Gene Expression - Biology of Cell - Lecture Notes PDF

Lecture 8 - Gene expression 7 layers of control that occur to form a protein from DNA ;...

Lecture 8 - Gene expression 7 layers of control that occur to form a protein from DNA ; 1. Transcriptional control 2. RNA processing 3. Transport and localisation. Translational 4 control proteins 5. RNA degradation - RNA is broken down to stop producing certain ↑.. Protein 6 degradation - Proteins are broken down , amino acids reused.. Protein 7 activation The control of gene expression contributes to variability. DNA content is identical in cells differential expression allows all , gene for different types of cells to form. certain genes have key functions in an cells e. g. RNA geres/Atpase other genes are only needed in specific tissues s some yeres are temporally regulated (in stages) ; Developmental stages (e. g. during embryo developments Differentiation stages cell cycle stage Inducible expression stages of DNA regulation ; Stage 1) Transcriptional Control ; · Transcription factors - bind to promoters ; guide and activate RNA Polymerase 11. ↳ Trans-acting ; bind and cis-acting elements. recognise Inducible transcription factors in · Occurs response to something e signalling -. g. or surroundings (Hormones (  Alternative · Alternative Promoters region of gene where transcription - starts. Produces the same protein , different protein /N shape. Epigenetic Control-Affect DNA and genome but not the code ; indirectly affect DNA function. Heterochromation - chromatin thats inactive and not transcribed. Enchromatin chromatin thats active and be transcribed. going to - Regulating Histores can activate or inhibit transcription. G Histore acetylation activates transcription ; makes DNA less tightly packed. Histone Methylation inhibits transcription ; by silencing the DNA. This produces our pre-mRNA. Stage 2) RNA processing ; · Alternative splicing - different exon combinations are able to produce tissue specific protein isoforms. Negative Control - Repressor comes in and prevents splicing. Positive control - Activator comes in and splices RNA ; making it shorter. o Alternative polyadenylation - Different site for poly-A tails. polyadenylation - Addition of AAA to 33 end of RNA. mRNA editing · - different RNA edits in different tissues Deamination of adehire to inosine (AtOI) within the intro causes RNA to told ; removing RNA. Deamination of Cytosine to uracil to form a stop codon. This is less common. Step 3) Transport and localistation ; mRNA is transported to ER or ribosure dependent upon 3'UTR. Step 4) Protein Translation ; cis-acting regulatory sequences in mRNA are targeted by trans-acting RNA for RNA longevity OR targeted by mirroRNA (miRNAs to inhibit translation/degrade MRNA. step 5) RNA degradation ; The longer the mRNA remains the more protein is made. mRNA is unstable and rapidly degraded. Eukaryotic Cells destroy MRNA via ; 5' cap removal or 3' degradation. Step 6) Protein degradation ; o ubiquitin - proteasome degradation pathway kills the protein. ephosphorylation · · unmasking cleave and destabilise & Reveal as areas ubiquitin of bind protein to. that enzymes such step 1) Protein activity control (post-translational control) ; The acids o cleavage - removal of amino o Presence of inhibitors of protein activity · Binding and activation - transport. Binding & Activation example ; RhoA-protein crucial for actin polymerisation. RhoA-Not made ; already present and regulated by other proteins to inhibit / activate it. GEF + RhoA - Active GTP  kinases phosphorylate proteins - leads to active/inactive form of proteins. Protein make up signalling networks where proteins bind to activate inhibit each other. DNA methods ; o cloning DNA using PCR. [cell based = in vivo] [in vitro = cell free]. · Nucleic acid hybirdisation - using DNA molecule to form complementary strand of DNA. DNA cloned for ; o studying DNA. Expressing Proteins Mutating o DNA Gaining understanding