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Dinucleotide Repeats in Z-DNA:- Dinucleotide repeats are short DNA sequences in which a pair of nucleotides is repeated many times. In Z-DNA, the most common repeat is:

(CG)n = CGCGCGCG...

where n represents the number of repeated CG units.

Key Features:-

> Z-DNA is a left-handed DNA double helix.

> It forms most easily in regions containing alternating purine and pyrimidine bases, especially CG repeats.

> Common sequences that form Z-DNA:

    i. (CG)n (most stable)

    ii. (CA/TG)n (under certain conditions)

Why Dinucleotide Repeats Favor Z-DNA:-

> Alternating purine (G) and pyrimidine (C) bases allow the DNA backbone to adopt the characteristic zigzag shape of Z-DNA.

> Negative supercoiling, high salt concentration, or cytosine methylation further stabilize these repeats in the Z-DNA conformation.

Biological Importance:-

> Helps regulate gene expression.

> Involved in DNA recombination.

> Plays a role in genome stability and DNA repair.

> Z-DNA-forming repeats are often found near promoter regions of actively transcribed genes.

Topoisomerases:- These are enzymes that regulate the topology (supercoiling) of DNA. They prevent DNA from becoming excessively twisted during DNA replication, transcription, recombination, and chromosome segregation.

Functions:-

> Relieve positive and negative DNA supercoils.

> Prevent DNA tangling during replication and transcription.

> Facilitate chromosome condensation and separation during cell division.

> Maintain DNA structural integrity.

CAP in Lac Gene Regulation:- CAP (Catabolite Activator Protein), also called CRP (cAMP Receptor Protein), is a positive regulatory protein that enhances transcription of the lac operon in E. coli when glucose is scarce.

Role of CAP:-

> CAP binds to cyclic AMP (cAMP) to form the CAP–cAMP complex.

> This complex binds to the CAP-binding site located upstream of the lac promoter.

> Binding of the CAP–cAMP complex helps RNA polymerase bind more efficiently to the promoter, increasing transcription of the lac operon.

Nurse Culture Technique:- It is a microbiological method in which a fastidious (difficult-to-grow) microorganism is grown in the presence of another microorganism (called the nurse organism) that supplies essential growth factors or modifies the environment to support its growth.

Principle:-

> Some bacteria cannot grow alone on ordinary culture media.

> A nurse organism secretes essential nutrients (e.g., growth factors such as NAD or hemin) or alters the medium, allowing the fastidious organism to grow nearby.

Procedure:-

> Prepare a suitable agar medium.

> Streak the nurse organism on the agar.

> Inoculate the fastidious organism near the nurse organism.

> Incubate under suitable temperature and conditions.

> Observe the growth of the fastidious organism around the nurse culture.

Genomic library:- 

> Size of some genomes and chromosomes:-

> The human genome contains approximately 50,000 unique genes within 3-4 billion base pairs of DNA, scattered about in 23 pairs of chromosomes.

> Fragmentation of genomic DNA:- Genomic DNA is too large to be incorporated into a vector and must be fragmented into desired sizes. RE enzymes are used for digestion.

> Insertion into lambda phage vectors:- 

- lambda phage is an E. coli phage with a type of icosahedral phage particle which contains the viral genome.

- During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid.

It was found that internal regions of the phage genome, which were not essential to phage replication, could be removed and replaced with DNA of interest.

This hybrid DNA could be efficiently packaged, and form an infective phage.

Replication Enzymology in Eukaryotes:- DNA replication in eukaryotes requires several enzymes and proteins that work together to ensure accurate duplication of the genome.

Enzymes / Proteins and their Functions:-

Origin Recognition Complex (ORC):- Recognizes and binds to the origin of replication.

Helicase (MCM2–7 complex):- Unwinds the DNA double helix by breaking hydrogen bonds.

Topoisomerase I & II:- Relieve torsional stress and prevent DNA supercoiling ahead of the replication fork.

Replication Protein A (RPA):- Binds to single-stranded DNA and prevents reannealing.

