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Molecules and Cells notesIntro to cellsCells can be solitary or part of a communityIndividual cells of multicellular organisms are specialized and have intricate functions and forms and communicationsCells are broken down into prokaryotes and eukaryotesProkaryotesCan be many shapesSphericalRodCorkscrew/spiralReproduces quickly (about 20 min)Most diverse cellsExplore many habitats that eukaryotes cannot tolerateBroken down into bacteria and archaeaThe archaea are the prokaryotic cells that live in extreme places (extremophiles)Eukaryotic cellsLarger defined membrane bound organelles with different functionsCytoskeleton that directs movementModel organismsUsed by researchers because a part of them is similar to usMoleculesCovalent bonds=sharing of electronsNonpolar covalent-equalPolar covalent-unequal and partial chargesNoncovalent bondsIonic No sharingLife itself is carbon basedCarbon forms 4 covalent bonds to 4 different atomsHydrogen bonds are vital to lifeFound in waterFound in DNAStabilize the shape of lysozymesHydrophilic interactionsMolecules that tend to interact with water via hydrogen bondTend to be charged or polarHydrophobic interactionsUncharged nonpolar molecules that do not interact with waterVan der waal interactionsWeaker than H-bondsThe dipole interactions are very weak and transientDistance dependantpH=-log[H+]concentration of hydronium and hydroxide ionsmore hydronium more acidicmore hydroxide more basic0-7 acidic7-14 basicpH of 7.4 is blood…slightly basicbuffers can act as acids or basesamino acids4 elements make up 99% of humansH, C, N, O7 make up .9%Na, Mg, K, Ca, P, S, Cl4 major families of organic compundsSugarsFatty acidsAmino acidsNucleotidesProteins are the most abundant and versatile of the macromoleculesMacromolecules are constructed by polymerization via condensation reactions where a molecule of water is given offBreak downadd waterAmino AcidsAmino groupActs as a baseCarboxylic acid groupAcidAlpha hydrogen off the main carbonSide chain (R group)The only thing that differs in each of the 20 AAIt exists in an L and a D form (enantiomers)Our bodies only use the L formThree groupsPolarNonpolarElectrically charged (ionic)FunctionsProtectionTransportReceptorsContractileAnd many moreR groups differ in shape, size, and reactivity with waterPKAIt is the pH where it is half protonated and half deprotonatedOnly the R group matters (amino and carboxy groups always dissociate)Proteins themselves are made up of clusters of amino acidsConnected by a peptide bondRigid and not very flexible at the bond pointDehydration synthesisHydrolysis (break down)Repeat of NCCThe beginning amino and the end carboxy are the only charged part of the backboneN-teminus and C-terminus5 levels of protein structurePrimaryAmino acid sequenceSecondaryFolding alpha helix and beta pleated sheetStabilized by hydrogen bondsTertiaryFolding as the unitAll the secondary structuresStabilized by:Disulfide bondsH-bondsCovalent bondsVan der waalsAll these bonds are between side chainsQuaternaryProtein has multiple tertiary structuresDomainDomains are protein substructures that can fold intependently into compact, stable structuresDifferent domains have different functionsPart of a larger proteinsSpecific functionFunction can operate independently of the rest of the proteinProtein foldingProtein folding is often spontaneousDoesn’t need any helpSome proteins need molecular chaperones which give them an environment to foldDenatureProteins unfold due usually to heat or pH changesDisulfide bonds are hardest to breakMisfolded proteins cause human diseasesPrionsβ-mercaptoethanol breaks disulfide bondsSDS (sodium dodecyl sulfate) totally denatures a proteinLipid, Membranes, first cellsPlasma membrane is a lipid bilaryerSeparates the cell from the outside environmentChemical reactions are more efficientSelective barrierLocalized specific functionsTwo types of lipidsFatty acidsHydrocarbon tailIsopreneLipids have a major hydrocarbon component and are mostly nonpolar and hydrophobicSteroidsRing structurePhospholipidsCholine, Phosphate, glycerol, 2 fatty acid tailsEster linkage between the glycerol and the fatty acidAmphipathaticPolar and nonpolar componentHydrophilic headHydrophobic tailsKinks in the fatty acid chains caused by cis double bondsCreates more space, allows the membrane to be more fluidFound in cold environmentsShorter tails also increase mobilityLess forcesTemperature increases, fluidity increasesVice versaVery hot conditions? Not many kinks and the chains are longerVice versa for coldLong and saturated=less permeableShort and unsaturated=more permeableAmphipathatic molecules form micelles or bilayers depending on the shapeCone shapemicellesCylinderbilaryerPermeabilitySmall nonpolar molecules get in easy (hydrophobic)Small uncharged polar molecules get inLarge uncharged polar don’t get inAny ions cant get in (charged)Eukaryotic cells add cholesterol to change permeabilityDisrupts van der waal forcesReduces membrane fluidityFluid mosaic