of how works · gene · Producing DNA copies to be used in lab. changing · organisms characteristics. cell based cloning (in-viru) ; · Desired DNA cut out : Inserted into plasmid o plasmid introduced into bacteria/ yeast · Bacteria/yeast containing plasmid replicates 1 is identified cell free cloning (in-vitro ( e. g. PCR ; · Heat DNA to separate strands (H+ bonds broken). · Cool DNA ; annealing primers o Temperature increased to allow DNA polymerase to work. o DNA polymerase forms complementary DNA strand. Nucleic acid hybirdisation ; Method used to combine single stranded DNA RNA molecules to form double-stranded molecules , (homologas DNA comes from different source). uses of DNA hybirdisation ; o PCR · Gene expression analysis · Gene editing e. g CRESPR. CRISPR gene editing ; used to cut DNA at specific site. & Watch It vid + Notes · Lecture 9-cell differentiation & development. Stomatic cells - All types of cells except gametes. Huploid cells = gamets egg + sperm : Zygote. that develops into a multicellular organism. model organisms - used to study development of an organism in a lab. model organisms must ; oproduce lots of eggs · Easy to work with · Large , visible , transparent embryo Xenopus luevis-African clawed trog ; · Easy to handle · produce all year round · Quick & Easy development oocyte development ; Froy egg = radially asymmetrical. Top half pigmented Bottom half , while. · Anterior/posteriora borsal ventral asymmetry All the stages of frog development is dependent upon where the sperm enters the egg. The sperm binds to receptors on cell surface ; most of the receptors are located just above the equator. SEP = Sperm entry point. The point opposite the SEP forms the dorsal end of the organism. As sperm enters oocyte ; causing the formation of a grey cresent 300 from the equator.  embryo development ; sperm enters well and migrates towards nucleus. ↓ Blastula formed With hole in Centre (All cells in blastula are the same ↓ Gastrulation causes differential transcription to occur ; forming different cells (Grastula Gastrulation - causes cells to rearrange and differentiate to form layers of tissues. Grastula made of 3 layers ; Ectoderm-outer of cells e Skin cells. g. mesoderm-middle of cells e. Bore g cells. Endoderm-Inner liver cells of cells e. g mRNA localisation role ; produces Vegt protein that VeyT mRNA acts as transcription a protein. mesoderm veyt protein activates genes to code for s endoderm. Determining cell fate ; 3. 1 Inject cells in blusua with marker / dye.. 2 Animal is allowed to develop Experiment 1. 3 Blastomere observed.. 4 Fate map created. 1 Same blastomere is selected and injected with marker I dye.. 2 The marked cell is then moved within the blastula.. 3 Identify location marked animal cell. of - Se.. g stained Kidney cell moved to epidermis still part of epidermis. Fate of blastomere = determined by plastula position Even if cells more into the wrong location early self generated positive , feedback occurs to form the correct sell type after. Human vs Xenopus deveolpment ; similarities ; Differences ; o start with zygotes · More complicated in humans · Early doubling o constrained area breaks down earlier Chumans ( s constrained area · Endoderm , mesoderm ectoderm form as , · differentiation based on embryo grows. location o more steps after Endoderm , mesoderm , ectoderm · Endoderm , mesoderm , ectoderm formation. foundation is crucial. Totipent - can differentiate into all cell types become ↓ pluripotent - can most cell types multipotent - can only become several types of cells G terminally differentiated - one purpose. ↳ · oocytes = polarity o Development of cel $ cell fate = determined by blustula position  Lecture 10 - The cell cycle Increase Hypertrophy in cell size (Hypertrophy). - Proliferation - Increase in cell number by cell division Most of cell cycle time is interphase. Major events in cell cycle ;. s-Phase 1 (synthesis phase) : chromosomes duplicate 