26. Apr 2020

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  1. DNA & RNA : MOLECULAR BIOLOGY Prepared by : Pratishtha Sharma M.Pharm (Pharmacology) Kota College of Pharmacy, Kota
  2. Genetic Codon ■ The Three nucleotide base sequence in mRNA that act as code words for amino acids in protein constitute the genetic code or codons. ■ There are 64 different combinations of three base codons composed of Adenine (A), Guanine (G), Cytosine (C) and Uracil (U). ■ Written from the 5-’ end to 3’ end. ■ UAA,UAG & UGA do not code for amino acid. They are called as stop codon or non sense codon. ■ Characteristics of Genetic Code are: 1. University: same codon for same amino acid in all living organism. 2. Specificity: A particular codon will code for the same amino acid,highly specific or unambiguous. 3. Non overlapping : read from a fixed point as a continuous base sequence. 4. Degenerate: Most of the amino acids have more than one codon. 61 codons available to code for only 20 amino acids.
  3. Fig: The genetic code along with respective amino acids
  4.  Deoxy Ribonucleic Acid (DNA) ■ DNA stands for Deoxy Ribonucleic acid. ■ It’s the genetic code that determines all the characteristics of living organism. ■ DNA is a double stranded molecule, made up of two chains of nucleotides. Nucleotides consist of three subunits : a sugar, a phosphate group and a nitrogen base pair. ■ Sugar present is Deoxyribose and Nitrogen bases are : Adenine (A) Guanine (G) Cytosine (C) Thymine (T)
  5. Fig: (a) Structure of Nucleotide (b) Structure of Deoxyribose sugar (c) Structure of DNA (a) (b) (c)
  6. Structure of DNA ■ Double helical structure of DNA was proposed by James Watson and Francis Crick in 1953. ■ Features of model of DNA are: 1. DNA is a right handed double helix, have two polydeoxyribonucleotide chains twisted around each other on a common axis. 2. Two strands are antiparallel i.e., one strand runs in the 5’ to 3’ direction while the other in 3’ to 5’ direction. 3. The diameter of helix is 20 A° (2nm). 4. Each turn of the helix is 34 A° (3.4 nm) with 10 pairs of nucleotides, each pair placed at a distance of about 3.4A°. 5. The two strands are held together by Hydrogen bonds formed by complementary base pairs.The A-T pair has 2 hydrogen bonds while G-C pair has 3 hydrogen bonds. 6. The complementary base pairing in DNA helix proves Chargaff’s rule.The content of adenine equals to that of thymine (A=T) and guanine equals to that of the cytosine (G≡C).
  7. Chargaff’s Rules: Chargaff (1950) made observations on the bases and other components of DNA.These observations or generalizations are called Chargaff’s base equivalence rule. (i) Purine and pyrimidine base pairs are in equal amount, that is, adenine + guanine = thymine + cytosine. [A + G] = [T + C], i.e., [A+G] / [T+C] = 1 (ii) Molar amount of adenine is always equal to the molar amount of thymine. Similarly, molar concentration of guanine is equalled by molar concentration of cytosine. [A] = [T], i.e., [A] / [T] = 1; [G] = [C], i.e., [G] / [C] = 1 (iii) Sugar deoxyribose and phosphate occur in equimolar proportions. (iv)A-T base pairs are rarely equal to С—G base pairs. (v)The ratio of [A+T] / [G+C] is variable but constant for a species (Table 6.2). It can be used to identify the source of DNA.The ratio is low in primitive organisms and higher in advanced ones.
  8. Fig : Watson and Crick Model of DNA
  9. Functions of DNA ■ DNA is the reserve bank of genetic information. ■ DNA is responsible for the hereditary characters of an organism. ■ DNA is the fundamental units of genetic information. ■ DNA forms RNA by the process calledTranscription. ■ DNA is the precursor of proteins.
  10. Ribonucleic Aid (RNA) ■ RNA is a single strand polymer of ribonucleotide held together by 3’5’-phosphodiester bonds. ■ It contains Ribose sugar and pyrimidine Uracil in place ofThymine (as in DNA). ■ It doesn’t followChargaff’s rule. ■ There are three types of RNA: 1. Messenger RNA (mRNA): synthesize in nucleus as heterogenous nuclear RNA (hnRNA).After modification helps in protein synthesis and mRNA has high molecular weight with short half life. 2. Transfer RNA (tRNA) : structure resemble to clove leaf. tRNA has four arms, each arm contains a base paired stem. i. The acceptor arm :This arm is capped with a sequence CCA (5’ to 3’).The amino acid is attached to the acceptor arm. ii. The Anticodon arm: It has three specific nucleotide bases(anticodon), which is responsible for the recognisation of triplet codon of mRNA.