DNA Polymerase α (with Primase):- Synthesizes a short RNA primer followed by a short DNA segment to initiate replication.

DNA Polymerase ε (Epsilon):- Synthesizes the leading strand continuously in the 5′→3′ direction.

DNA Polymerase δ (Delta):- Synthesizes the lagging strand discontinuously, forming Okazaki fragments.

PCNA (Proliferating Cell Nuclear Antigen):- Sliding clamp that increases the processivity of DNA polymerases.

RFC (Replication Factor C):- Clamp loader that loads PCNA onto DNA.

RNase H & FEN1 (Flap Endonuclease 1):- Remove RNA primers and process primer remnants.

DNA Ligase I:- Joins Okazaki fragments by sealing nicks in the sugar-phosphate backbone.

Telomerase:- Extends telomeres at chromosome ends to prevent DNA shortening during replication.

DNA sequencing:-

> It is the process of determining the sequence of nucleotides in a piece of DNA.

> Methods:-

a. Maxam - Gilbert method

b. Sanger's method

a. Maxam - Gilbert method:-

- Also called as chemical cleavage method.

- Discovered by American molecular biologists Allan M. Maxam and Walter Gilbert in 1973.

Procedure:- The steps of Maxam Gilbert Sequencing are: 

i. Radioactive labeling of the 5′ end by a kinase reaction using gamma-32P ATP and purification.

  

ii. Four chemical Treatments for A+ G,  G, C + T, C bases (Depurination of purines (A+G) by  formic acid, Methylation of guanine (G) by  dimethyl sulfate, Hydrolyization of pyrimidines  (C+T) by hydrazine, and Inhibition of the hydrazine  reaction for thymine by the addition of sodium chloride, hydrolyzing  only cytosine (C).

iii. Cleavage of the modified DNA by hot piperidine;  (CH2)5NH  at the position of the modified base producing a series of labeled fragments from the radiolabeled end to the first ‘cut’ site.  

iv. Size fractionation of the fragments on a PAGE (polyacrylamide gel electrophoresis).  

v. Visualization by autoradiography, inferring the sequence.

b. Sanger's method:-

- Also called as dideoxy method.

- Discovered by English biochemist Frederick Sanger in 1977. He got Nobel Prize in Chemistry in 1980.

Procedure:- The Sanger sequencing method consists of 6 steps:

i. The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ssDNA).

ii. A primer that corresponds to one end of the sequence is attached.

iii. Four polymerase solutions with four types of dNTPs but only one type of ddNTP are added.

iv. The DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated.

v. The resulting DNA fragments are denatured into ssDNA.

vi. The denatured fragments are separated by gel electrophoresis and the sequence is determined.

Lac operon in E. coli:-

·  The operon controlling lactose metabolism. It consists of -

a) A regulatory or inhibitor (i) gene]:- It codes for the repressor.

b) 3 structural genes:-

i. z gene:- It codes for β-galactosidase which hydrolyze lactose to galactose and glucose.

ii. y gene:- It codes for permease which increase permeability of the cell to lactose.

iii. a gene:- It codes for transacetylase.

·  The genes present in the operon function together in the same or related metabolic pathway. There is an operator region for each operon.

·  In the absence of inducer:- If there is no lactose (inducer), Lac operon remains switched off. The regulator gene synthesizes mRNA to produce the repressor protein; this protein binds to the operator genes and blocks RNA polymerase movement. So the structural genes are not expressed.

·  In the presence of inducer:- If lactose is provided in the growth medium, the lactose is transported into the E. coli cells by the action of permease. Lactose (inducer) binds with repressor protein. So repressor protein cannot bind to operator gene. The operator gene becomes free and induces the RNA polymerase to bind with promoter gene. Then transcription of structural genes starts. It is also known as indusible operon. Regulation of lac operon by repressor is called negative regulation.