2. M-phase (mitotic phase) : Duplicated chromosomes. segregate a phases of cell cycle ; o S-phase o M-phase Gap phases = G $G2 (separates and M phases and allow for growth/duplication. order of phases : 1 GI phase 2 3 S G2 phase phase - DNA duplicated I - Interphase Y M Division Phase - In interphase the following is constantly duplicated (except s phase) ; · Proteins o RNA · chromatin · organelles In early embryos cell cycles are free running and also occurs as quickly as possible. whereas in stomatic cells , the cell cycle is high regulated and controlled. stamatic cells double in size after each generation , causing organisms to get bigger  cell cycle control ; The cell cycle progresses from one stage to another if it meets a certain criteria e.. Is g all DNA replicated , Is environment favorable ? etc. Most cells are not dividing constantly and are in a resting state known as Quiescence/Go phase. cells require stimulation to move out of Go into 61. - ↳ Growth factors signal transduction · the M-phase of the cell cycle is dominant ; the signal to go through mitosis will override all other stages of the cell cycle. CdR (cyclin-dependent Rinases) - Master regulators of the cell cycle. Kinase enzyme that adds a phosphate group to amino acid. CDK / LDK 2 CDK CDKY 3 I 4 that different CDK's the bind cell to cycle. cyclin driving cycling - undergo synthesis and degradation cycles Four classes of cyclins ; G, 13 [cyclin ES Activates CdR in G. · - late o S Cyclin [CyclinA] binds Cdk start mitosis - - ; to o M-Cyclin [cyclin BS - Activates (dK for G2 > - M transition , o G-cyclin [cyclin DJ - controls Cyclin E. Cyclin activates (dle but also positions the kinase next to the protein that is going to be phosphorylated for the next stage  Lecture 11 - cell division Mitosis - Division of somatic cells Meiosis - Division of germ cells to produce gametes Phases of mitosis ; Prophase - chromosomes condense , centrosomes form at opposite ends Prometaphase - Nuclear envelope breaks down , chromosomes can now more Metaphase chromosomes - more onto metaphase plate(equator ( Anaphase - spindle fiber joins to centromere ; sister chromatids pulled to opposite ends Telophase - chromosomes reach opposite poles and decondense , Nuclear membrane reforms ; cytoplasm splits due to clearage furrow. cytokinesis = Anaphase & Telophase to form a daughter cells. mitotic spindle-microtubules that pull sister chromatids apart in Anaphase. Astral microtubules anchor centrosome to cell membrane ; allowing for pulling. Kinetochore microtubules - Attach to chromosome Motor proteins use ATP to pull chromosomes Apart. chromosomes twist and turn to attach to Rinetochore microtubules to then be pulled apart. As all diploid haploids. organisms are , gametes are 4 embryo (46) Sperm (23) + egg(23) =  Meiosis - produces haploid gametes Meiosis = 2 division : Meiosis 1 Meiosis 11 Meiosis 1 chromosome pairs separate after DNA replication No s- Phase J = Meiosis 11 = chromatids segregate before During meiosis 1 , recombination occurs , this allows for the exchange of DNA between pairs of homologous chromosomes. [ Prophase 1 - Homologous chromosomes pair up and crossing over occurs Metaphase 1 Homologous pairs of chromosomes line up at metaphase plate - meiosis ( Anaphase 1- Homologous Pairs of chromosomes more to opposite ends of cells. Telophase 1 - clearage furrow forms ; newly formed cells = Haploid cells Prophase 11-cells from Prophase / used chromosomes condense I are ; Meiosis 11 Metaphase/1- chromosomes line up at metaphase plate. Anaphase 11-sister chromatids are separated to opposite ends of the cell. Telophuse 11 - Newly formed gametes are haploids. Random assortment - occurs during meiosis 1. The random assortment of2 chromosomes 1 = possible gametes. 