  11. iii.The D arm : It has dihydrouridine. vi.TheTΨC arm: contains a sequence ofT, pseudouridine (represented by psi,Ψ) and C. v.TheVariable arm :This arm is the most variable arm in tRNA. 3. Ribosomal RNA (rRNA) :The function of rRNA in ribosome is not clearly known. It is believed that they play a significant role in the binding of mRNA to ribosome and protein synthesis.
  12. Fig: Structure of tRNA
  13. DNA Replication ■ Central Dogma :The biological information flow from DNA to RNA, and RNA to proteins.This is termed as Central Dogma. It is ultimately that DNA controls every function of the cell through protein synthesis. ■ Replication is the process in which DNA copies itself to produce identical daughter molecules of DNA. ■ DNA replication is semiconservative sine half of the original DNA is conserved in the daughter DNA.This was explained by Meselson and Stahl (1958).
  14. Steps of DNA Replication 1. Initiation 2. Chain Elongation 3. Termination Initiation ■ Replication starts at a specific point of DNA called origin of chromosomal replication (Ori C). In prokaryotic cell, there is one Ori whereas in Eukaryotic cell number of Ori is more than one. ■ Ori C is a 245 base pair region of the chromosome and bears DNA sequence elements i.e. 2 types of repeated sequence. i. Four 9 mer motif which is the binding site for DnaA. ii. Three 13 mer morif which is the initial site of single stranded DNA formation. ■ There is a formation of replication bubble due to the presence of origin of replication. ■ At first, double stranded DNA starts to separate and uncoil due to breaking of hydrogen bond present between nitrogen bases by helicase. The unwinding DNA molecule takes place once in every ten nucleotide pair in eukaryotic DNA. ■ Each uncoiled parental DNA strand acts as template DNA strand for the synthesis of new complementary strands. ■ When two strands unwind and separate incompletely, they form Y-shape where active synthesis occurs. This region is called replication fork. ■ Each separated strands are stabilized by single stranded binding protein. ■ As the two strands are separated, supercoiling occurs which is removed by DNA topoisomerase. ■ Initiation of replication requires RNA primer, which is a small strand of RNA, synthesized by primase.
  15. Chain Elongation ■ Chain elongation proceeds from the initiation site by the addition of deoxyribonucleotides at 3’-OH end of the primer by DNA polymerase III. ■ DNA Polymerase III forms continuous strand of DNA on 3’→5’ template. The continuous strand of DNA is called leading strand. Since the direction of movement of replication fork and direction of leading strand synthesis are same, leading strand is synthesized continuously after its initiation. ■ However, in other template strand 5’→3’, there is discontinuous formation of DNA and thus more RNA primer are required for the formation of whole strand. Due to discontinuous formation, smaller fragments are formed, which are called Okazaki fragments. DNA ligase joins these Okazaki fragments to form complete lagging strands. Since the direction of movement of replication fork is opposite to direction of lagging strand synthesis, it cannot be synthesized continuously. ■ After completion of chain elongation RNA primer is removed by exonuclease activity of DNA polymerase I and the gap is filled with complementary bases. Termination ■ Replication must be terminated to produce two daughter DNA molecule and to regulate and co-ordinate replication with cell division. ■ When two replication fork meets Ter-Tus complex, DNA synthesis stops. And the daughter DNA are produced. ■ In bacteria DNA is circular. Therefore two interlinked daughter DNA are obtained at completion of replication. Such interlinked DNA are called catenanes. Finally DNA topoisomerase IV cuts one DNA, removes it out of other and finally reseals it. So that two daughter DNA are separated. This process is known as decatenation.
  16. Fig : DNA Replication (Semiconservative )
  17. Transcription ■ Transcription is the first step in gene expression, in which information from a gene is used to construct a functional product such as a protein. The goal of transcription is to make a RNA copy of a gene's DNA sequence. For a protein-coding gene, the RNA copy, or transcript, carries the information needed to build a polypeptide (protein or protein subunit). Eukaryotic transcripts need to go through some processing steps before translation into proteins. ■ The main enzyme involved in transcription is RNA polymerase, which uses a single-stranded DNA template to synthesize a complementary strand of RNA. Specifically, RNA polymerase builds an RNA strand in the 5' to 3' direction, adding each new nucleotide to the 3' end of the strand. ■ Transcription of a gene takes place in three stages: 1. Initiation 2. Elongation 3. Termination.
