Genes and Central Dogma PDF

Molecular Biology - Dr. Sonali Patil Syllabus  Content Prokaryotes Structure and Central and Nucleic acids Replication function of Dogma Euka...

Molecular Biology - Dr. Sonali Patil Syllabus  Content Prokaryotes Structure and Central and Nucleic acids Replication function of Dogma Eukaryotes DNA Transcription Eukaryotes RNA Translation Prokaryotes Viruses Difference between Prokaryotes and Eukaryotes  DNA is organized into informational units called genes Chromosomes contain hundreds to thousands of genes  Friedrich Miescher in 1869 Isolated what he called nuclein from the nuclei of pus cells Nuclein was shown to have acidic properties, hence it became called nucleic acid https://www.youtube.com/watch?v=TNKWg cFPHqw  Deoxyribonucleic acid, DNA Nucleic acid Ribonucleic acid, RNA  DNA as genetic material: The circumstantial evidence 1. Present in all cells and virtually restricted to the nucleus 2. The amount of DNA in somatic cells (body cells) of any given species is constant (like the number of chromosomes) 3. The mutagenic effect of UV light peaks at 253.7nm. The peak for the absorption of UV light by DNA The distribution of nucleic acids in the eukaryotic cell DNA is found in the nucleus with small amounts in mitochondria and chloroplasts RNA is found throughout the cell  1. The components of DNA and RNA DNA and RNA are polymers of nucleotide units. DNA (RNA) consists of 4 kinds of ribonucleotide units linked together through covalent bonds. Each nucleotide unit is composed of a nitrogenous base a pentose sugar a phosphate group NUCLEOTIDE STRUCTURE PHOSPHATE SUGAR BASE Ribose or PURINES PYRIMIDINES Deoxyribose Adenine (A) Cytocine (C) Guanine(G) Thymine (T) Uracil (U) NUCLEOTIDE 1.2 Ribose (in RNA) and deoxyribose (in DNA) Ribose and deoxyribose predominantly exist in the cyclic form.  1.1 Bases Purines : – Adenine (A) – Guanine (G) Pyrimidines : – Cytosine (C) – Uracil (U) – Thymine (T) DNA: A,G,C,T RNA: A,G,C,U Thymine (T) is a 5-methyluracil (U) 1.3 Nucleosides =ribose/deoxyribose + bases The bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside Glycosidic bond 1 R Ribose or 2’-deoxyribose Adenosine, guanosine, cytidine, thymidine, uridine 1.4 Nucleotides = nucleoside + phosphate A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up to three phosphates may be attached. Phosphate ester bonds Deoxynucleotides Ribonucleotides (containing deoxyribose) (containing ribose)  P G ADDING IN THE BASES P C The bases are attached to P the 1st Carbon C Their order is important P It determines the genetic A information of the molecule P T P T BASES NUCLEOSIDES NUCLEOTIDES Adenine (A) Adenosine Adenosine 5’-triphosphate (ATP) Deoxyadenosine Deoxyadenosine 5’-triphosphate (dATP) Guanine (G) Guanosine Guanosine 5’-triphosphate (GTP) Deoxyguanosine Deoxy-guanosine 5’-triphosphate (dGTP) Cytosine (C) Cytidine Cytidine 5’-triphosphate (CTP) Deoxycytidine Deoxy-cytidine 5’-triphosphate (dCTP) Uracil (U) Uridine Uridine 5’-triphosphate (UTP) Thymine (T) Thymidine/ Thymidine/deoxythymidie Deoxythymidie 5’-triphosphate (dTTP)  phosphate nucleic acid nucleotides pentose nucleosides bases NH2 N O HO P O CH2 N O O OH OH OH  Composition of DNA and RNA Nucleic base ribose acid DNA A、G、C、T deoxyribose RNA A、G、C、U ribose  2. Structure and function of DNA 2.1 Primary structure ❖ Definition: the base sequence (or the nucleotide sequence) in polydeoxynucleotide chain. ❖ The smallest DNA in nature is virus DNA. The length of φX174 virus DNA is 5,386 bases (a single chain). ❖ The DNA length of human genome is 3,000,000,000 pair bases. 5’end Phosphodiester bond 3’ end: free hydroxyl (-OH) group 3’,5’ phosphodiester bond link nucleotides together to form polynucleotide chains The structure of a DNA chain can be concisely represented An even more abbreviated notation for this chain is – pApCpGpTpA – pACGTA The base chain is written in the 5’ →3’ direction 2.2 Secondary structure The secondary structure is defined as the relative spatial position of all the atoms of nucleotide residues. Secondary structure — DNA double helix structure Francis H.C. Crick Watson and Crick , 1953 The genetic material of all organisms except for some viruses. The foundation of the molecular biology. James D. Watson  The discovery of DNA double helix Chargaff's Rule (A=T, G=C in DNA) A+T does not have to equal G+C Franklin, Wilkins: X-ray Diffraction Refined Structure  DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE The sister strands of the DNA molecule run in opposite directions (antiparallel) They are joined by the bases Each base is paired with a specific partner: A is always paired with T G is always paired with C Purine with Pyrimidine Thus the sister strands are complementary but not identical The bases are joined by hydrogen bonds, individually weak but collectively strong.  