23 homologous pairs = 8 , 388 , 608 possible gametes. When meiosis doesn't occur as it should , it can cause ; · Euploidy Complete set of chromosomes formed - · Aneuploidy - One chromosome has a Extra copy or missing copy (45/47 chromosomes). polyploidy contain · - more than 2 homologous sets of chromosomes. Non-disjunction = Failure of paired chromosomes to separate in Meiosis 1 , or Sister chromatics to separate at meiosis 11/ mitosis. meiosis males = spermatocyte > - 4 smaller and similar spermatids. Meiosis females = egy cell doesn't divide. Oocyte- > legg and 3 polar bodies that then disintegrate. before meiosisconstantlyoccuringin males , only birth in fers a Mitosis is more prevalent for everyday growth I repair.  Lecture 12 - Membranes cells have membranes to keep their inside · Separate from their outside. cell surrounded by plasma membrane. All organelles have membranes membranes ↳ a selective pures to get Nutrients in / out. · Able to flow · made of phospholipid bilayer Biological membranes involve both proteins & Lipids. Lipid bilayer = Inner outer membranes that act as a barrier to water soluble molecules. Lipid bilayer made up of lipid molecules that are Amphipathic : Both fails and hydrophilic heads. hydrophobic one of the hydrocarbon tails bent allow is to the lipid bilayer to form and prevent the formation of a micelle. ↳ spontaneously aggregate to form bilayer. Lipids ↳ Fluid I o · Laterally diffuse properties · Rotate o Flex the plasma membrane of a cell differs across the cell due to the accumulation of membrane in certain areas. Proteins within the membrane add functionality ↳ Transport structural, links , receptors , sensing environment control process/control what · goes in $ out Membrane Proteins = hydrophobic regions that are embedded into the hydrophobic core via attachment/embedded proteins. Types of membrane proteins ; some cells are coated with sugar residues that act as a protective layer. 4 epithelial/endothelial cells. carbohydrate layer - glycocalyx and glycoprotein layer that surrounds the cell. Involved in cell adhesion : Not all cells have it. Membrane transport occurs vic : currier proteins OR Ion channels. Lipid bilayer has to be crossed to ingest nutrients a secrete metabolic waste , alongside regulating intracellular ion concentration / charge , Hydrophobic molecules - can pass through bilayer small uncharged polar molecules - Mostly repelled large uncharged polar molecules - mostly repelled Ions-repelled from bilayer carrier proteins ; solute binds to side of membrane , initiating conformational change in protein come side is shut when the other side g. GLUTs). opens e. Channel Proteins ; contain pores that allow certain solutes in lout of cells. uniport - only one molecule is transported in at a time. symport - A molecule is transported in alongside a co-transported ion. Antiport - One molecule is moved in WHEN another molecule leaves. ATP driven pumps - use ATP hydrolysis to move 2kt into the neurone and more out 3 Nat. ↳ Needed for muscle function & maintain neurone charge. Ion channels - proteins open / close hydrophylic pores for transport of ions. ↳ can be opened mechanically due voltage or to , alongside several other mechanisms. Ion channels = useful for neurones & Skeletal muscle cells. ↳ Malfunctioning = Epilepsy & Neuromuscular conditions  Lecture 13 - Introduction to organelles Organelles have compartments to isolate different functions within the cell. compartmentalisation allows for organisation ; the sorting movement of material. Eukaryotic cells divided into ↳ & membranes. organelles Nucleus -DNA BRNA Synthesis ↳ cytosol-jelly surrounding organelles Mitochondria - Energy production SER-Produces lipids Golgi) · Important acid 3 amino signal sequences; o N-terminal : Transport to target membrane target insert · Internal stop-transfer : stop threading across membrane · Proteins into different · Internal second signal : similar to N-terminal. membranes. * Endoplasmic Rough ER Reticulum 4 cystolic membrane bound Ribosomes Lots of membrane folds = ↑ SA ER · membrane forming continuous internal space. Smooth ER ↳ No ribosomes vesicle formation site Protein import to ERi cotranslational sumpled o ; N-terminal for motif by (SRP). Mutif Present = mRNA/ Protein directed to RER membrane. o post-translational , protein made in cytosol & prevent from folding = import signal used.  