1 CONTENTS Central dogma of molecular biology Basics
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CONTENTS ØCentral dogma of molecular biology ØBasics of Replication ØBasics of Transcription ØGenetic code ØMutations – types and their effects ØTranslation 2
Of Molecular Biology 3
Medical Importance 1. 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. 4
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. 5
REPLICATION 6
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. 7
Chromosomes after replication Sister chromatids 8
Type of DNA Replication Each daughter DNA = one parental strand + one newly synthesised strand 9
Semi conservative replication over two generations Parental DNA 2 strands Parental DNA 2 /4 strands Parental DNA 2 /8 strands 10
Process of replication Each strand of parent DNA serves as a template DNA. Each template strand its complementary strand forms the daughter DNA. The deoxyriboucleotides of the daughter strand are aligned as per the base pair rule. 11
TRANSCRIPTION 12
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 13
Types of RNA m. RNA Messenger RNA carries information on how to construct a protein. r. RNA Ribosomal RNA is not a code carrier but a structural part of ribosomes. t. RNA Transfer RNA has a coding section and an amino acid carrying section. 14
Types of RNA. . . . hn. RNA Heterogenous nuclear RNA – high MW – synthesised from DNA. sn. RNA Small nuclear RNA. si. RNA Small interfereing RNA 15
Transcription has 4 stages I stage : Initiation of transcription II stage : Elongation III stage : Termination IV stage : Post-transcriptional modifications 16
Post Transcriptional Modifications hn. RNA 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 mature m. RNA 17
TRANSLATION A cytoplasmic process where the genetic information present in the form of nucleotide sequences in m–RNA is translated into the language of amino acid sequence (proteins). 18
Translation……. Site of protein biosynthesis Sub-cellular site : Cytosol. : Ribosomes (free), rough endoplasmic reticulum. Template for protein synthesis : m. RNA. Amino acids – all the 20 amino acids should be present at the same time in the cytosol (amino acid pool). 19
1. m. RNA Requirements for translation……. A U G Cap codons Poly A tail Other requirements • Amino acids – 20 amino acids form the cytosolic amino acid pool. 20
Other requirements of Translation……. 2. t RNA – Adapter RNA Acceptor arm Activated amino acid Amino acid (amino acyl t RNA) Anticodon arm 32 different t RNA’s to transport 20 amino acids 21
3. Ribosomes : • 2 subunits Other requirements of Translation……. Large subunit PROKARYOTES Whole ribosome EUKARYOTES 50 S 60 S 30 S 40 S Small subunit Subunits are made up of r RNA and proteins 22
Other requirements of Translation • Enzymes • Protein factors – Initiation factors, Elongation factors, Termination factors • Energy ---- ATP, GTP • Mg++ 23
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 codons coding for 20 amino acids. 24
Genetic Code U Second base C A G Third base First base U UUC UU family UUA UUG C A G CUU CUC CU family CUA CUG AUU AUC AU family AUA AUG GUU GUC GU family GUA GUG UCU UCC UC family UCA UCG CCU CCC CC family CCA CCG ACU ACACC family ACA ACG GCU GCC GC family GCA GCG UAU UAC UA family UAA UAG UGU UGC UG family UGA UGG U C A G CAU CAC CA family CAA CAG AAU AAC AA family AAA AAG GAU GAC GA family GAA GAG CGU CGC CG family CGA CGG AGU AGAGC family AGA AGG GGU GGC GG family GGA GGG U C A G 25
GENETIC CODE Termination / non sense / stop codons UAA Amber UAG Ochre UGA Opal UNMIXED FAMILIES 1 FAMILY OF CODONS CODES FOR 1 AMINO ACID MIXED FAMILIES AUG Initiation codon, codes for methionine 1 FAMILY OF CODONS CODES FOR 2 AMINO ACID 26
GENETIC CODE m. RNA Cap Poly A tail A UG codons CODON Set of three consecutive nucleotides on m RNA - Triplet codons ØNon-overlapping ØNo punctuations ØUniversal ØDegenerate - One codon codes only for one amino acid but one amino acid can have more than one codon. ØUnambiguous ØAmino acids with multiple codons the difference lies in the third base – GCU, GCC, GCA, GCG all code for Alanine 27 WOBBLE HYPOTHESIS
GENE MUTATIONS SINGLE BASE CHANGES Transition Pyrimidine Purine Mutations by base substitution Pyrimidine Purine Transversion Pyrimidine Purine Gene transcription single base change m-RNA translation (appropriate complementary base change) Protein (mutant protein) 28
EFFECT OF SINGLE BASE CHANGES v Silent mutation No on the function of the protein Hbeffect Milwaukee, Hb Sydney v Missense mutation Different amino acid is incorporated in the mutant protein 3 types – based on the location of the amino acid replacement in the protein Acceptable mutation Mutant protein is not recognizable from the native protein Hb Hikari Partially acceptable mutation Unacceptable mutation v Mutation leading to nonsense codon Hb Sprotein (Sicklehas cellpartial, hemoglobin) Mutant abnormal function Mutant protein Hb is incapable of doing its assigned function M Premature termination of the protein which may or may not be functional FRAME SHIFT MUTATIONS Cause : Insertion or deletion of bases Result : Altered reading frame beyond the point of deletion / insertion Garbled protein beyond the point of mutation Terminated protein if a nonsense codon appears 29
EFFECT OF FRAME SHIFT MUTATIONS 1 amino acid is missing Garbled 30
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 31
Post Translational Modifications Nascent protein (inactive ) Proteolysis Addition of groups Disulfide bridge formation Modification of amino acids Protein folding Protein (active) 32
Thank you 33
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