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Chromosomal Basis Of Inheritance

A. Chromosomal structure

On the basis of the following information, chromosomal Structure have been prepared.

(1) The genes are linearly arranged on a chromosome and therefore, the gene order should be known.

(2) The percentage of crossing over between two genes is directly proportional to their distance. It is infact the index of their distance. The unit of crossing over has been termed as by Haldane as centi Morgan (cM). One unit of map distance (cM) is therefore, equivalent to 1% crossing over. When chiasma is organised in between two gene loci, only 50% meiotic products shall be crossovers and 50% non-crossovers. Thus, the chiasma frequency is twice the frequency of cross over products i.e., chiasma % = 2 (cross over %) or crossover %= ½ (chiasma %).

(3) Accordingly, Sturtevant,1911 prepared the first chromosomal map. Infact this map is a line representation of a chromosome where the location of genes has been plotted as points at specific distances. These distances are proportional to their crossing over percentage. Suppose there are three genes on a chromosome say, A Band Cwhich could be arranged as A, B, C; A, C, B or B, A, C. A three point test cross confirms as to which gene is located in the centre. By determining the crossing over value between A and B, B and C as also between A and C, the linkage maps can be prepared. Broadly speaking, a chromosomal map can be prepared from the following results of crossing over between the genes A, B and C:

(i) 4% crossing over taking place between A and B.   (ii) 9% crossing over taking place between A and C.

Hence the genes be located as above and there should be 13% crossing over between B and C and the genes may be arranged as under:

If there is 5% crossing over between Band C, the genes are arranged in the following manner and there should be 9% crossing over between Aand C.

(4) Uses of chromosomal map

(i) Finding exact location of gene on chromosomes.

(ii) Knowing recombination of various genes in a linkage group of chromosomes.

(iii) Predicting result of dihybrid and trihybrid cross.

B. Nucleic acids

Two types of nucleic acids are found in the cells of all living organisms. These are DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid). The nucleic acid was first isolated by Friedrich Miescherin 1868 from the nuclei of pus cells and was named nuclein.The term nuclein was given by Altman.

C. DNA (Deoxyribonucleic Acid)

Introduction : Term was given by Zacharis, which is found in the cells of all living organisms except plant viruses,where RNA forms the genetic material and DNA is absent. In bacteriophages and viruses there is a single molecule of DNA, which remains coiled and is enclosed in the protein coat. In bacteria, mitochondria, plastids and other prokaryotes, DNA is circular and lies naked in the cytoplasm but  in eukaryotes it is found in nucleus and known as carrier of genetic information and capable of self replication. Isolation and purification of specific DNA segment from a living organism achieved by Nirenberg H.Harries is associated with DNA-RNA hybridization technique.

(1) Chemical composition : The chemical analysis has shown that DNA is composed of three different types of compound.

(i) Sugar molecule :Represented by a pentose sugarthe deoxyribose or 2-deoxyribose which derived from ribose due to the deletion of oxygen from the second carbon.

(ii) Phosphoric acid : H3PO4that makes DNA acidicin nature.

(iii) Nitrogeneous base :These are nitrogen containing ring compound. Which classified into two groups:

(a) Purines : Two ring compound namely as Adenine and  Guanine.

(b) Pyramidine : One ring compound included Cytosine and Thyminein RNA uracil is present instead of Thymine.

Nucleosides : Nucleosides are formed by a purine or pyrimidine nitrogenous base and pentose sugar. DNA nucleosides are known as deoxyribosenucleosides.

Nucleotides :In a nucleotide, purine or pyrimidine nitrogenous base is joined by deoxyribose pentose sugar (D), which is further linked with phosphate (P) group to form nucleotides.

                             Composition of nucleoside and nucleotides of DNA and RNA

                           (D= Deoxyribose sugar, R = Ribose sugar, P = Phosphoric acid)

Base with its symbol

Nucleoside

Nucleotide

Formula

Name

Formula

Name

DNA

Adenine = A

D – A

Deoxyandenosine

Deoxyandenosine monophosphate or Adenine deoxyribose nucleotide

Guanine = G

D – G

Deoxyguanine

Deoxygunine monophosphate or Guanine deoxyribose-nucleotide

Thyamine = T

D – T

Thymidine

Thymidine monophosphate or Thymidine nucleotide

Cytosine = C

D – C

Deoxycitidine

 