ER targeting sequence = membrane - spanning domain = Stop Transfer sequence Glycoproteins = proteins synthesised in RERI glycoslated by addition of n-linked oligosaccharides. Glycolysation = quality control i ligands for receptors. * Mitochondria MDNA - = encodes ax RNA , 22XtRNA &13 proteins = respiratory chain subunits. · Double membrane ; proteins have to cross both membranes Mitochondrial · proteins transported from cytosol. protein translocators - Transport proteins into mitochondrial matrix. Types of Protein translocators ; · TOM complex - Found in Outer - membrane o TIM complexes - Found in Inner membrane - o OXA complex - Moves proteins Synthesised within Mitochondria Lecture 14/seminar 2- Cytoskeleton I cell movement mechanical functions in cells dependent upon cytoskleton. ↳ o Pull chromosomes part in mitosis dividing · splits cells Supports o plasma membrane o Drives intracellular traffic to organelles cytoskeleton filaments ↳ o Actin filaments o Microtubules · Intermediate filaments Actin-2 stranded polymer of actin. 4 determine cell shape · Attach to membrane · Allows for cell movement/muscle contraction. Microtubules - Long hollow cylinder Fubulin of made of a $ heterodimer that determine organelle position& Intracellular transport ↳ Ostar arrays - Microtubules grow from the centrosome o centrosome forms during mitotic division · Allows for Lillia movement Intermediate filuments - rope like fibres. that provide mechanical strength and resistance. ↳ Form mesh to provide mechanical strength o Lamins line inner surface of nuclear membrane · Found in cytoplasm ; Neurore axon Nails/hair ; Keratin Lamins , Keratin , Neurofilaments , Desmin , vimentins] type of Intermediate filements Actin Nucleation- ↳ controlled by actin related proteins (ARPs) and forming Microtubule nucleation - the production of microtubules at the microtubule - organising centre (MOT) · ↳ occurs at centrosome near Nucleus Microtubules = made of 2 subunits and are unstable I constant state of flux). Microtubules undergo cell division causing centrosome to duplicate form 2 pole of mitotic spindle. Tubulin = GTPase ↳ Hydrolyses GTP + GDP Tubulin-GTP = Stable Tubulin-GDP = unstable Filaments = organised into higher order structures that are cross- linked and ordered into parallel arrays 3 types order actin of higher structures ; o contractile bundles - a activin , Loose packing · Gel-like network-loose meshwork , Filmin right parallel bundle Fimbrin - · MAP (microtuble accessory proteins) form binding bridge structures to bundle microtubules together. Accessory proteins are responsible for determining overall cell shape. ↳ Make cytoskeleton more fluid ; sever cytoskeleton filaments. Accessory proteins also make cell membrane more rigid ; Bundling , cross-linking B attaching filaments to membrane. Myosin-motor protein that 'walk' along actin filament. Dyneins Kinesin = walk along microtubules , carrying cargo. cell migration important for ; · Development wound healing s · Immune response · Metastatic profession : Cancer Rho - outer membrane ; ↓ · consists of Purin transport channels Inner membrane ; · Main part working · Impermeable · proteins generate ATP (Respiratory chain · cristae that folds = ↑ SA Intermembrane space = Proton (H) gradient Matrix= Mitochondrial DNA , Protein synthesis & Krebs cycle. Mitochondria input = Acetyl CoA + O2 BADP Mitochondria Output = CO2 + H20 P ATP  ETC ; o Imbedded in Intermembrane space. uses e'energy to create proton gradient as produce ATP. · · starts at NADH Dehydrogenase complex ↑ affinity for = e. ATP released as e-travels along ET ! NADH Dehydrogenase Higher affinity ↓ ubiquinone for Cytochrome b- 21 complex ↓ e cytochrome Cytochrome Oxidase complex + cycle repeats until 4 