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Molecular Biology

Molecular Biology

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Molecular Biology

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  1. Molecular Biology

  2. CONTENTS • Central dogma of molecular biology • Basics of Replication • Basics of Transcription • Genetic code • Mutations – types and their effects • Translation

  3. Of Molecular Biology

  4. Medical Importance • One of the causes of mutation, is a change in the nucleotide sequence of DNA, which maybe due to faulty replication and may result in a genetic disease. • E.g. Sickle cell disease.

  5. Medical Importance 2. Many compounds interfere with DNA replication, transcription and translation . They are used therapeutically as drugs Eg. : anti-cancer, anti-viral, anti-bacterial or immune suppressants. They may be poisons also.

  6. REPLICATION

  7. Replication Parent DNA 2 Daughter DNA Definition : Replication is the formation of two daughter DNA molecules from one parent DNA. Replication is template dependant process.

  8. Chromosomes after replication Sister chromatids

  9. Type of DNA Replication Each daughter DNA = oneparental strand + one newly synthesised strand

  10. Semi conservative replication over two generations Parental DNA 2 strands Parental DNA 2 /4 strands Parental DNA 2 /8 strands

  11. Process of replication Each strand of parent DNA serves as a template DNA. Each template strand and its complementary strand forms the daughter DNA. The deoxyriboucleotides of the daughter strand are aligned as per the base pair rule.

  12. TRANSCRIPTION

  13. Transcription Definition : Transcription is the synthesis of RNA using DNA as a template. In Eukaryotes, DNA is nuclear but proteins synthesized in the cytoplasm RNA intermediate between DNA and proteins RNA synthesised in the nucleus is exported to the cytoplasm

  14. Types of RNA • mRNA • Messenger RNA carries information on how to construct a protein. • rRNA • Ribosomal RNA is not a code carrier but a structural part of ribosomes. • tRNA • Transfer RNA has a coding section and an amino acid carrying section.

  15. Types of RNA....... • hnRNA • Heterogenous nuclear RNA – high MW – • synthesised from DNA. • snRNA • Small nuclear RNA. • siRNA • Small interfereing RNA

  16. Transcription has 4 stages I stage : Initiation of transcription II stage : Elongation III stage : Termination IV stage : Post-transcriptional modifications

  17. Post Transcriptional Modifications hnRNA mature mRNA 1. Capping at 5’ end - 7 methyl GTP 2. Splicing : Excision of introns and joining of exons 3. Removal of extra RNA from the 3’ end 4. Tailing at the 3’ end – Poly A tail 5. Modification of certain bases

  18. TRANSLATION A cytoplasmic process where the genetic information is translated into the language of amino acid sequence (proteins). present in the form of nucleotide sequences in m–RNA

  19. Translation……. Site of protein biosynthesis : Cytosol. Sub-cellular site : Ribosomes (free), rough endoplasmic reticulum. Template for protein synthesis : mRNA. Amino acids – all the 20 amino acids should be present at the same time in the cytosol (amino acid pool).

  20. Requirements for translation……. 1. mRNA A U G Cap codons Poly A tail Otherrequirements • Amino acids – 20 amino acids form the cytosolic amino acid pool.

  21. Otherrequirements of Translation……. 2. t RNA – Adapter RNA Activated amino acid (amino acyl t RNA) Acceptor arm Amino acid Anticodon arm 32 different t RNA’s to transport 20 amino acids

  22. Otherrequirements of Translation……. PROKARYOTES EUKARYOTES50S60S30S40S 3. Ribosomes : • 2 subunits Large subunit Whole ribosome Small subunit Subunits are made up of r RNA and proteins

  23. Otherrequirements of Translation • Enzymes • Protein factors – Initiation factors, • Elongation factors, • Termination factors • Energy ---- ATP, GTP • Mg++

  24. GENETIC CODE m-RNA : codes for numerous amino acids. directs the amino acid sequences in a protein. There are 4 nitrogenous bases – A, G, U, C. If Codon has 2 bases 42 = 16 codons is possible. If Codon has 3 bases 43 = 64 codons is possible Each codon is a triplet of three bases. Genetic code contains 64 codonscoding for 20 amino acids.

  25. U C A G Second base U C A G Third base First base UGU UGC UGA UGG UAU UAC UAA UAG UA family UG family U U C A G CUU CUC CUA CUG CCU CCC CCA CCG CAU CAC CAA CAG CGU CGC CGA CGG U C A G C CU family CC family CA family CG family AAU AAC AAA AAG AUU AUC AUA AUG ACU ACC ACA ACG AGU AGC AGA AGG A AU family AC family AA family AG family GAU GAC GAA GAG GUU GUC GUA GUG GCU GCC GCA GCG GGU GGC GGA GGG G GU family GC family GA family GG family Genetic Code U C A G UUU UCU UUC UCC UU family UC family UUA UCA UUG UCG

  26. GENETIC CODE UNMIXED FAMILIES Termination / non sense / stop codons UAA UAG UGA Amber Ochre Opal 1 FAMILY OF CODONS CODES FOR 1 AMINO ACID MIXED FAMILIES 1 FAMILY OF CODONS CODES FOR 2 AMINO ACID Initiation codon, codes for methionine AUG

  27. GENETIC CODE mRNA Poly A tail Cap A U G codons CODON Set of three consecutive nucleotides on m RNA - Triplet codons • Non-overlapping • No punctuations • Universal • Degenerate - One codon codes only for one amino acid but one amino acid can have more than one codon. • Unambiguous • Amino acids with multiple codons the difference lies in the third base – GCU, GCC, GCA, GCG all code for Alanine WOBBLE HYPOTHESIS

  28. GENE MUTATIONS SINGLE BASE CHANGES Pyrimidine  Pyrimidine Transition Purine  Purine Mutations by base substitution Pyrimidine  Purine Transversion Purine  Pyrimidine translation transcription Gene m-RNA (appropriate complementary base change) Protein (mutant protein) single base change

  29. EFFECT OF SINGLE BASE CHANGES No effect on the function of the protein Hb Milwaukee, Hb Sydney • Silent mutation Different amino acid is incorporated in the mutant protein • Missense mutation 3 types – based on the location of the amino acid replacement in the protein Acceptable mutation Mutant protein is not recognizablefrom the native protein Hb Hikari Hb S (Sickle cell hemoglobin) Mutant protein has partial, abnormal function Partially acceptable mutation Unacceptable mutation Mutant protein is incapable of doing its assigned function Hb M • Mutation leading to nonsense codon Premature termination of the protein which may or may not be functional FRAME SHIFT MUTATIONS Cause : Insertion or deletion of bases Result : Alteredreading frame beyond the point of deletion / insertion Garbled protein beyond the point of mutation Terminated protein if a nonsense codon appears

  30. EFFECT OF FRAME SHIFT MUTATIONS Garbled 1 amino acid is missing

  31. TRANSLATION Direction of protein synthesis : N-terminal to C-terminal Direction in which m-RNA is read : 5’ to 3’ direction STAGES OF TRANSLATION – 5 stages • AMINOACYL-t RNA SYNTHESIS – • Activation of amino acids. INITIATION ELONGATION TERMINATION POST-TRANSLATIONAL MODIFICATIONS

  32. Post Translational Modifications Nascent protein (inactive ) Protein (active) Proteolysis Addition of groups Disulfide bridge formation Modification of amino acids Protein folding

  33. Thank you