CONTENTS • Central dogma of molecular biology • Basics of Replication • Basics of Transcription • Genetic code • Mutations – types and their effects • Translation
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.
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.
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.
Chromosomes after replication Sister chromatids
Type of DNA Replication Each daughter DNA = oneparental strand + one newly synthesised strand
Semi conservative replication over two generations Parental DNA 2 strands Parental DNA 2 /4 strands Parental DNA 2 /8 strands
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.
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
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.
Types of RNA....... • hnRNA • Heterogenous nuclear RNA – high MW – • synthesised from DNA. • snRNA • Small nuclear RNA. • siRNA • Small interfereing RNA
Transcription has 4 stages I stage : Initiation of transcription II stage : Elongation III stage : Termination IV stage : Post-transcriptional modifications
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
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
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).
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.
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
Otherrequirements of Translation……. PROKARYOTES EUKARYOTES50S60S30S40S 3. Ribosomes : • 2 subunits Large subunit Whole ribosome Small subunit Subunits are made up of r RNA and proteins
Otherrequirements of Translation • Enzymes • Protein factors – Initiation factors, • Elongation factors, • Termination factors • Energy ---- ATP, GTP • Mg++
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.
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
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
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
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
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
EFFECT OF FRAME SHIFT MUTATIONS Garbled 1 amino acid is missing
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
Post Translational Modifications Nascent protein (inactive ) Protein (active) Proteolysis Addition of groups Disulfide bridge formation Modification of amino acids Protein folding