electron produced ↳ O2 takes up e- and forms H20. ↳ (final electron acceptor ( Mitochondria energy production process ; Chemiosmotic Proton Coupling - cone gradient generated by etc drives ATP production via ATP Synthuse channels , Stage 1 - Energy produced by movement of electrons along ETC generates proton gradient. Stage 2 - Electrochemical gradient used to produce ATP Protein-motive force (PMF) = Force due to membrane potential of force of pf ( ++ ion) gradient. proton gradient = energy store for ATP Synthesis. Electron Transport ; NADH carries from Krebs ETC. - e > - ETC Ara respiratory chain. ↳ 3 proton pumps ; Complex · 1 - CNADH Dehydrogenase · complex 11- (cytochrome - c reductase( · complex 111-(Cytochrome - -Oxidase ATP synthase Converts energy into chemical bond ADP + ATP energy -. Mitochondrial respiratory chain (MRC) Consists of 5 complexes ; 1) NADH-ubiquinone oxidoreductase 2) succinate dehydrogenase uniquinore oxidoreductase 3) uniquinone - Cytochrome Coxidoreductase 4) cytochrome - causes inflammation ↳ programmed = No inflammation Cytochrome - C released by Mitochondria ↳ activates programmed cell death. Microbiology Week 7 Prokaryotic vs Eukaryotic cells ; Proj - No Nucleus ; Bacterial DNA forms nucleoid one single chromosome - -Less complex ribosomes - No membrane bound organelles Relatively small - Evi - Nucleus with double membrane Multiple linear chromosomes - 5 rRNA species a 80 different proteins - Membrane enclosed organelles - Large cells. - open flask with broth would become contaminated = Groth whereas if the open flack was placed over bunsen burner the broth would remain sterile. Pasteur discovered spoiling of food was due to bacteria & microbial cells can't be spontaneously generatedB come from the air. Disproving spontaneous generation of bacteria allowed development of sterilisation techniques e. g. milk pasteurisation.  Koch experiment ; 1) Pathogen present in diseased organismB absent from healthy organism. 2) suspected pathogenic is in organism grow culture on agar plates ↳ o spread inoculum over small area on plate edge. o sterilise wire loop I make several streaks. · Sterilise loop again I repeat procedure x2 · Incubate plates at 372 overnight. 3) cells from pure culture should cause disease in a healthy animal. 4) Organism is isolatedB shows same results. Koch's findings ; 1) Bacteria causes diseases 2) Purification procedures for microorganisms 3) TB was caused by Mycobacterium TB. 4) PurifiedB grew +B mylobacterium on agar plates · 5) staining procedure for mycobacterium TB. Bacteria = Gram-positive or gram-negative Microbiology : · Research tools for life processes · All cells have many common things obacteria can be easily manipulated = Advances in genetic biochemical understanding of metabolism in calls. Week & Bacterial cell cycle I DNA replication ; Lecture Bacterial cells undergo fission to divide. 4 exponentially (N No2"(7 grow ; = division Number of cells = cells to begin with x a viable titre - flow many living cells are present within a culture. ↳ helps calculate doubling time. Semi-Lograthmic curve - time axis remains linear Stationary Phase Death rate metabolic activity is reduced. · - = Growth rate , Bacterial cellular growth ; small cell is elongated. Elongated cell is split into two cells via Fts2 protein that forms a septal ring. septal ring is constricted to form2 cells.  chromosome duplication Bacteria have chromosomes that are replicated by replicones. The two halves of the chromosome are called replichores single origin of bacteria undergo two replication forks ; counter-clockwiseB Clockwise until they meet the origin fully opposite , duplicating the chromosome. > Replisome Replication - machinery. Helicase first protein = opens parental duplex Primase = Leading strand requires 1 primer to start. lagging strund = opposite direction $ is completed in Okazaki fragments that require their own primers SSB Binds to single stranded DNA to prevent unwanted reactions from occurring. 