Deoxycytidine monophosphate or Cytosine deoxyribose nucleotide

RNA

Adenine = A

R – A

Adenoside

Adenosine monophosphate or Adenine ribose nucleotide

Guanine = G

R – G

Guanosine

 

Guanosine monophosphate or Guanine ribose nucleotide

Uracil = U

R – U

Uridine

Uridine monophosphate or Uracil ribose nucleotide

Cytosine = C

R – C

Cytidine

Cytidine monophosphate or Cytosine ribose nucleotide

(2) Watson and Crick’s model of DNA : In 1953 Watson and Crick suggested that in a DNA molecule there are two such polynucleotide chains arranged antiparallal or is opposite directions i.e. one polynucleotide chain runs in 5’ →3’direction, the other in 3’→ 5’ direction. It means the 3’end of one chain lies beside the 5’ end of other in right handed manner.

(i) Important features of Watson and Crick double helical model of DNA

There are important features of DNA double helix.

(a) The double helix comprises of two polynucleotide chains.

(b) The two strands (polynucleotide chains) of double helix are anti-parallel due to phosphodiester bond.

(c) Each polynucleotide chain has a sugar-phosphate ‘backbone’with nitrogeneous bases directed inside the helix.

(d) The nitrogenous bases of two antiparallel polynucleotide strands are linked through hydrogen bonds. There are two hydrogen bonds between A and T, and three between G and C.The hydrogen bonds are the only attractive forces between the two polynucleotides of double helix. These serve to hold the structure together.

(e) The two polynucleotides in a double helix are complementary. The sequence of nitrogenous bases in one determines the sequence of the nitrogenous bases in the other. Complementary base pairing is of fundamental importance in molecular genetics.

(f) Erwin Chargaff (1950) made quantitative analysis of DNA and proposed “base equivalence rule” starting that molar concentration of A = T & G ≡ C or which is constant for a species.

(g) Ten base pairs occur per turn of helix (abbreviated 10bp). The spacing between adjacent base pairs is 10Å. The helix is 20Å in diameterand DNA molecule found 360oin a clockwise.

(3) Forms of DNA : Five different morphological forms of DNA double helix have been described. These are A, B, C, D and Z forms. Most of these forms (except B, and Z) occur in rigidly controlled experimental conditions. Watson and crick model represents commonest form, Biotic-form (B-form or B-DNA) of DNA. Some DNA forms are inter convertible also. The differences in these DNA forms are associated with:

(i) The numbers of base pairs, present in each turn of DNA helix.

(ii) The pitch or angle between each base pair.

(iii) The helical diameter of DNA molecule.

(iv) The handedness of double helix. Which is mentioned in table.

(4) Characteristics of DNA

(i) Denaturation or melting : The phenomenon of separating of two strand of DNA molecule by breaking of hydrogen bond at the temp. 900C.

(ii) Renaturation or annealing : Separated strands reunite to form double helix molecule of DNA by cooling at the room temp. i.e. 250C.

These properties help to form hybrid from different DNA or with RNA.

(5) Evidences of DNA as the genetic material : The following experiments conducted by the molecular bioloigists provide direct evidences of DNAbeing the genetic material bacterial transformation, bacterial recombination and bacteriophage infection.

(i) Bacterial transformation or Griffith's Experiments : Griffith  (1928) injected into mice with virulent and smooth (S-type, smooth colony with mucilage) form of Diplococcus pneumoniae. The mice died due to pneumonia. No death occurred when mice were injected with nonvirulent or rough (R-type, irregular colony without mucilage) form or heat- killed virulent form. However, in a combination of heat killed S-type and live R-type bacteria, death occurred in some mice. Autopsy of dead mice showed that they possessed S-type living bacteria, which could have been produced only by transformation of R-type bacteria. The transforming chemical was found out by O.T.Avery, C.M. Mc. leod and M. Mc. Carty (1944). They fractionated heat-killed S-type bacteria into DNA, carbohydrate and protein fractions. DNA was divided into two parts, one with DNAase and the other without it. Each component was added to different cultures of R-type bacteria. Transformation was found only in that culture which was provided with intact DNA of S-type. Therefore, the trait of virulence is present in DNA. Transformation involves transfer of a part of DNA from surrounding medium or dead bacteria (donor) to living bacteria (recipient) to form a recombinant.

