permease and transacetylase.
(v) The lac operon is switched on.
23. Human Genome Project (HGP)
n Genetic make-up of an organism lies in DNA sequences. If two individuals differ, then their DNA sequences will also vary, at least at some places if not all.
n The human genome project was a 13-year project by the U.S. Department of Energy and the National Institute of Health. It was launched in 1990 and completed is 2003.
Goals of HGP
(i)To identify the 20,000–25,000 genes in human DNA .
(ii)To determine all the 3 billion chemical base pair sequences that make up human DNA.
(iii)To store the above information in databases.
(iv)To improve the tools for data analysis.
(v)To transfer the technologies to other sectors such as industries.
(vi)To address the ethical, legal and social issues (ELSI) that may arise from the project.
n Bacteria, yeast, caenorhabditis elegans (free living non-pathogenic nematode), Drosophila (fruit fly), plants (rice and Arabidopsis), etc. have also been sequenced as of today.
Advantages of HGP
(i)The effect of DNA variation can be studied among individuals which can lead to revolutionary new ways to diagnose and treat many disorders or diseases.
(ii)Provides clues to understand human biology.
(iii)More information can be obtained about non-human organisms like bacteria, yeast, nematode, fruit fly, plant, rice, etc.
Methodologies of HGP
l The methods involve two major approaches:
(i) Expressed sequence tags (ESTs): This method focusses on identifying all the genes that are expressed as RNA.
(ii) Sequence annotation: It is an approach of simply sequencing the whole set of genome that contains all the coding and non-coding sequences, and later assigning different regions in the sequence with functions.
l For sequencing, the total DNA from cell is first isolated and broken down in relatively small sizes as fragments.
l These DNA fragments are cloned in suitable host using suitable vectors. When bacteria is used as vector, they are called bacterial artificial chromosomes (BAC) and when yeast is used as vector, they are called yeast artificial chromosomes (YAC).
l Frederick Sanger developed a principle according to which the fragments of DNA are sequenced by automated DNA sequences.
l On the basis of overlapping regions on DNA fragments, these sequences are arrangedaccordingly.
l For alignment of these sequences, specialised computer-based programmes were developed.
l These sequences were annotated and were assigned to each chromosome. Sequence of chromosome 1 was completed only in May 2006. It was the last chromosome be sequenced).
l Finally, the genetic and physical maps of the genome were constructed by collecting information about certain repetitive DNA sequences and DNA polymorphism, based on endonuclease recognition sites.
24. Salient Features of Human Genome
(i) The human genome contains 3164.7 million nucleotide bases.
(ii) The average gene consists of 3000 bases; the largest known human gene being dystrophin at 2.4 million bases.
(iii) The total number of genes is estimated to be 30,000 and 99.9 per cent nucleotide bases are exactly the same in all people.
(iv) The functions are unknown for over 50 per cent of the discovered genes.
(v) Less than 2 per cent of the genome codes for proteins.
(vi) The human genome contains large repeated sequences, repeated 100 to 1000 times.
(vii) The repeated sequence is thought to have no direct coding functions but they throw light on chromosome structures, dynamics and evolution.
(viii) Chromosome 1 has most genes (2968) and the Y has the fewest genes (231).
(ix) Scientists have identified about 1.4 million locations where single base DNA sequence differences called SNPs or single nucleotide polymorphism (pronounced as ‘snips’) occur in humans. This information promises to revolutionise the processes of finding chromosomal locations for disease—associated sequences and tracing human history.
Rice Genome Project
n The International Rice Genome Sequencing Project (IRGSP) began in September 1997, at a workshop held in conjunction with the International Symposium on Plant Molecular Biology in Singapore.
n Rice genome sequencing is being conducted along the same lines as numerous other large-scale genome sequencing projects. Large insert genomic libraries, used as the primary sequencing templates, are constructed in bacterial artificial chromosomes (BACs).
n One of the initial motivators for sequencing rice, besides the relatively small genome size, was that it could be used as a model for other cereal crops with larger genomes, such as maize and wheat. This was predicated somewhat on rice’s small genome size and the realization from molecular mapping, e.g., RFLPs, of conserved markers and marker order.
n The availability of the rice genome, together with the community annotation and other resources that added functionality, transformed genetics research and rice breeding.
25. DNA Fingerprinting
n Dr. Alec Jeffreys developed the technique of DNA fingerprinting in an attempt to identify DNA marker for inherited diseases.
n Human genome has 3 × 109 bp. 99.9% of base sequences among humans are the same, which makes every individual unique in phenotype.
Polymorphism
l The genome consists of small stretch of DNA which are repeated many times. These are called repetitive DNA and comprise of satellite DNA. Satellite DNA does not code for any proteins but form large portion of human genome. These sequences show high degree of polymorphism. As polymorphisms are inheritable from parents to children, DNA finger printing is the basis of paternity testing. Polymorphism arises due to mutations. New mutations may arise in somatic cells or in germ cells. If mutation occurs in germ cells; it is passed on to offsprings. If an inheritable mutation is observed in a population at high frequency, it is called as DNA polymorphism. Polymorphism ranges from single nucleotide change to very large scale changes.
n DNA fingerprinting uses short nucleotide repeats called Variable Number Tandem Repeats (VNTRs) as markers. VNTRs vary from person to person and are inherited from one generation to the next. Only closely related individuals have similar VNTRs. Satellite DNA that shows very high degree of polymorphism are called Variable Number of Tandem Repeats (VNTR) VNTRs are used in DNA fingerprinting as probes. Number of repeat shows very high degree of polymorphism. Thus, size of VNTR varies from 0.1 to 20 kb.
Methodology and Technique
(i) DNA is isolated and extracted from the cell or tissue by centrifugation.
(ii) By the process of polymerase chain reaction (PCR), many copies are produced. This step is called amplification.
(iii) DNA is cut into small fragments by treating with restriction endonucleases.
(iv) DNA fragments are separated by agarose gel electrophoresis.
(v) The separated DNA fragments are visualised under ultraviolet radiation after applying suitable dye.
(vi) The DNA is transferred from electrophoresis plate to nitrocellulose or nylon membrane sheet. This is called Southern blotting.
(vii) VNTR probes are now added which bind to specific nucleotide sequences that are complementary to them. This is called hybridisation.
(viii) The hybridised DNA fragments are detected by autoradiography. They are observed as dark bands on X-ray film.
(ix) These bands being of different sizes, give a characteristic pattern for an individual DNA. It differs from individual to individual except in case of monozygotic (identical) twins.
Applications of DNA Fingerprinting
(i) It is used as a tool in forensic tests to identify criminals and criminal investigations.
(ii) It is used to settle paternity disputes and maternity disputes.
(iii) It is used to determine population and genetic diversities to study evolution.
(iv) It is used in the study of evolutionary biology.
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