4 RPC is that stranded DNA * the protein binds to single in Eukaryotics. Proteins fall off DNA. To prevent polymerase floating off ; the B-sliding clump tethers RNA to the DNA ensuring RNA polymerase stability. * Eukaryotic cells also have PCNA instead of B-sliding clamp. Okazaki fragments - Primer + synthesised parts of DNA on the discontinuous strand. chemical reaction inside of DNA Polymerase occurs in one direction. B clamp/sliding clamp = Catalyses reactions for DNA replication without releasing the substrate. sliding clamp lightly encircle DNA must be forced open to allow DNA to slide through. ATP clamp loaders open clamps and place clamp onto DNA. clamp loaders grab onto clamps ; open the clamp and allow DNA through.  Clamp loading mechanism ; 1. clamp loader = low affinity for clamp & Primer template DNA in ATP absence. 2 ATP presence : clamp loader opens DNA sliding clamp. 3. Primer template DNA binding causes hydrolysis of ATP into ADP + pi u. Clamp loader is ejected. 5. DNA Polymerase binds to clamp : DNA replication starts. clamp 3. 5. ATP-powered > - clamp loader 2. 4. Primers ; Primers - 30 nucleotide RNA sequences that act as the start point for DNA replication : by binding to the 3' end of DNA. Primers are synthesised by primases , and are composed of RNA's that are more abundant than DNA DNA replication and orazari maturation ; 1) Topoisomerase uncoils /unwinds DNA double helix. 2) DNA relicase breaks down H + bonds between strands. 3) clamp proteins stabalise replication forc allowing primers to attuch to send of DNA. 4) primer signals and acts as starting point for 31t5' replication. 5) DNA polymerase attaches complementary nucleotides onto newly synthesised strand. 6) 2 strands of DNA being replicated ; o continuous strand-DNA polymerase just runs along. o DiscontinuousStrand-DNA Polymerase runs antiparallel 7. Primace synthesises new primer for each section of DNA ; okazaki forming fragments. 7) RNase H-Removes primers from the okazaki fragments in the discontinuous strand. 8) Gaps are left between orazaki fragments due to removal of primers. 9) Gaps between okazaki fragments are replaced by DNA polymerase. 20) DNA ligase , joins orazaki fragments = complete DNA strand. DNA regulation ; DNA methylation and sequestration ; origin region contains GATC in high proportion of sequences. Adenine methylates adenine in GATC methyl-transferase , sequences. J unmethylated I methylated A new replication form comes along and methylates full sequences instead of just nucleotides. CH3-Act as methyl tags. Parental strand is methylated X ↑ New Strand is unmethylated (Hemimethylated ( Hemimethylated GATL's at the origin prevent the origin from being used. SegA binds to hemimethylated GATC. Sequences , preventing methylation. ↳ origin = unmethylated for 113 of Cycle. mmmmmm Methylation Allows the MMR system to fix errors on the newly synthesised DNA strand by looking for and matching the sequences in the methylated parent strand.  methylated parent strand. I & Hemi-methylated+ New strand. prevention of methylation leads to ; · Blocks initiation of replication as Drak can't bind to the origin. · prevents reinitiation of replication. · Prevents Mismatch repair Mechanism from working ; there are no methyl tags to differentiate between original and new strand. unfixed errors build up overtime = ↑ DNA mutations = Cancer , Replication - Termination ; Termination occurs when two converging replication forks meet. This converging causes Topoisomerase (Topo 1) to leave the cycle due to to space. tus protein binds to ter-sites = Replication fork trap , allowing forks to enter but not leave. co-directionality between transcription and replication in bacteria prevents accidents between the two and ensures the cell works efficiently.  Replicative DNA polymerase = responsible for DNA replication. Roles of Replicative DNA polymerase ; 1. Copy both DNA strands. 