परागकोष संवर्धन (Anther Culture):-

1. परिचय (Introduction):-

·    परिभाषा (Definition):- यह एक कृत्रिम तकनीक है जिसके द्वारा परिवर्धित हो रहे परागकोषों को एक बन्द पुष्प कालिका से एक ठीक व नाजुक अवस्था पर निकाला जाता है और पोषक माध्यम पर संवर्धित किया जाता है। जहाँ परागकोष के अंदर उपस्थित लघुबीजाणु कैलस ऊतक या भ्रूण समान संरचना में विकसित हो जाते हैं जो अंग निर्माण या भ्रूण निर्माण के माध्यम से अगुणित पौधों का निर्माण करते हैं।

(It is an artificial technique by which the developing anthers are collected from a closed floral bud at a fine and delicate stage and cultured on a nutrient medium. Where the microspores develop into callus tissue or embryo like structure, that develop haploid plants through organ formation or embryo formation.)

·    अभी तक लगभग 250 पादप जातियों में परागकोष संवर्धन किया गया है। तीन कुलों में अधिक सामान्य है –

(So far anthers have been cultured in about 250 plant species. The three families are more common -)

i. Solanaceae

ii. Criciferae

iii. Poaceae

2. इतिहास (History):-

·    W. Tulecke (1953):- इसने सबसे पहले देखा कि अनावृतबीजी पौधे Ginkgo biloba के परिपक्व परागकणों को संवर्धन में अगुणित कैलस निर्माण के लिए प्रेरित किया जा सकता है।

(He first observed that mature pollens of the gymnosperm plant Ginkgo biloba can be induced to form haploid callus in culture.)

·    S. Guha and P. Maheshwari (1964):- इन्होने सबसे पहले देखा कि Datura innoxia के निकाले गए परागकोषों के संवर्धन से लघुबीजाणुओं से भ्रूणो का प्रत्यक्ष विकास होता है।

(They first observed that the embryos are developed from the microspores directly in anther culture of Datura innoxia.)

·    J. P. Bourgin and J. P. Nitsch (1967):- इन्होने तंबाकू (Nicotiana tabacum) के परागकोष संवर्धन द्वारा पूर्ण रूप से अगुणित पौधे प्राप्त किए।

[They obtained fully haploid plants by the anther culture of tobacco (Nicotiana tabacum).]

·    Niizeki and Oono (1968):- ये जापान के वैज्ञानिक थे। इन्होने धान में परागकोष संवर्धन करके पादप प्रजनन के लिए अगुणित पौधे विकसित किए।

(He was a Japanese scientist. He developed haploid plants for plant breeding by anther culture in paddy.)

3. सिद्धान्त (Principle):-

·    लघुबीजाणु की पूर्णशक्तता के उपयोग से अगुणित पौधे का निर्माण किया जाता है।

(The haploid plant is produced using the totipotency of the microspore.)

·    लघुबीजाणु में गुणसूत्रों का केवल एक समुचय उपस्थित होता है।

(Only one set of chromosomes is present in the microspore.)

·    अगुणित पादप निर्माण की प्रक्रिया में लघुबीजाणु का नर युग्मक निर्माण का सामान्य विकास व कार्य रुक जाता है। कायिक कोशिका विभाजन के लिए इसे बलपूर्वक नए उपापचय पथ की ओर मोड़ दिया जाता है।

(In the process of haploid plant development, the normal growth and functioning of the microspore of developing male gametes is halted. It is forced into a new metabolic pathway for somatic cell division.)

·    परागकोष संवर्धन में परागकोष के अंदर उपस्थित लघुबीजाणु के प्राकृतिक आवास व वातावरण को परिवर्तित नहीं किया जाता है। संवर्धन परिस्थितियों में माध्यम पर परागकोष का द्विगुणित ऊतक बिना विभाजन किए जीवित रहता है और पोषण प्रदान करके लघुबीजाणु के कायिक विभाजन को प्रेरित करता है।

(Anther culture does not alter the natural habitat and environment of the microspores present inside the anther. In culturing conditions, the diploid tissue of the anther survives on the medium without cell division and induces somatic cell division of the microspore by providing nutrition.)