(ii) Evidence from genetic recombination in bacteria or bacterial conjugation : Lederberg and Tatum (1946) discovered the genetic recombination in bacteriafrom two different strains  through the process of conjugation. Bacterium Escherichia colican grow in minimal culture medium containing minerals and sugar only. It can synthesize all the necessary vitamins from these raw materials. But its two mutant strains were found to lack the ability to synthesize some of the vitamins necessary for growth. These could not grow in the minimal medium till the particular vitamins were not supplied in the culture medium.

(a) Mutant strain A : It (used as male strain) had the genetic composition Met, Bio, Thr+, Leu+, Thi+. It lacks the ability to manufacture vitamins methionine and biotin and can grow only in a culture medium which contains these vitamins in addition to sugar and minerals.

(b) Mutant strain B : It (used as female strain or recipient) has a genetic composition Me++, Bio+, Thr, Leu,  Thi. It lacks the ability to manufacture threonine, leucine and thionine and can grow only when these vitamins are added to the growing medium.

These two strains of E.coli are, therefore, unable to grow in the minimal culture medium, when grow separately. But when a mixture of these two strains was allowed to grow in the same medium a number of colonies were formed. This indicates that the portion of donor DNA containing information to manufacture threonine, leucine and thionine had been transferred and incorporated in the recipient’s genotype during conjugation.

This experiment of Lederberg and Tatum shown that the conjugation results in the transfer of genetic material DNA from one bacterium to other. During conjugation a cytoplasmic bridge is formed between two conjugating bacteria.

(iii) Evidence from bacteriophage infection : Hershey and Chase (1952) conducted their experiment on T2 bacteriophage,which attacks on E.colibacterium. The phage particles were prepared by using radioisotopes of 35S and 32Pin the following steps.

(a) Few bacteriophages were grown in bacteria containing 35S. Which was incorporated into the cystein and methionine amino acids of proteins and thus these amino acids with 35 S formed the proteins of phage.

(b) Some other bacteriophages were grown in bacteria having 32P. Which was restricted to DNA of phage particles. These two radioactive phage preparations (one with radioactive proteins and another with radioactive DNA) were allowed to infect the culture of E.coli.The protein coats were separated from the bacterial cell walls by shaking and centrifugation.

The heavier infected bacterial cells during centrifugation pelleted to bottom. The supernatant had the lighter phage particles and other components that failed to infect bacteria. It was observed that bacteriophages with radioactive DNA gave rise to radioactive pellets with 32 P in DNA. However in the phage particles with radioactive protein (with 35 S) the bacterial pellets have almost nil radioactivity indicating that proteins have failed to migrate into bacterial cell. So, it can be safely concluded that during infection by bacteriophage T2, it was DNA,which entered the bacteria. It was followed by an eclipse period during which phage DNA replicates numerous times within the bacterial cell. Towards the end of eclipse period phage DNA directs the production of protein coats assembly of newly formed phage particles. Lysozyme (an enzyme) brings about the lysis of host cell and release, the newly formed bacteriophages. The above experiment clearly suggests that it is phage DNA and not protein, which contains the genetic information for the production of new bacteriophages. However, in some plant viruses (like TMV), RNA acts as hereditary material (being DNA absent).

(6) DNA replication : Watson and Crick suggested a very simple mechanism of DNA replication or DNA transcription on the basis of its double helical structure. During replication the weak hydrogen bondsbetween the nitrogeneous bases of the nucleotides separate so that the two polynucleotide chains of DNA also separate and uncoil. The chains thus separated are complementary to one another. Because of the specificity of base pairing, each nucleotide of separated chains attracts it complementary nucleotide from the cell cytoplasm. Once the nucleotides are attached by their hydrogen bonds, their sugar radicals unite through their phosphate components, completing the formation of a new polynucleotide chain.

The method of DNA replication is semi-discontinuous and described as semi-conservative method,because each daughter DNA molecule is a hybrid conserving one parental polynucleotide chain and the other one newly synthesized strand. DNA replication occur in S-phage in cell cycle.

(i) Mechanism of DNA replication    

The entire process of DNA replication involves following steps in E.coli.

(a) Recognition of the initiation point : First, DNA helix unwind by the enzyme “Helicase” which use the energy of ATP and replication of DNA begin at a specific point, called initiation point or origin where replication fork begins.