2 Distinguish if the nucleotide present is correct / Incorrect. Replicative Polymerase resembles right hand. ↳ - In the active centre DNA runs into the palm. The send needs be that to elongated is at the bottom of the paim. The DNA template runs into the palm as is then kinked upwards. selection of incorrect nucleotide = Geometric problem. J Primer longer no held in active centre I wrong nucleotide present = primer end of DNA strands bends downwards into the Exonucleate + basepairs are removed DNA synthesis continues. , DNA Mismatch Repair (MMR) Mechanism ; MMB = wrong base is added causing a mismatch nucleotide and forming a distorted DNA strand.  3 proteins needed for MMR ; 1) Muts Recognises Mismatch nucleotides Act as J - Dimers 2) Mut L-Binds to Muts protein 3) Mut H-Binds to Mut SB Mut L ; forming complex Enzymes within MMR detect i recognise methyl groups to differentiate between Methylated & unmethylated strands. Step 1) Mut L activates Mut H(Endonuclease) activity. Step 2) Mut H searches for GATC with methylated adenine. step 3) Mut H cleaves daughter strand at GATC. Step 4) Helicase 11 (UVRD) - unwinds cleared daughter strand. MMR in humans can causeTumors in; colon , skin , ovaries and stomach. weeks The inside of a cell ; seminar consists of organelles e. g: Nucleus , Ribosomes , membrane , ER , Golgi etc. Biogenomes generated & can be within labs and they are then intro- 4 duced into Bioterrorism = host Potential cells to viruses produce With virus Rey particles. sequences aren't made by manufacturers unless justification s proof is provided. chromosomes can be removed from cells and new chromosomes can be introduced into bacterial cells to cause the cell to produce new proteins & cells that also produce the new protein. The cell replicates chromosomes. There are multiple subunits within the chromosome to yet DNA replication going. Week & Transcription & Regulatory Networks ; Lecture The central dogma -unidirectional flow of DNA to produce proteins. /C transcription INA replication RNA polymerase = Transcribes RNA from DNA. RNA polymerase structure in E. Coli ; · RNA 3' end · 2 x a subunits · w comegal subunit · BBB' (Beta prime) subunits o O-factor. The Active site is located within RNA polymerase. Triophosphate within the active centre is activated by 2x Mg ions that pull electrons away. MgA My ; · Mg A = activates RNA 3' co-ordinates a phosphate for NTP. · My B = Stabilises-ve charge during transition stage. RNA polymerase moves into. the cytoplasm via diffusion. ↳ Binds unspecifically to DNA. ↳ Complex slides along DNA molecule to locate promoter. complex sections. Intersegment transfer = can skip  Promoter&Initiation : promoter = nides RNA polymerace to start of gene identifies gene to be transcribed. O-factors do following : > Bind to RNA polymeruse to form holoenzyme · Direct holvenzyme to specific promoters · Meet DNA region ; create single strand complex O 70 (sigma factor 70) ; o Directs RNA polymerace to specific promoter elements. · Responsible for general transcription in bacteria O 54 = transcribes genes for Nitrogen starvation. O 32 transcribes for neat shock and starvation. = genes Elongation : RNA Polymerase synthesises RNA (30-50 n/s). Termination Types : Rho-dependent : 1) Rno-factor binds to binding site in RNA. 2) Rho-factors climbs to RNA polymerate. 3) Rho RNA strand apart pulls transcript DNA 4) RNA molecule released = Transcription stops. & a 3 1  Rho-independent : 1) Region rich in 16 nucleotides is transcribed. 2) RNA transcribed = folds back & Complementary CIG bind = C1G hairpin. 3) Enzyme instability made. = falls off $ New RNA transcript Pauses & Mis-incorporation Backtracking = of RNA polymerase complexes = Abouts elongation B leads to extremely stable complexes. Transcriptional Activation : Activator proteins bind near promoters : RNA polymerase recruited. Transcriptional Repression : Repressor proteins block RNA polymerase binding. linked that regulate expression operon? other geres the of Lac-