4. विधि (Procedure):-

·    पुष्पन पर तंबाकू की बन्द पुष्पीय कलिकाओं को एकत्रित करते हैं। 17 – 22 mm लंबाई की पुष्पीय कालिका का चयन करते हैं जब बाह्यदलों की लंबाई दलों की लंबाई के बराबर होती है। खुल रही सभी पुष्पीय कलिकाओं को त्याग देते हैं।

(Collect the closed floral buds of tobacco upon flowering. A floral bud of length 17 - 22 mm is selected when the length of the sepals is equal to the length of the petals. Discards all floral buds are to be opened.)

·    चयनित पुष्पीय कलिकाओं को LAF कैबिनेट के अन्दर ले जाते हैं। प्रत्येक पुष्पीय कलिका में 5 परागकोष होते हैं और बन्द कलिकाओं के अन्दर इनकी सतह स्वत: निर्जमित होती है। पुष्पीय कलिकाओं के सतही निर्जमीकरण के लिए इन्हें पहले 10 सेकंड के लिए 70% ऐथेनोल में डुबोकर रखते हैं और फिर 10 मिनट के लिए 20% सोडियम हाइपोक्लोराइट विलयन में डुबोकर रखते हैं। अतिरिक्त रसायन को सतह से हटाने के लिए पुष्पीय कलिकाओं को निर्जमित आसुत जल से 3 बार धोते हैं। अंत में इन पुष्प कलिकाओं को निर्जमित पेट्रीडिश में स्थानांतरित कर देते हैं।

(Selected floral buds are carried inside the LAF cabinet. Each floral bud has 5 anther and its surface is sterilized automatically inside the closed buds. For surface sterilization of the floral buds, first of all dip them in 70% ethanol for 10 seconds and then in 20% sodium hypochlorite solution for 10 minutes. To remove excess chemicals from the surface, the floral buds are washed 3 times with sterilized distilled water. Finally these floral buds are transferred to the sterilized patridish.)

·    अब एक तीखे चाकू के द्वारा कालिका के एक तरफ कट लगाते हैं और चिमटी की सहायता से दलों व बाह्यदलों को हटा देते हैं। अब एक अन्य चिमटी की सहायता से 5 पुंकेसरों को पुतन्तु सहित उखाड़कर एक अन्य निर्जमित पेट्रीडिश में स्थानांतरित करते हैं। अब 5 पुंकेसरों के पुतन्तुओं को चाकू या ब्लेड से काटकर अलग कर देते हैं जिससे केवल परागकोष रह जाते हैं। क्षतिग्रस्त परागकोषों को भी त्याग देते हैं।

(Now with a sharp knife, cut one side of the floral bud and remove the petals and sepals with the help of forceps. Now with the help of another forceps, collect 5 stamens along with their filaments and transfer them to another sterilized patridish. Now cut the filaments of 5 stamens with a knife or blade, leaving only the anthers. Damaged anthers are also discarded.)

·    अब इन स्वस्थ व साबुत परागकोषों को ठोस अगार माध्यम पर स्थापित करते हैं। परागकोष संवर्धन के लिए निम्न में से कोई एक माध्यम उपयोग किया जाता है-

(Now these healthy and intact anthers are placed on solid agar medium. One of the following mediums is used for anther culture -)

i. MS माध्यम (MS medium)

ii. White माध्यम (White's medium)

iii. Nitsch & Nitsch माध्यम (Nitsch and Nitsch medium)

·    प्रारम्भ में संवर्धन को अंधेरे में रखा जाता है। 3 – 4 सप्ताहों में परागकोष के अंदर उपस्थित लघुबीजाणु भ्रूणजनन करते हैं और संवर्धित परागकोषों से अगुणित भ्रूण बन जाते हैं। कुछ पौधों में परागकोष के प्रवर्धन द्वारा कैलस ऊतक का निर्माण हो सकता है जिन्हें अगुणित प्लांटलेट्स बनाने के लिए प्रेरित किया जा सकता है।

(Initially the culture is kept in the dark. In 3–4 weeks, the microspores present inside the anther show embryogenesis and haploid embryos are developed from cultured anther. In some plants anther culture can lead to the formation of callus tissue that can be induced to form haploid plantlets.)