(b) Unwinding of DNA :The unwinding proteins bind to the nicked strand of the duplex and separat the two strands at DNA duplex. Topoisomerase(Gyrase is a type of topoisomerase in E.coli)helps in unwinding of DNA.

(c) Single stranded binding protein (SSB) : Which remain DNA in single stranded position and also known as helix destabilising protein (HDP).

(d) RNA Priming :The DNA directed RNA polymerase now synthesizes the primer strands of RNA (RNA primer).  The priming RNA strands are complementary to the two strands of DNA and are formed of 50 to 100 nucleotides.

(e) Formation of DNA on RNA primers :The new strands of DNA are formed in the 5′→3′ direction from the 3′→5′ templateDNA by the addition of deoxyribonucleotides to the 3′  end of primer RNA.    

Addition of nucleotide is done by DNA polymerase III. The leading strand of DNA is synthesized continuously in 5′→3′direction as one piece. The lagging strand of DNA is synthesized discontinuously in its opposite direction in short segments.  These segments are called Okazaki fragments.

(f) Excision of RNA primers : Once a small segment of an okazaki fragment has been formed. The RNA primers are removed from the 5′by the action of 5′→3′exonuclease activity of DNA polymerase I.  

(g) Joining of okazaki fragments :The gaps left between Okazaki fragments are filled with complimentary deoxyribonucleotide residues by DNA polymerase-I. Finally, the adjacent 5′ and 3′ ends are joined by DNA ligase.

(ii) DNA polymerase enzymes  : There are three DNA polymerase enzymes that participate in the process of DNA replication.

(a) DNA polymerase-I : This enzyme has been studied in E. coli in detail. It possesses a sulphydryl group, single interchain disulphide and one zinc molecule at the active site. DNA polymerase-I was discovered by Kornberg and his colleagues in 1955. It was considered to carry out DNA replication and also participates in the repair and proof reading of DNA by catalyzing the addition of mononucleotide units (the deoxyribonucleotide residues) to the free 3′ -hydroxyl end of DNA chain. A pure DNA polymerase-I can add about 1,000 nucleotide residues per minute per molecule and catalyses 5′ →3′ exonuclease activity and removes nucleotide residues of primer RNA at 3′.

(b) DNA polymerase-II : The biological role of polymerase II is not yet known.

(c) DNA polymerase-III : This enzyme was discovered by T. Kornberg and M.L. Gefter (1972). It is the most active  enzyme and responsible for DNA chain elongation.

(iii) DNA repair : When DNA damaged by mutagen, a system is activate to repair damage DNA. Say for example UV light induced thymidine dimers in DNA and repair mechanism of that DNA called photoreactivation. Many enzyme involved in repair mechanism in which endonuclease (Chemical knives) cut the defective part of DNA then gap is filled with DNA polymerase I and finally DNA ligase seals that repaired part.

(iv) Evidence in support of semiconservative mode of DNA replication (Meselson and Stahl’s experiment) :Meselson and Stahl (1958)cultured (Escherichia coli) bacteria in a culture medium containing N15 were isotopes of nitrogen. After these had replicated for a few generations in that medium both the strands of their DNA contained N15 as constituents of purines and pyrimidines. When these bacteria with N15were transferred in cultural medium containing N14, it was found that DNA separated from fresh generation of bacteria possesses one strand heavier than the other. The heavier strand represents the parental strand and lighter one is the new one synthesized from the culture indicating semiconservative mode of DNA replication. circular form of replication on as characteristic of prokaryotes is theta replication discovered by J. Cairns.

D. RNA (Ribonucleic acid)  

Introduction : RNA is found in the cytoplasm and nucleolus.Inside the cytoplasm it occurs freely as well as in the ribosomes. RNA can also be detected from mitochondria, chloroplasts and associated with the eukaryotic chromosomes. In some plant viruses RNA acts as hereditary material.

(1) Structure of RNA : More commonly RNA is a single stranded structure consisting of an unbranched  polynucleotide chain, but it is often folded back on itself forming helices. DNA is a double stranded structure and its two polynucleotide chains are bounded spirally around a main axis. It is made up by:

(i) Sugar : Ribose

(ii) Phosphate : In the form of H3PO4.

(iii) Nitrogenous base : Two types: (a) Purine,  (b) Pyramidine

(a) Purine is further divided into Adenine and Guanine.

(b) Pyramidindivided into Cytosineand Uracil.