modelProteins span across the membranePredictions of the fluid mosaic modelMembranes are made up of 2 layers with hydrophobic chains on the interior and polar head groups exposed to the exteriorMobility of lipids is allowed only lateral and rotational and never a flip flopBilayers are asymmetric in natureDiffer in lipid concentrationMembrane proteins and either integral or peripheralRBC it is no cytoplasmCan see the bilayer asymmetry through an antibody reaction with both sidesPhospholipase CDegrades the phospholipid membraneUse a detergent to gain access to the insideCan see the mobility of the phospholipidsFusion experimentTagging with florescence Raise the tempmore mobilityTypes of membrane proteinsTransmembrane and membrane associatedTransmembrane –all acrossMembrane associated-only one sideLipid linked-inside or outside, linked to phospholipidsProtein attached-one transmembrane protein and another attached proteinIf there are 18-21 nonpolar proteins in a row, there is most likely a transmembrane domainUsually an alpha helixMost proteins have a minimum of 3 domainsPassive vs active transportPassive is diffusing without energy down a concentration gradientActive requires an input of energy and against a concentration gradientLinear graph=passive transportPassive across membranesIons (need a channel)Aquaporins (allow water)Carrier proteinsIon channelsHighly specialized for only allowing a certain ion inConcentrationOr chargeGated or passive channelsSaturation rate with transportEventually reach a maximum and it levels offCan only diffuse so fastInside the CellProkaryotesNot too many organelles (no membranous ones)RibosomesCircular DNAIn a nucleoid regionCytoskeletonPlays a role in cell division, shape determination, and polarity determinationCell wallMade of peptidoglycanGram positiveThick peptidoglycan wallGram negative2 membranesPeptidoglycan and lipopolysaccharide and protein membraneReleases endotoxinsHarder to defendPlasmidsAble to be shared with other prokaryotesCircularEukaryotic cell wallsFungi, algae, plantsStiff cell wall for cellular supportSemi permeablePermits small molecules and small proteins (30-60 kda)Plants and algae have a cell wall made of celluloseFungi=chitin cell wallEukaryotic cells have membrane bound organellesNucleusERGolgiPeroxisomesSecretory cellsSecrete a product (can be good or bad)Make thingsHighly specializedProduces material and secrete into a duct or the bloodExocrine gland=ductEndocrine gland = bloodSecretion is regulatedProducts are released from secretory granulesNucleusSurrounded by a double membrane nuclear envelopeNucleolus=site of RNA/ ribosome assembleFibrous proteins form a latticeNuclear laminaStructure and shape of the nucleusFunctionInformation, storage, and processingContains the cells chromosomes (code)Ribosomal RNA synthesisNuclear membrane is an extension of the rough ERThings can get through a nuclear pore complexRegulates proteins going in and outSelective based on sizeNucleus has a distinct set of proteins that work with itEach protein has a targeting sequence within the primary sequenceMolecular “zip code”Nuclear localization signal (NLS)17 AA signal that tells a protein it belongs in the nucleusRough ERNetwork of membrane bound tubes and sacs studded with ribosomesInterior is the lumenRough ER continuous with nuclear envelopeHugs nucleus tightlyFunctionRibosomes in rough ER synthesizes proteinsProteins are folded and processed and assembled in the lumenProteins are glycosylated in the ER lumenBegin protein modificationMajority occurs in the golgiSmooth ERRough ER without ribosomesEnzymes within smooth ER produces fatty acids and phospholipids or breaks down poisonous lipidsCalcium ion reservoirGolgiGolgi apparatus is formed by a series of flat membranous sacs called cisternaeFunctionGolgi processes, sorts, and ships proteins synthesized in the rough ERAlso site of lipid synthesisMembranous vesicles carry products to destination (either inside the cell or out)Carried in vesicles to the golgi too3 facesCis golgi is closest to the nucleusMedial golgiTrans golgi-closest to the PMThe proteins can have destinationsResident in the cis golgiContinue to go on to inside or outside the cellProteins in the lumen are dumped outside the cellMembrane sequence can allow a protein to be transmembraneDestinations?LysosomesPlasma membraneSecretory vesiclesAdd oligosaccharides to proteins that are secreted out of the cellMitochondria2 membranesOuterInner-cristaeFoldedHas its own DNA and ribosomesSupports the belief that they used to be cellsFunctionATP productionPeroxisomesGlobular organelles bound by a single membraneFunctionCenter of oxidation reactions (contains them)Catabolize long chain fatty acids, branched fatty acids, polyamines, and creates plasmolagensSpecialized peroxisomes in plants called glyoxysomesPacked with enzymes that oxidize fats to be used in energy storageLysosomesMembrane bound structures containing approximately 40 different digestive enzymesFound in animal cellsFunctionDigestionWaste processing40 different enzymes