·   अब इन अगुणित भ्रूणो को नये ताजा अगार माध्यम पर संवर्धन ट्यूब में स्थापित करते हैं।  इस अवस्था पर संवर्धनों का 24 – 28°C ताप पर ऊष्मायन किया जाता है। 14 घंटे का प्रकाश व 10 घंटे का अंधकार दिया जाता है। प्रकाश की तीव्रता 2000 लक्स रखी जाती है।

(Now these haploid embryos are established on a new fresh agar medium in culture tube. At this stage the cultures are incubated at 24 - 28 ° C temperature. 14 hours of light and 10 hours of darkness are given. Light intensity is maintained at 2000 lux.)

·    जब अगुणित प्लांटलेट्स की लंबाई 50 mm हो जाती है तो अगार माध्यम से स्वतंत्र करने के लिए नल के बहते हुए पानी में धोया जाता है। अब तुरन्त इन्हें औटोक्लेवित कम्पोस्ट युक्त छोटे गमलों में रोपित कर दिया जाता है। शुष्कन को रोकने के लिए प्रत्येक पौधे को काँच के बीकर से ढक देते हैं और आगे के परिवर्धन के लिए नम ग्रीन हाउस में रख देते हैं। कुछ सप्ताहों पश्चात काँच के बीकरों को हटा देते हैं और पौधों को मृदा युक्त बड़े गमलों में रोपित कर देते हैं जहाँ ये पौधे परिपक्व होकर अंत में पुष्पन करते हैं।

(When the length of haploid plantlets becomes 50 mm, they are washed in running tap water to free them from the agar medium. Now they are immediately planted in small pots containing autoclaved compost. To prevent drying, cover each plant with a glass beaker and place it in a moist greenhouse for further growth. After a few weeks, the glass beakers are removed and the plants are planted in large pots containing soil where these plants mature and eventually flowering.)

5. लाभ (Advantages):-  

·    आधारभूत अनुसंधान के लिए परागकोष संवर्धन की उपयोगिता 

(Utility of Anther Culture for Basic Research)

·    सरल (Simple)

·    कम समय लेता है 

(Less Time Consuming)

·    उत्तरदायी (Responsive)

·    उत्परिवर्तन का अध्ययन (Mutation Study):-

Ø  अगुणित पौधों में प्रत्येक जीन का केवल एक ही युग्मविकल्पी पाया जाता है। इसलिए कोई भी अप्रभावी उत्परिवर्तन या लक्षण स्पष्ट रूप से दिखाई देता है।

(In haploid plants, only one allele of each gene is found. Therefore any recessive mutation or trait is clearly visible.)

Ø  घातक जीनों युक्त पौधे जीन पूल से निष्कासित हो जाते हैं।

(Plants containing lethal genes are expelled from the gene pool.)

Ø  इस प्रकार उत्परिवर्तन का अध्ययन आसानी से किया जा सकता है।

(Thus mutation can be studied easily.)

·    क्रायोजेनिक अध्ययन के लिए अगुणितों का उपयोग 

(Use of Haploids for Cryogenic Study)

·    पादप प्रजनन व फसल सुधार के लिए उपयोग:-

(For Plant Breeding and Crop Improvement:-)

Ø  समयुग्मजी वंशक्रमों को उत्पन्न किया जा सकता है। इसके लिए अगुणित पौधों को कोल्चिसीन रसायन से उपचारित किया जाता है।

(Homozygous lines can be produced. For this, haploid plants are treated with colchicine chemical.)

Ø  उत्परिवर्तन प्रजनन विधि का उपयोग फसल सुधार में किया जाता है।

(Mutation breeding method is used in crop improvement.)

·    बागवानी पौधों के लिए अगुणित संवर्धन का अनुप्रयोग 

(Application of Haploid Culture for Horticultural Plants)

·    द्वितीयक मेटाबोलाइट्स के अध्ययन के लिए 

(For Study of Secondary Metabolites Content)

6. हानियाँ (Disadvantages):-

·    बिना क्षति पहुंचाए परागकोषों को निकालने के लिए कौशल की आवश्यकता होती है।

(Skill is required to remove anthers without damage.)