(2) Types of RNA :  RNA can be classified into two types.

(i) Genetic RNA : Which established by Conrat. In most of the plant viruses, some animal viruses and in many bacteriophages DNA is not found and RNA acts as hereditary material. This RNA may be single stranded or double stranded.

(ii) Nongenetic RNA : In the all other organisms where DNA is the hereditary material, different types of RNA are nongenetic. The nongenetic RNA is synthesized from DNA template. In general, three types of RNAs have been distinguished:   

(a) Messenger RNA or nuclear RNA (mRNA)        (b) Ribosomal RNA (rRNA)     (c) Transfer RNA (tRNA)

(a) Messenger RNA or Nuclear RNA : mRNA is a polymer of ribo-nucleotide as a complementary strand to DNA and carries genetic information in cytoplasm for the synthesis of proteins. For this reason only, it was named messenger RNA (mRNA) by Jacob and Monod is 5% of total RNA. It acts as a template for protein synthesisand has a short life span.

(b) Ribosomal RNA : rRNA constitutes redundant nature upto 80% of total RNA of the cell. It occurs in ribosomes, which are nucleoprotein molecules.

Types of rRNA : Inside the ribosomes of eukaryotic cells rRNA occurs in the form of the particles of three different dimensions. These are designated 28S, 18S, and 5S. The 28S and 5S molecules occur in large subunit  (60S subunit) of ribosome, whereas 18S molecules is present in the small subunit (40S subunit) of ribosome. In prokaryotic cells there are only 23S and 16S rRNA are found. Which are synthesized in Nucleolus / SAT region.

(c) Transfer RNA (tRNA) : The transfer RNA is a family of about 60 small sized ribonucleic acids which can recognize the codons of mRNA and exhibit high affinity for 21 activated amino acids, combine with them and carry them to the site of protein synthesis. RNA molecules have been variously termed as soluble RNA or supernatant RNA or adapter RNA. It is about 0-15% of RNA of the cell. tRNA molecules are smallest, containing 75 to 80 nucleotides. The 3 ′ end of the polynucleotide chain ends in CCA base sequence. This represents site for the attachment of activated amino acid. The end of the chain terminates with guanine base. The bent in the chain of each tRNA molecule contains a definite sequence of three nitrogenous bases, which constitute the anticodon. It recognizes the codon on mRNA.

Four different region or special sites can be recognised in the molecule of tRNA.These are :

Amino acid attachement site : It occurs at the 3′end of tRNA chain and has OH group combines with specific amino acid in the presence of ATP forming amino acyl tRNA.

Site for activating enzymes : Dihydrouridine or DHU loop dictate activation of enzymes.

Anticodon or codon recognition site : This site has three unpaired bases(triplet of base) whose sequence is complementary with a codon in mRNA.

Ribosome recognition site () : This helps in the attachment of tRNA to the ribosome.  

E. Genetic code

Introduction: Defined as structure of nitrogen bases(nucleotides) in mRNA molecule which contain the information for the synthesis of protein molecule. It is discovered by frame shift mutation by Crick.

Codon is the sequence of nitrogen bases (nucleotides) in mRNA, which codes for a single amino acid. Nirenberg and Mathaei (1961) experimentally proved that a single amino acid is determined by a sequence of three nitrogen bases which is known as triplet code. Khorana has got Nobel prize on genetic code.

Salient Features

(i) Triplet : A single amino acid is specified by a sequence of three nucleotides in mRNA i.e.called codon. Due to triplet nature, it consist 64 codon.

(ii) Universal :A codon specifies the same amino acid in all organisms from viruses to human beings.

(iii) Commaless :There is no pause, so it reads continously.

(iv) Non-overlapping :No overlapping between adjacent nucleotide.

(v) Initiation codon :The synthesis of polypeptide chain initiated by initiation codon, which located beginning the cistron i.e.,AUG or GUG, which codes to methionine and valine amino acid respectively.

(vi) Termination codon : Termination is done by codon. These are UAA, UGA or UAGwhich does not code to any amino acid. These are also called nonsense codon.

(vii) Degeneracy :A single amino acid may be specified by many codon i.e., called degeneracy. Degeneracy is due to the last base in codon, which is known as wobble base. Thus first two codon are more important to determining the amino acid and third one is differ without affecting the coding i.e.,known wobble hypothesis, which establishes a economy of tRNA molecule and put forwarded by Crick.Degeneracy of genetic code was discovered by Berrfield and Nirenberg.