DNA double helix Essential for replicating DNA and Two separate strands transcribing RNA Antiparellel (5’→3’ direction) 3’ Base pairing: hydrogen 5’ bonding that holds two strands together Complementary (sequence) Sugar-phosphate backbones (negatively charged): outside Base pairs (stack one above the other): inside 3’ 5’  4 3 2 1 7 6 8 5 1 9 4 3 2 A:T G:C Base pairing  B form of DNA double helix Right-handed helix; The diameter of the double helix：2 nm The distance between two base pairs: 0.34 nm; Each turn of the helix involves 10 bases pairs, 3.4 nm. ✓ Stable configuration can be maintained by hydrogen bond and base stacking force (hydrophobic interaction).  Groove binding Small molecules like drugs bind in the minor groove, whereas particular protein motifs can interact with the major grooves.  Watson, Crick, and Wilkins shared the Nobel Prize in medicine or physiology in 1962 for this brilliant accomplishment. The discovery of the DNA double helix revolutionized biology: it led the way to an understanding of gene function in molecular terms (their work is recognized to mark the beginning of molecular biology). Conformational variation in double-helical structure B-DNA A-DNA Z-DNA  B-form: the duplex structure proposed by Watson and Crick is referred as the B-form DNA. It is the standard structure for DNA molecules. A-form: at low humidity the DNA molecule will take the A-form: The A-form helix is wider and shorter, with a shorter more compact helical structure, than the B-form helix. Z-form: the Z-form DNA is adopted by short oligonucleotides. It is a left-handed double helix in which backbone phosphates zigzag. 2.3 Tertiary structure : Supercoils: double-stranded circular DNA form supercoils if the strands are underwound (negatively supercoiled) or overwound (positively supercoiled). Increasing degree of supercoiling Relaxed supercoiled  If the strands are overwound, form positively supercoiled; If the strands are underwound, form negatively supercoiled.  The DNA in a prokaryotic cell is a supercoil. Supercoiling makes the DNA molecule more compact thus important for its packaging in cells. 2.4 Eukaryotic DNA DNA in eukaryotic cells is highly packed. DNA appears in a highly ordered form called chromosomes during metaphase, whereas shows a relatively loose form of chromatin in other phases. The basic unit of chromatin is nucleosome. Nucleosomes are composed of DNA and histone proteins. The importance of packing of DNA into chromosomes Chromosome is a compact form of the DNA that readily fits inside the cell To protect DNA from damage DNA in a chromosome can be transmitted efficiently to both daughter cells during cell division Chromosome confers an overall organization to each molecule of DNA, which facilitates gene expression as well as recombination.  2.5 Functions of DNA The carrier of genetic information. The template strand involved in replication and transcription. Gene: the minimum functional unit in DNA Genome: the total genes in a living cell or living beings. 3. Structures and functions of RNA Conformational variability of RNA is important for the much more diverse roles of RNA in the cell, when compared to DNA. Types : mRNA: messenger RNA, the carrier of genetic information from DNA to translate into protein tRNA: transfer RNA , to transport amino acid to ribosomes to synthesize protein rRNA: ribosomal RNA, the components of ribosomes hnRNA: Heterogeneous nuclear RNA snRNA: small nuclear RNA Classes of eukaryotic RNAs  RNA structure RNA molecules are largely single-stranded but there are double-stranded regions. 