·    धान्य फसलों में यह बहुत अधिक सफल विधि नहीं है।

(This is not a very successful method in cereal crops.)

·    परागकोष की भित्ति द्विगुणित कोशिकाओं की बनी होती है जिससे द्विगुणित कैलस निर्माण व काइमेरा निर्माण का रिस्क रहता है।

(The anther wall is made up of diploid cells, which has the risk of developing diploid callus and chimera.)

Various Methods of Gene Transfer:- Gene transfer is the process of introducing foreign DNA (gene) into a host cell to produce desired traits or proteins. It is an essential step in genetic engineering and biotechnology.

1. Biological (Vector-Mediated) Methods:-

i. Agrobacterium-mediated Gene Transfer:-

> Uses the bacterium Agrobacterium tumefaciens.

> The Ti (Tumor-inducing) plasmid transfers the desired gene into plant cells.

> Most commonly used for dicot plants (e.g., tobacco, tomato, cotton).

> Advantages: High efficiency, stable gene integration.

ii. Viral Vector-mediated Gene Transfer:-

> Uses modified viruses as carriers.

> Common viral vectors include:

- Retroviruses

- Adenoviruses

- Lentiviruses

- Plant viruses (e.g., Cauliflower mosaic virus)

> Used in gene therapy and research.

2. Physical Methods:-

i. Gene Gun (Biolistic Method):-

> DNA-coated gold or tungsten particles are shot into cells using high pressure.

> Suitable for monocots (rice, maize, wheat) and plants difficult to transform by Agrobacterium.

ii. Electroporation:-

> Short electrical pulses create temporary pores in the cell membrane.

> DNA enters through these pores.

> Used for bacteria, yeast, plant protoplasts, and animal cells.

iii. Microinjection:-

> DNA is directly injected into the nucleus using a fine glass micropipette.

> Commonly used in animal embryos and cultured cells.

iv. Silicon Carbide Whisker Method:-

> DNA is mixed with needle-like silicon carbide fibers.

> Fibers create tiny openings in cells, allowing DNA entry.

v. Laser-mediated Gene Transfer:-

> A laser beam creates microscopic pores in the cell membrane.

> DNA enters through these pores.

3. Chemical Methods:-

i. Calcium Phosphate Method:-

> DNA forms a calcium phosphate precipitate.

> Cells take up the DNA by endocytosis.

> Common in mammalian cell transformation.

ii. Polyethylene Glycol (PEG)-mediated Transfer:-

> PEG increases membrane permeability.

> Mainly used for plant protoplasts and fungal cells.

iii. Liposome-mediated Gene Transfer (Lipofection):-

> DNA is enclosed in lipid vesicles (liposomes).

> Liposomes fuse with the cell membrane and deliver DNA.

> Widely used in animal cell culture.

iv. DEAE-Dextran Method:-

> DNA binds to the positively charged DEAE-dextran.

> Facilitates DNA uptake into animal cells.

4. Direct DNA Uptake Methods:-

i. Transformation:- Uptake of naked DNA by competent bacterial cells. Common in E. coli.

ii. Transfection:- Introduction of DNA into eukaryotic (animal) cells.

iii. Transduction:- Transfer of DNA from one bacterium to another through bacteriophages.

iv. Conjugation:- DNA transfer between bacterial cells through direct cell-to-cell contact using a sex pilus.

Transcription:-

Introduction:-

•   It is the process of copying genetic information from one strand of the DNA into RNA.

•   Here, adenine pairs with uracil instead of thymine.

•   Both strands are not copied during transcription, because:-

-   The code for proteins is different in both strands. This complicates the translation.

-   If 2 RNA molecules are produced simultaneously this would be complimentary to each other, hence form a double stranded RNA. This prevents translation.

Transcription Unit:-

•   It is the segment of DNA between the sites of initiation and termination of transcription. It consists of 3 regions:-

-   A promoter (Transcription start site):- Binding site for RNA polymerase.

-   Structural gene:- The region between promoter and terminator where transcription takes place.

