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Dr. S.Chakravarty MBBS, MD MD

Components of Molecular biology. Dr. S.Chakravarty MBBS, MD MD. Learning objectives . Define the central dogma of molecular biology Describe the basic components of nucleotides and its nomenclature

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Dr. S.Chakravarty MBBS, MD MD

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  1. Components of Molecular biology Dr. S.Chakravarty MBBS, MDMD

  2. Learning objectives • Define the central dogma of molecular biology • Describe the basic components of nucleotides and its nomenclature • Define Chargaff Rule and apply it in calculating the percentage of nucleotides in an unknown sample of DNA or RNA • Outline the features of Watson and crick model of DNA • List the various types of RNA and its functions and compare each RNA • Describe the special features and modifications of each subtype of RNA • Describe the functions of nucleolus and P-bodies • Describe the Structure and components of Ribosome – and differences between prokaryotic and eukaryotic ribosome

  3. Central Dogma of molecular biology Transcription Translation DNA RNA Protein Replication Reverse transcription DNA The flow of genetic information in a biological system

  4. DNA • Molecular blue prints – identity of a person • Storage of Genetic information Replication : • Inheritance of characteristics in the family • Maintenance of rapidly metabolizing cells • Growth and differentiation

  5. Expression of Genetic message • Transcription - synthesis of RNA from DNA • Reverse transcription :- synthesis of DNA from RNA • Translation – synthesis of Proteins from RNA • Information is encoded in the order of the nitrogenous bases.

  6. Components of Nucleic acids • A nitrogenous heterocyclic base -Purine or Pyrimidine • A Pentose sugar -Ribose (RNA) or Deoxyribose (DNA) 3) A phosphate group in ester linkage to the 5’ carbon of the pentose

  7. Purines • 9 membered Rem :-(smaller pyrimidine molecule has the longer name )

  8. Purines Mnemonic – PUreAs Gold Adenine and Guanine are present in both DNA and RNA

  9. Pyrimidines • 6 membered

  10. Pyrimidines Mnemonic - CUT Cytosine and thymine are present in DNA Cytosine and uracil are present in RNA

  11. Ribose and Deoxyribose: • A pentose sugar composed of five carbon atoms • RIBOSE (found in RNA):Hydroxyl (-OH) group attached to the 2’-carbon • DEOXYRIBOSE (found in DNA): the hydroxyl (-OH) group attached to the 2’-carbon is replaced by a hydrogen (-H) group

  12. Nomenclature • Bases = (Purines or Pyrimidines) • Nucleosides = Base + sugar • Nucleotides = Base + sugar + phosphate

  13. Structure of a Nucleotide Attachment of phosphate to sugar is at 5’ carbon Attachment of nucleic acid to the sugar is at 1’ carbon

  14. Modification of nucleotides Can Generate Additional Structures DNA BACTERIA & HUMAN DNA BACTERIA & VIRAL NUCLEIC ACIDS MAMMALIAN MESSENGER RNA Harper

  15. Hypoxanthine , Xanthine and Uric acid Harper

  16. Caffeine – trimethyl xanthine (The dimethylxanthinestheobromine and theophylline are similar but lack the methyl group at N-1 and at N-7, respectively. ) Harper

  17. Synthetic Nucleotide analogs are used in chemotherapy Harper

  18. Chemotherapy of cancer Metabolized to 6-mercaptopurine  Immunosuppressant for organ transplantation Harper

  19. Nucleotides absorb UV light @ 260 nm • The conjugated double bonds of purine and pyrimidine derivatives absorb ultraviolet light. • The mutagenic effect of ultraviolet light is due to its absorption by nucleotides in DNA that results in chemical modifications . • While spectra are pH-dependent, at pH 7.0 all the common nucleotides absorb light at a wavelength close to 260 nm. • The concentration of nucleotides and nucleic acids thus often is expressed in terms of "absorbance at 260 nm."

  20. FUNCTIONS OF NUCLEOTIDES • CONSTITUENTS OF DNA and RNA • HIGH ENERGY COMPOUNDS – ATP, GTP etc • SECOND MESSENGERS :- cAMP • Coenzymes – Many are derivatives of Adenosine Monophosphate • ALLOSTERIC REGULATOR OF PROTEIN SYNTHESIS :- GTP( also provides energy ) • SULFATE DONORS – Adenosine 5 phosphosulphate • METHYL DONORS – SAM( S-adenosyl methionine) • UDP –sugars  Glycogen synthesis, synthesis of glycoproteins and proteoglycans

  21. DNA and RNA are nucleotide polymers • 3’, 5’ phosphodiester bonds  to form single stranded DNA

  22. The 3' C of a sugar molecule is connected through a phosphate group to the 5' C of the next sugar. • This linkage is also called 3'-5' phosphodiester linkage. • All DNA strands are read from the 5' to the 3' end where the 5' end terminates in a phosphate group and the 3' end terminates in a sugar molecule.

  23. Chargaff’s rules • Purines always base pairs with Pyrimidines: • % Purines = % Pyrimidines • %A = %T • %G = %C • Purines + Pyrimidines = 100% • Rem – Rule only for ds-DNA • Ss-DNA , RNA will not obey !!