4. Physical and Chemical Properties of Nucleic Acids  General properties Acidity – Amphiphilic molecules; normally acidic because of phosphate. Viscosity – Solid DNA: white fiber; RNA: white powder. Insoluble in organic solvents, can be precipitate by ethanol. Optical absorption – UV absorption due to aromatic groups. Thermal stability – Disassociation of dsDNA (double-stranded DNA) into two ssDNAs (single-stranded DNA).  4.1 UV Absorption Specific absorption at 260nm. This can be used to identify nucleic acid. The UV absorption spectra of the common ribonucleotides  4.2 Denaturation Concept: The course of hydrogen bonds broken, 3-D structure was destroyed, the double helix changed into single strand irregular coil. Results: (1) the value of 260nm absorption is increased; (2) biological functions are lost.  Heat denaturation and Tm When DNA were heated to certain temperature, the absorption value at 260nm would increased sharply， which indicates that the double strand helix DNA was separated into single strand. Tm (melting temperature of DNA): The temperature of UV absorption increase to an half of maximum value in DNA denaturation.  Factors affect Tm: G-C content: There are three hydrogen bonds between G-C pair. The more G-C content, the higher Tm value. Less G+C Tm of Higher G+C two DNA molecules with different G+C content Temperature  4.3 Renaturation of DNA When slowly cooling down (Annealing) the denatured DNA solution, the single strand DNA can reform a double strands helix to recover its biological functions.  Molecule hybridization During the course of lowing down denaturing temperature, between different resource DNAs or single stand DNA and RNA with complementary bases will repair into a double strands to form a hybrid DNA or DNA-RNA. This course is called molecule hybridization.  Summary The components of DNA and RNA – Nucleotide: base (A,G,C,T,U), pentose sugar (Ribose and deoxyribose), phosphate group Structure and function of DNA – Primary structure: 3’,5’ phosphodiester bond – Secondary structure: DNA double helix – Tertiary structure: supercoil – Eukaryotic chromosomes: nucleosome Structures and functions of RNA – mRNA, tRNA, rRNA Properties of nucleic acid – UV absorption, denaturation and renaturation, molecule hybridization Central Dogma  Central Dogma Replication – DNA making a copy of itself Making a replica Transcription – DNA being made into RNA Still in nucleotide language Translation – RNA being made into protein Change to amino acid language  Replication in Eukaryotic Organism Remember that DNA is self complementary Replication is semiconservative – One strand goes to next generation – Other is new Each strand is a template for the other – If one strand is 5’ AGCT 3’ – Other is: 3’ TCGA 5’ Replication is Semiconservative  Replication Roles of enzymes – Topoisomerases – Helicase – DNA polymerases – ligase DNA binding proteins – DNA synthesis Leading strand Lagging strand Replication  Replication Leading strand – 3’ end of template – As opens up, DNA polymerase binds – Makes new DNA 5’ -→ 3’ Same direction as opening of helix Made continuously  Replication Lagging strand – 5’ end of template Can’t be made continuously as direction is wrong – RNA primer – New DNA made 5’ → 3’ Opposite direction of replication Discontinuous – Okazaki fragments Ligase closes gaps  DNA Replication DNA Replication  Transcription DNA template made into RNA copy – Uracil instead of Thymine One DNA strand is template – Sense strand Other is just for replication – Antisense (not to be confused with nonsense!) In nucleus – nucleoli Transcription  Translation RNA --→ Protein – Change from nucleotide language to amino acid language On ribosomes Vectorial nature preserved – 5’ end of mRNA becomes amino terminus of protein – Translation depends on genetic code  Genetic Code Nucleotides read in triplet “codons” – 5’ -→ 3’ Each codon translates to an amino acid 64 possible codons – 3 positions and 4 possiblities (AGCU) makes 43 or 64 possibilities – Degeneracy or redundancy of code Only 20 amino acids Implications for mutations Genetic Code  Genetic Code Not everything translated AUG is start codon – Find the start codon Also are stop codons To determine aa sequence – Find start codon – Read in threes – Continue to stop codon  Translation