-   A terminator:- The site where transcription stops.

•   The DNA- dependent RNA polymerase catalyzes the polymerization only in 5’→3’direction.

•   3’→5’ acts as template strand and 5’→3’ acts as coding strand.

3’-ATGCATGCATGCATGCATGCATGC-5’ template strand.

5’-TACGTACGTACGTACGTACGTACG-3’ coding strand.

Transcription unit and gene:-

•   Gene:- Functional unit of inheritance. It is the DNA sequence coding for RNA molecule.

•  Cistron:- A segment of DNA coding for a polypeptide.

•  Structural gene in a transcription unit is of 2 types:-

i. Monocistronic structural genes (split genes):- It is seen in eukaryotes. Here, the coding sequences  exons are interrupted by introns.

ii. Polycistronic structural genes:- It is seen in prokaryotes. Here, there are no split genes.

Reverse Transcription:- It is the process in cells by which an enzyme makes a copy of DNA from RNA. The enzyme that makes the DNA copy is called reverse transcriptase and is found in retroviruses, such as the human immunodeficiency virus (HIV).

Steps of transcription in prokaryotes:-

i. Initiation:- Here, the enzyme RNA polymerase binds at the promoter site of DNA. This causes the local unwinding of the DNA double helix. An initiation factor (σ factor) present in RNA polymerase initiates the RNA synthesis.

ii. Elongation:- The RNA chain is synthesized in the 5’-3’ direction. In this process, activated ribonucleoside triphosphates (ATP, GTP, UTP & CTP) are added. This is complementary to the base sequence in the DNA template.

iii. Termination:- A termination factor (ρ factor) binds to the RNA polymerase and terminates the transcription.

NOTE:- In bacteria (Prokaryotes) transcription and translation can be coupled (Translation can begin before mRNA is fully transcribed) because:-

-   mRNA requires no processing to become active.

-   Transcription and translation take place in the same compartment (no separation of cytosol and nucleus).

Polysome or Polyribosome or Ergosome:- It is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides.

Eukaryotic transcription:- 

In eukaryotes, there are 2 additional complexities-

i. There are 3 RNA polymerases:-

•  RNA polymerase I:- Transcribes rRNAs (28S, 18S & 5.8S).

•  RNA polymerase II:- Transcribes mRNA.

•  RNA polymerase III:- Transcribes tRNA, 5S rRNA and snRNAs.

ii. The primary transcripts (hnRNA):- It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes splicing process.

Control at Initiation:-

i. Steric hindrance for RNA polymerase binding:- If the operator and – 35 sequence overlap, repressor could prevent the formation of a closed complex by preventing polymerase binding. It is also possible that repressor binding can prevent the activator's interaction with polymerase and/or DNA.

ii. Inhibition of open complex formation:- Isomerization is a step in which RNA polymerase conformation is changed and a more stable complex is formed spanning the region of + 5 to beyond – 35 sequence. The exact boundary of nucleotides protected by RNA polymerase in footprinting

experiments varies depending on the promoter used in an experiment. The repressor when bound at or near the – 10 region of the promoter (the site of complex formation) could distort the DNA and as a consequence, formation of stable open complexes may be prevented. The Arc repressor involved in bacteriophage P22 lysogeny is believed to repress the Pant promoter in this fashion.

iii. Inhibition of initiation complex formation:- RNA polymerase initiates RNA synthesis with the formation of nascent RNA of 3-8 nucleotides. During this stage, polymerase is still bound to the promoter. The repressor which is bound to the operator may make direct contacts with the enzyme present in open complex state, thus preventing the subsequent step, i.e. oligoribonucleotide formation. Studies with the gal repressor support this mechanism. The gal repressor does not block open complex formation at the gal operon promoter and hence is believed to interfere at the stage of oligoribonucleotide formation.

iv. Increased abortive initiation:- Once an initiation complex is formed, RNA polymerase moves out of the promoter and gets locked into the elongation mode. The repressor could make contacts with RNA polymerase and block promoter clearance. 