F. Transcription.

Formation of mRNA from DNA is called as Transcription. It is heterocatalytic function of DNA. Template of DNA called sense strand (Master Strand) is involved. The segment of DNA involved in transcriptions is cistron, which have a promoter region where initiation is start and terminator region where transcription ends. Enzyme involved in transcription is RNA polymerase-II. Which consist five polypeptide (constitute core enzyme) and (sigma factor). Sigma actor recognise promoter site while remaining core enzyme takes part in chain elongation. After transcription, DNA molecule reassociates to form its original structure. In eukaryotes hn RNA (heterogenous nuclear RNA) which consist exon (coded region) and introns(non coded region or intervening sequences) formed in nucleus and diffuse in cytoplasm is also known as split gene which goes to transcription changes for removing the introns and later formed mRNA.

It consist three phenomenon: –     

(1) Initiation : Initiation start with help of (sigma) factor of RNA polymerase enzyme. At the cap region which have 7 methyl guanosine residue at the 5′.

(2) Elongation : Elongation is done by core enzyme, which moves along the sense strand.

(3) Termination : In prokaryotes termination is done by rho () factor while in eukaryotes poly A tail is responsible for termination at the 3′.

G. Translation.

Formation of protein from mRNA is called translationis also known as polypeptide synthesis or protein synthesis. It is unidirectional process. The ribosomes of a polyribosome are held together by a strand of mRNA. Each eukaryotic ribosome has two parts, smaller 40S subunit (30S in prokaryotes) and larger 60S subunit (50S in prokaryotes). Larger subunit has a groove for protection and passage of polypeptide, site A (acceptor or aminoacyl site), enzyme peptidyl transferase and a binding site for tRNA. The smaller subunit has a point for attachmentof mRNA. Along with larger subunit, it forms a P-site or peptidyl transfer (donor site). There are binding sites for initiation factors, elongation factors, translocase, GTPase, etc. The raw materials for protein synthesis are amino acids.mRNA, tRNAs and amino acyl tRNA synthetases.

Amino acids : Twenty types of amino acids and amides constitute the building blocks of proteins.

mRNA : It carries the coded information for synthesis of one (unicistronic) or more polypeptides (polycistronic). Its codons arerecognised by tRNAs.

tRNAs : They picks up specific amino acid from amino acid pool and carrying over the mRNA strand.

Amino Acyl tRNA Synthetases : The enzymes are specific for particular amino acids and their tRNAs.

(1) Activation of Amino Acids : An amino acid combines with its specific aminoacyl tRNA synthetaseenzyme (AA-activating enzyme) in the presence of ATP to form aminoacyl adenylate enzyme complex (AA-AMP-E). Pyrophosphate is released. Amino acid present in the complex is activated amino acid. It can attach to CCA or 3′end of its specific tRNA to form aminoacyl or AA-tRNA (charged tRNA / adaptor molecule)

Amino Acid (AA) + ATP + Aminoacyl tRNA Synthetase (E)

AA-AMP-E + tRNA AA—tRNA + AMP + Enzyme.

(2) Initiation : It is accomplished with the help of initiation factors. Prokaryotes have three initiation factors – IF3, IF2and IF1. Eukaryotes have nine initiation factors – eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF4D, eIF5, eIF6,,mRNA attaches itself to smaller subunit of ribosome with its cap coming in contact with 3′end of 18 S rRNA (16S RNA in prokaryotes). It requires eIF2(IF3 in prokaryotes). The initiation codon AUG or GUGcomes to lie over P-site. It produces 40S – mRNA complex. P-site now attracts met tRNA (depending upon initiation codon). The anticodon of tRNA (UAC or AUG) comes to lie opposite initiation codon. Initiation factor eIF3 (IF2 in prokaryotes) and GTPare required. It gives rise to 40S-mRNA - tRNAMet. Methionine is nonformylated (tRNA) in eukaryoticcytoplasm and formylated (tRNA in case of prokaryotes. The larger subunit of ribosome now attaches to 40S-mRNA-tRNAMetcomplex to form 80S mRNA -tRNA complex. Initiation factors eIF1 and eIF4(A, B and C) are required in eukaryotes and IF1 in prokaryotes. Mg2+is essential for union of the two subunit of ribosomes. A-site becomes operational. Second codon of mRNA lies over it.