Initiation – Ribosomal subunits assemble on mRNA – rRNA aids in binding of mRNA Elongation – tRNAs with appropriate anticodon loops bind to complex – have aa attached (done by other enzymes) – Amino acids transfer form tRNA 2 to tRNA 1 – Process repeats Termination – tRNA with stop codon binds into ribosome – No aa attached to tRNA – Complex falls apart Translation DNA to Protein  Prokaryotic DNA Replication DNA replication is perfomed by a multienzyme complex >1 MDa DNA Nucleotides Replisome: DNA polymerases Helicase Primase SSBs DNA ligase Clamps (Topoisomerases)  Replication is semiconservative, accurrate and fast Accuracy 1 error in 1 billion bases Speed 500 nt/s in bacteria 50 nt/s in mammals Each original strand functions as template for DNA synthesis After each replication cycle, DNA is doubled DNA is synthesized in 5´to 3´direction DNA is synthesized by DNA polymerase DNA polymerase III is a protein complex Subunit function not  known  3’ exonuclease  polymerase clamp  dimerisation      clamp loader DNA polymerase I Found by Arthur Kornberg, mid 1950’s Three enzymatic activities: Polymerase activity 3’ to 5’ exonuclease activity 5’ to 3’ exonuclease activity Klenow enzyme is lacking one subunit responsible for the 5’ to 3’ exonuclease activity  DNA polymerase requires A free 3’-OH group supplied by RNA Primer for start of polymerisation Mg2+ ions for activity in active site A template to copy DNA replication initate at origin of replication Bacterial chromosome doubles in 40 min DNA replication is bidirectional  The replication origin OriC in E.coli 245 base pairs AT-rich Initiation proteins bind to 9 bp consensus sequence  Initiation of replication at the replication origin Regulation of initiation of replication DNA is synthesized in the replication fork in 5’ to 3’ direction  Leading strand synthesis is continuous whereas lagging strand is synthesized in fragments Length of Okazaki fragments in prokaryotes are 1000- 2000 nt, in eukaryotes 100-200 nt  Mistakes during DNA synthesis are edited This results in a very low error rate of 1 in 1 billion nucleotides  3’ to 5’ exonuclease activity corrects errors Requirements for proofreading mechanism Addition of nucleotides to RNA primer Absolute requirement for a match at the 3’ end of the extended strand 3’ to 5’ exonuclease activity of DNA polymerase Template DNA is identified by methylation (E. coli) or absence of nicks (eukaryotes)  5’ to 3’ exonuclease activity causes strand displacement/nick translation No net synthesis Helicase unzips double-helix Single strand binding proteins keep strands single stranded Each SSB bind to 7-10 nt Bind in clusters Cooperative binding Lowers Tm of template Binding of SSBs to DNA DNA pol. is attached to strand by Clamp loader and Sliding clamp  DNA primase Makes the 10 nt RNA primer required for start of replication In beginning of each Okazaki- Fragment RNA primer is later erased and replaced with DNA by DNA Pol I Apol I  DNA ligase Seals the nicks between Okazaki fragments Requires close and free 3’- OH and 5’-P and proper base-pairing NAD+ required in prokaryotes ATP required in eukaryotes  Nick sealing by DNA ligase  Topoisomerases Relieves torsional stress caused by rotation of DNA ahead of the fork 10 nucleotides = 1 turn  Topoisomerase I Breaks one strand of the duplex  Mechanism of topoisomerase I  Topoisomerase II (DNA gyrase) Breaks both strands of the duplex Introduces negative superhelices ATP dependent Summary of replication DNA is bent duing replication process DNA is proofread during the process Termination of replication The two replication forks are synchronized by 10 23 bp Ter sequences that bind Tus proteins Tus proteins can only be displaced by replisomes coming from one direction  Resolvation of replication products by decatenation  Comparison prokaryotic vs eukaryotic replication Prokaryote (E.coli) Eukaryote (Human) # Origins of 1 1000-10000 in replication replicons Speed of replication 500 nt/s 50 nt/s Time for replication 40 min 8 hours Okazaki fragments 1000-2000 nt 100-200 nt Polymerases 3 (5) 5 (10) Chromosomes 1, circular 46, linear Other Telomeres, histones  Reverse transcription Retroviruses are mobile genetic elements RNA-dependent DNA polymerase THANK YOU