Control at termination:-

Attenuation:- Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism.

i. Transcriptional-attenuation:- It is characterized by the presence of an attenuator within the DNA sequence that results in formation of mRNA-stem loops that prevent further transcription from occurring. The non-functional RNA produced prevents proper transcription.

ii. Translational-attenuation:- It is characterized by the misfolding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence, responsible for ribosomal binding to allow proper translation, is inaccessible because it is folded into a hairpin-loop structure, thus, translation cannot occur.

Heterogenous RNA Processing:- The hnRNA is the collective term for the unprocessed mRNA (pre-mRNA) molecules in the nucleus. It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes the following processes-

i. Splicing:- From hnRNA introns are removed by the spliceosome and exons are joined together.

ii. Capping:- Here, a nucleotide methyl guanosine triphosphate (cap) is added to the 5’ end of hnRNA.

iii. Tailing (Polyadenylation):- Here, adenylate residues (200-300) are added at 3’-end. It is the fully processed hnRNA, now called mRNA.

Production of useful compounds through cell-culture:- Biotransformation is used to production of secondary metabolites. The genetic information required for the manufacture of sec. 

1. Biotransformations of Steroids:- 

> Digitoxin and Digitoxigenin can be extracted from the leaves of Digitalis lanata. 

> In the process of biotransformation of Digitoxin many compounds are produced:

Methyl digitoxin →→→→ Purpurea glycoside A + Deacetyl lanotoside C + Lanatoside C

> Purpurea glycoside A is major product, while Deacetyl lanotoside C and Lanatoside C are minor products.

2. Biotransformation of Alkaloids:-

> A variety of alkaloids have been used, as pharmaceuticals & most of them are plant metabolites. 

> Research of production of useful alkaloids by plant cell cultures has been carried out for more than 25 years. 

> Industrial production has not yet succeeded because of low producing ability of the cultured of the cultured cells. 

> Plant used for their studies are mainly, Atropa belladonna, Hysocyamus niger, Datura metalloids.

> Some of the examples of the compounds in these groups include:

a. Morphine alkaloids

b. Berberine

c. Tropane alkaloids

d. Vincristine

a. Morphine alkaloids:- 

- Papaver somniferum is a traditional commercial source of Codeine & Morphine. This Morphine can be converted to codeine.

- Codeine is an analgesic and cough-suppressing drug.

- Mature capsule of Papaver bracteatum accumulates up to 3.5 % of the baine, which also can be converted to codeine. 

b. Berberine:- 

- Berberine is an isoquinoline alkaloid, which is distributed in roots of Coptis japonica, &

cortex of Philodendra anurans. 

- Addition of a polyamine, sperm dine, was found to stimulate the production of berberine by Thalictrum minus cell suspension cultures.

c. Tropane alkaloids:- 

- Scopolamine & Hyosyamine are tropane alkaloids. 

- The conc. of scopolamine & hyoscyamine in cultured cells are generally very low.

d. Vincristine:- 

- It is reported biotransformation of a Hydrovincritin to Vindolin by a cell extract of Catharanthus-roseus cell suspension culture. 

Hydrovincritin →→→→ Vindolin

- Catharanthus roseus plants produce two highly valuable drugs that are used for the treatment of cancer:-

i. Vinblastin 

ii. Vincristine

3. Biotransformation of Glycoside:- The Rue plant (Ruta graneolens) is rich in these kinds of secondary metabolites. One of its examples is furanocoumarins, derived from the substrate 7 hydroxy coumarin (A).

7 hydroxy coumarin (A) →→→→ Furanocoumarin

4. Biotransformation of Terpenoids:- 

> Biotransformation has the ability to transform mentha cell lines to:-

i. isomenthone

ii. (-)- Mentone 

iii. (+) neomenthol 

5. Biotransformations of Paclitaxel (Taxel):-

> Incubation of Eucalyptus citriodora, Azadirachta indica & Capsicum annum cell cultures with paclitaxel is a useful procedure to extract Texans from the cultures for commercial applications.

Paclitaxel →→→→ Texan

> It is carried in a refrigerated shaker incubator at 120 rpm & 25 ± 1°C for 48 hr.