(3) Elongation/chain formation : A new AA-tRNA comes to lie over the A site codon by means of GTP and elongation factor (eEF1in  eukaryotes, EF-Tu and EF-Ts in prokaryotes). Peptide bond (–CO.NH–) is established between carboxyl group (–COOH) of amino acid of P-site and amino group (–NH2) of amino acid at A-site with the help of enzyme peptidyl transferase/synthetase.

Connection between tRNA and amino acid of P-site and A-site tRNA comes to bear a dipeptydl. Freed tRNA of P-site slips away. By means of translocase(eEF2 in eukaryotes and EF-G in prokaryotes) and GTP, ribosome moves in relation to mRNA so that peptidyl carrying tRNA comes to lie on P-site and a new codon is exposed at A-site.Incorporation of an amino acid in polypeptide chain thus requires one ATP and two GTP molecules. Peptide formation and translocation continue uninterrupted till the whole m-RNA code is translated into polypeptide. In a polyribosome, when a number of ribosomes are helping in translation of same mRNA code, the ribosome nearest the 5′end of mRNA carries the smallest polypeptide and the one towards the 3′ end the longest. Of course, ultimately the whole polypeptide is formed by each.

(4) Termination :Polypeptide synthesis stops when a nonsense or termination codon [UAA, (ochre), UAG (Amber) or UGA (opal)] reaches A-site. It does not attract any AA-tRNA, P-site tRNA seperates from its amino acid in the presence of release factor eRF1in eukaryotes (RF1for UAG and UAA, RF2for UAA and UGA in prokaryotes).  The completed polypeptide is released, mRNA and ribosome separate. The two subunits of ribosome also dissociate with the help of dissociation factor.

(5) Modification : Formylated methionine present at the beginning of polypeptide in prokaryotes and organelles is either deformylated (enzyme deformylase) or removed from chain (enzyme exopeptidase). Initially the polypeptide is elongated having only primary structure. As soon as the polypeptide comes out the groove of larger ribosome sub-unit, it forms -helix (secondary structure) which coils further forming a number of linkages (tertiary structure). Two or more polypeptides may get associated to become -pleated which then coil to produce tertiary and quaternary structure.

H.  Genes expression and its regulation.

(1) Gene expression in prokaryotes : Gene expression refers to the molecular mechainism by which a gene expresses a phenotype by synthesizing a protein or an enzyme. Which determines the character. The gene contains the blue print or the information for the protein or an enzyme.

The category includes mechanism involved in the rapid turn-on and turn-off gene expression in response to environmental changes. Regulatory mechanism of this type are very important in microorganisms, because of the frequent exposure of these organisms to sudden changes in environment.

Gene concept can be studied by operon model. Operon are segment of genetic material which function as regulated unit that can be switched on and switched off, which was given by French scientists. Jacob and Monod (1961) working at Pasteur institute. They were studying lactose utilisation in mutants of E.coli.An operon consists of one to several structural genes (three in lac operon and five in tryptophan operon of Escherichia coli, nine in histidine operon of Salmonella typhimurium), an operator gene a promoter gene a regulator gene, a repressor and inducer or corepressor. Operons are of two types, inducible and repressible.

(i) Inducible operon system /lac operon system : An inducible operon system is that regulated genetic material which remains switched off normally but becomes operational in the presence of an inducer. It occurs in catabolic pathways.The components are :–

(a) Structural genes : They are genes, which produce mRNAs for forming polypeptides/proteins/enzymes. Lac operon of Escherichia colihas three structural genes-Z(produces enzyme -galactosidasefor splitting lactose/galactoside in to glucose and galactose) Y (produces enzyme galactoside permeaserequired in entry of lactose/galactoside) and A(produces enzyme galactoside acetylase/transacetylasewithout any function in E.coli). The three structural genes of lac operon produce a single polycistronic mRNA. The three enzymes are, however, produced in different concentration.

(b) Operator gene : It gives passage to RNA polymerase when the structural genes are to express themselves. Normally, it is covered by a repressor. Operator gene of lac operon is small, made of 27 base pairs.

(c) Promoter gene : It is recognition centre / initiation point for RNA polymerase of the operon.

(d) Regulator gene (i Gene) : It produces a repressor that binds to operator gene for keeping it nonfunctional (preventing RNA polymerase to pass from promoter to structural genes).