  24. DNA is double stranded • Due to formation of hydrogen bonds between the nucleotides of two strands: • Purine always pairs with a pyrimidine : complimentary base pairing

  25. Many conformations of DNA From left to right, the structures of A, B and Z DNA

  26. Watson and crick model of DNA • Double strands wind around a central axis to form helical structure. • Right handed helical structure - B DNA physiologically stable form. • Sugar and phosphate form the two sides of the twisted ladder and bases form the rungs of the twisted ladder.

  27. DNA is polar , anti-parallel and is a negatively charged

  28. Helix • One turn = 34 A • One turn = 1 major and 1 minor grove • 10 nucleotides(base pairs ) per turn • External diameter – 2nm(2A) • Importance of major and minor groves – • Protein and Drug binding • Initiation and elongation factors binding.

  29. MAJOR GROOVE MINOR GROOVE

  30. Strands of DNA

  31. Melting or Denaturation of DNA: The melting temperature Tm is the temperature at which 50% of the DNA becomes single stranded Tm increases with G-C content Tm is influenced positively by ionic strength Important for a number of molecular biology techniques

  32. Supercoils • Positive super coils – same direction as the DNA helix – forms tightly coiled DNA. • Negative super coils – loosens the structure of DNA helix- helps in replication and transcription. • Topoisomerase and Gyrases removes super coils

  33. Higher Organization of DNA

  34. Histones • Proteins having very high concentration of basic amino acids .(Lysine and arginine ) • 5 classes • H1– Linker histone • H2A, H2B, H3 and H4- Core histone • Help to forming Nucleosomes • 146 nucleotides /nucleosome Nucleosomes

  35. Credit: Felsenfeld and Groudine. Nature, 2003

  36. How long is the DNA? • Each diploid cell therefore has 2 meters of DNA • Your total body DNA would actually stretch to the sun and back about 300 times! http://www.nature.com/scitable/topicpage/dna-packaging-nucleosomes-and-chromatin-310

  37. 98% of chromosome doesn’t code • Constitutive heterochromatin(densely packed ) • Any genes contained within the constitutive heterochromatin will be poorly expressed at all times. • Facultative heterochromatin • The regions of DNA packaged in facultative heterochromatin will not be consistent between the cell types within a species, and thus a sequence in one cell that is packaged in facultative heterochromatin (and the genes within are poorly expressed) may be packaged in euchromatin in another cell (and the genes within are no longer silenced) • Example :-X-chromosome inactivation • Euchromatin – Active regions of chromosome that stains less densely

  38. Differences DNA RNA • 2- deoxy ribose sugar moiety • Pyrimidines – cytosine and thymidine • Double stranded • G= C and A=T • Cannot be hydrolyzed by alkali • Ribose sugar • Cytosine and Uracil • Single stranded • Not always equal • Can be hydrolyzed by alkali

  39. RNA Types of RNA: • m-RNA – template for protein synthesis • r-RNA – formation and function of ribosome • t-RNA – transfer of AA to growing polypeptide chain • sn-RNA – mRNA processing (ribozymes or SNURPS) • mi-RNA - inhibition of gene expression.

  40. Messenger RNA • Contains the genetic code for protein synthesis. It is the copy of DNA derived after transcription • 5’ end – has 7-methyl Guanosine triphosphate CAP • Polyadenylate tail – 20 to 30 residues • Hn RNA is converted to m-RNA by bysnRNA

  41. t-RNA

  42. Functions of various arms of t-RNA • Acceptor arm – ends with CCA nucleotides aminoacid specific • Anticodon arm - three base region that can base pair to the corresponding three base codon region on mRNA. • D-arm – recognition site for the enzyme aminoacyl t-rnasynthase. • T –arm – recognition site for ribosome to attach

  43. Special bases of t-RNA • TΨC loop contains  thymidine, pseudouridine and cytidine residues.  • tRNA is the only RNA species that contains the nucleoside thymidine. • D-loop – dihydrouridine

  44. Ribosomes :- Used for protein synthesis (Translation)

  45. Functions of rRNA in ribosomes • Ribosomal assembly • Binding of mRNA to ribosomes: • 16s – identifies Shine Dalgarno sequence in Prokaryotes • 18s – identifies Kozak consenses sequence in Eukaryotes • Maintain structural integrity of ribosomes • Peptidyltransferaseactivity: Ribozymes • 23s in prokaryotes • 28s in eukaryotes.

  46. Nucleolus • Dense region within the nucleus • Place for transcription of r-RNA and assembly

  47. P-bodies • Distinct foci present in the cytosol which contain a pool of enzymes. • Functions: • Storage of m-RNA in the cytosol • Degradation of unwanted m-RNAs • Repression by mi-RNAs

  48. Mcq 1 A sample of human DNA is subjected to increasing temperature until the major fraction exhibits optical density changes due to disruption of its helix (melting or denaturation). A smaller fraction is atypical in that it requires a much higher temperature for melting. This smaller, atypical fraction of DNA must contain a higher content of a. Adenine plus cytosine b. Cytosine plus guanine c. Adenine plus thymine d. Cytosine plus thymine e. Adenine plus guanine

  49. Mcq 2 In a given sample the amount of adenine nucleotide is 20%. Calculate the amount of cytosine content in DNA? • 20% • 30% • 40% • 50% • 60%

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