  18. Initiation. RNA polymerase binds to a sequence of DNA called the promoter, found near the beginning of a gene. Each gene (or group of co-transcribed genes, in bacteria) has its own promoter. Once bound, RNA polymerase separates the DNA strands, providing the single-stranded template needed for transcription. There are two base sequence on the coding DNA Strand which the sigma factor of RNA polymerase can recognize for initiation of transcription. 1. Pribnow box (TATA box): This consists of 6 nucleotide bases (TATAAT), located on the left side about 10 bases away (upstream) from the starting point of transcription. 2. The ‘-35’ sequence : It contains the base sequence TTGACA, which is located about 35 base (upstream) from the starting point of transcription. Elongation. One strand of DNA, the template strand, acts as a template for RNA polymerase. As it "reads" this template one base at a time, the polymerase builds an RNA molecule out of complementary nucleotides, making a chain that grows from 5' to 3'. The RNA transcript carries the same information as the non-template (coding) strand of DNA, but it contains the base uracil (U) instead of thymine (T). Termination. There are two types of termination signals 1. Rho (ρ) dependent termination : A specific protein, named ρ factor, binds to the growing RNAor weakly to DNA, and in bound state it acts as ATPase and terminates transcription and release RNA. Ρ factor is also responsible for the dissociation of RNA polymerase from DNA. 2. Rho (ρ) independent termination : Termination occurs due to formation of hairpins of newly formed RNA. This occurs due to the presence of palindromes (words that reads alike forward & backward).
  19. Fig : Transcription process
  20. Translation ■ Translation refers to the process of polymerisation of amino acid to form polypeptide. ■ It takes place in cytoplasm following the transcription. ■ The key components required for translation are mRNA, ribosomes, tRNA and aminoacyl- tRNA synthetases. During translation mRNA nucleotide bases are read as three base codons, each of which codes for a particular amino acid. ■ The genetic code is described as degenerate because a single amino acid may be coded for by more than one codon.There are also specific codons that signal the start and the end of translation. ■ Each tRNA molecule possesses an anticodon on the opposite end that is complementary to the mRNA codon. tRNA molecules are therefore responsible for bringing amino acids to the ribosome in the correct order ready for polypeptide assembly. ■ Aminoacyl-tRNA synthetases are enzymes that link amino acids to their corresponding tRNA molecules.The resulting complex is charged and is referred to as an aminoacyl-tRNA.
  21. Initiation ■ At the 5’ cap of mRNA the small 40s subunit of the ribosome (with methionyl-tRNA) binds. For translation to start the start codon 5’AUG must be recognised. This is a codon specific to the amino acid methionine (anticodon on tRNA=5’CAU). The large 60s subunit of the ribosome then binds for elongation to occur. ■ The ribosome has two tRNA binding sites; the P site which holds the peptide chain and the A site which accepts the tRNA. Elongation ■ While Met-tRNA occupies the P site, another aminoacyl-tRNA with an anticodon complementary to the next codon comes to occupy the A site. This process requires GTP. The enzyme peptidyl-transferase forms a peptide bond between methionine and the next aminoacyl-tRNA. ■ The tRNA molecule in the P site becomes uncharged and leaves the ribosome. The ribosome then translocates along the mRNA molecule to the next codon. This opens up the A site for the next aminoacyl- tRNA. The polypeptide chain is built up in the direction from the N terminal to the C terminal. Termination ■ One of the three stop codons enters the A site. No tRNA molecules bind to these codons so the peptide and tRNA in the P site become hydrolysed releasing the polypeptide into the cytoplasm. ■ The small and large subunits of the ribosome dissociate ready for the next round of translation. ■ The polypeptide produced undergoes post-translational modification before becoming the fully active protein.
  22. Fig :Translation process
  23. Protein synthesis Inhibitors ■ Majority of the antibiotics interfere with the bacterial protein synthesis and are harmless to higher organisms.This is due to the fact that the process of translation sufficiently differs between prokaryotes and eukaryotes. For eg : Streptomycin : Initiation of protein synthesis is inhibited by streptomycin. lt causes misreading of mRNA and interferes with the normal pairing between codons and anticodons. Tetracycline : It inhibits the binding of aminoacyl tRNA to the ribosomal complex. In fact, tetracycline can also block eukaryotic protein synthesis.This, however, does not happen since eukaryotic cell membrane is not permeable to this drug. Erythromycin : It inhibits translocation by binding with 50S subunit of bacterial ribosome. Diphtheria toxin : It prevents translocation in eukaryotic protein synthesis by inactivatingelongation factor eEF2. Chloramphenicol : It acts as a competitive inhibitor of the enzyme peptidyl transferase and thus interferes with elongation of peptide chain.