(e) Repressor : It is a small protein formed by regulator gene. Which binds to operator gene and blocks passage of RNA polymerase towards structural enzymes. Repressor has two allosteric sites, one for attaching to operator gene and second for binding to inducer. Repressor of lac operon has a molecular weight of 160,000 and 4 subunit of 40,000 each.

(f) Inducer : It is a chemical which attaches to repressor, changes the shape of operator binding site so that repressor no more remain attached to operator.

Lactose/galctoside is inducer of lac operon. As soon as the operator gene becomes free, RNA polymerase is recognised by promoter gene. cAMP is required, RNA  polymerase passes over the operator gene and then reaches the area of structural genes. Here it catalyses transcription of mRNAs.

Induction

Repression

It turns the operon on.

It turns the operon off.

It starts transcription and translation.

It stops transcription and translation.

It is caused by a new metabolite which needs enzymes to get metabolised.

It is caused by an excess of existing metabolite

It operates in a catabolic pathway.

It operates in an anabolic pathway.

Repressor is prevented by the inducer from joining the operator gene.

Aporpressor is enabled by a corepressor to join the operator gene

(ii) Repressible operon system/tryptophan operon system : A repressible operon system is that regulated genetic material, which normally remains active/operational and enzymes formed by its structural genes present in the cell till the operon is switched off when concentration of an end product crosses a threshold value. Repressible operon system usually occurs in anabolic pathways, e.g., tryptophan operon, argnine operon. Each has the following parts.

(a) Structural genes : They are genes, which take part in synthesis of polypeptides/proteins/enzymes through the formation of specific mRNAs. Tryptophan operon has five structural genes – E, D, C, B and A.

(b) Operator gene : It provides passage to RNA polymerase moving from promoter to structural genes. Operator gene of repressible operon is normally kept switched on as aporepressor formad by regulator gene is unable to block the gene.

(c) Promoter gene :It is initiation/recognition point for RNA polymerase.

(d) Regulator gene :The gene produces an aporepressor.

(e) Aporepressor :It is a proteinaceous substance formed through the activity of regulator gene. It is able to block operator gene only when a corepressor is also available.

(f) Corepressor : The nonproteinaceous component of repressor, which can be end product (feed back inhibition/repression) of the reaction mediated through enzymes synthesized by structural genes. Corepressor of tryptophan operon is tryptophan. It combines with aporepressor, form repressor which then blocks the operator gene to switch off the operon.

(2) Gene expression in eukaryotes : In regulation of gene expression in eukaryotes the chromosomal proteins play important role. The chromosomal proteins are of two types. They are histones and non-histones. The regulation of gene expression involves an interaction between histones and non-histones. Histones inhibit protein synthesis and non-histones induce RNA synthesis. There are four main steps in the expression of genes. Hence regulation is brought about by the regulation and modification of one or more of these steps. They are

(i) Replication        (ii) Transcription     (iii) Processing      (iv) Translation

(i) Regulation of replication : Differential gene expression is achieved by gene amplification.

(ii) Regulation of transcription : The regulation of the expression of gene is mainly done at transcription. Hybridization experiments clearly show that production of specialised protein is due to differential gene transcription.

(iii) Regulation of the processing level : Some of the RNA synthesized in the nucleus are destroyed without leaving the nucleus. 80% of the nuclear RNA has no equivalent in the cytoplasm and only 20% if the nuclear RNA is identical in the cytoplasm. All the genes in a cell are transcribed into mRNA at all times, but the mRNA produced by some genes is destroyed rapidly. But the mRNA modeled on other genes are stabilized and only these mRNAs are passed into the cytoplasm.

(iv) Regulation of translation : The control of mRNA-translation is a fundamental phenomenon. In sea-urchin eggs fertilisation is followed by a tremendous increase in protein synthesis; but in the unfertilised egg, there is no protein synthesis. Still the unfertilised egg has complete machinery (i.e.,amino acids, ribosomes, mRNA) for protein synthesis. There are two model for regulation in eukaryotes.

(a) Frenster's model :According to 1965, The histones act as repressor's during protein synthesis.

(b) Britten Davidson model :This is also called gene battery model or operon-operator model. It was proposed by Britten and Davidson in 1969. They have been proposed four type of genes namely integrator. sensor, producer and receptor.

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