break downNucleic acidsCarbsLipidsProteinsWorking pH is 5.0Has a pump to maintain a low pHEndocytosisPhagocytosisIngestion of large moleculesMacropinocytosisMembrane grows aroundFluid uptakeClathrin-coated vesiclesForms a membrane enclosed vesicleNon-coated vesicleCaveolaeSmaller vesicle (50-80 nm)LysosomesFour processesPhagocytosisAutophagyEndomembrane transportReceptor mediated endocytosisEarly endosome matures to late endosomeLysosomal storage diseasesSubstances accumulate inside of the cellEndomembraneER, golgi, and ribosomes work together to produce proteinsProteins synthesized in the rough ERMove to golgi for processingTravel to the cell membrane or other destinationsER signal hypothesisLife begins in cytoplasmic ribosomesSignal sequence is synthesized by ribosomesAbout 20 AASRP binds to signal, pauses translation, and brings it to the rough ERSRP receptor allows translation to continueSRP signal is removedTrans golgi networkForm a vesicleGet a mature lysosomeMaturation processCytoskeletonComposed of protein fibersGives the cell shape and structural stabilityAids cell movement and transport of materials within the cellOrganizes all of the organelles and other cell structures into a cohesive wholeDisrupt the cytoskeleton?Entire intermembrane system is destroyed3 types of cytoskeletal elementsActinAlong the outside (cortex) of the cellIntermediate filamentsMicrotubulesOrganize the organellesMicrotubulesMade of alpha and beta tubulin dimersHave polarityDynamic (can grow and shrink)Usually grow from the plus end of the cellFunctionStabilityMovementStructural framework for organelles and a track for intracellular transportOriginate from Microtubule Organizing centers (MTOC)Major one is the centriole in animals TaxolMolecule that stabilizes microtubules to study themColchicineDepolymerized MTMotor proteins that use ATPase enzymes to moveKinesinTowards the + endDyneinTowards the – endForm cilia and flagellaActinMicrofilamentsGrouped in bundlesFound just inside the cell membrane in the cortexTwo strands of actin are twisted together for strengthPlay a role in muscle movementIntermediate filamentsProvide structural support for the cellNot involved in movementConnected cell to cell or cell to ECM to help anchor the cellCell AdhesionCells are physically connected to the ECMECMEverything outside of the cellPeripheral proteins are usually anchored by actin filaments on the inside of the cellIntegrin (in the PM)Attaches to fibronectin in the ECM which binds to collagenA way for the cell to anchorCollagenPart of the ECM (main component)Most abundant protein in the bodyFibronectinDimerLinked by disulfide bondsBinds to integrinsTight junctionsForms between PM of adjacent animal cells to form barriers that allow adheringMembranes are pinched together and have membrane proteins to form a tight junctionUsually found in tissuesTight water lineDesmosomesAdhering of cell’s intermediate filamentsIf damaged, can lead to blistering diseasesProteins in between=cadherinsGap junctionsAllow cells to communicateAllow small molecules to pass throughCan be gated or ungatedSelective adhesionCell-cell connections are species and tissue specificSignal transductionEvery cell interacts with its environmentRelay signals from outside inReceptorsFound in the plasma membrane for signals that cannot pass through the PMFound in the cytosol for signals that can pass through the PM (lipids/steroids)Receptor usually becomes a transcription factor and goes into the nucleusBinding creates a shape change in the receptor, activating itTarget cells are very specificLong distance signaling uses the bloodShort distance signaling is local growth factorsTransmembrane receptorsShape change leads toActivation of cytosolic protein that trigger the production of intracellular messenger moleculesEnzyme linked receptors (Tyrosine kinase) that create a phosphorylation cascade that activates a series of proteins in cellG-proteinsActivated by GTPStepsHormone binds to receptorChange activates the G proteinG protein binds GTPG protein activates downstream enzymes which signal or generate second messengerscAMP, Ca, cGMP, DAG, IP3second messenger cascade amplifies the signal and increases the reactiondeactivationG proteins hydrolyze GTP to GDP2nd messengers degraded or pumped back into storage (Ca ion)Cascades are turned off by phosphatasesenzyme linked receptorsdirectly phosphorylize kinase by the receptor instead of using a 2nd messengerTK form a dimer when activatedStepsSignal bindsTriggers a receptor cross phosphorylationCauses Ras to bind to GTP and receptor through bridge proteinsRas triggers a phosphorylation cascadePhosphorylation by a kinaseDephosphorylation by a phosphataseCell cycleBasic detail (see cell bio notes for a complete chapter on cell cycle)Five steps of mitosisProphaseChromosomes condense and the spindle begins to formPrometaphaseNuclear envelope breaks downKinetochore microtubules connectMetaphaseChromosomes complete migration to middle of the cellAnaphaseSister chromatids separatePulled to opposite sides of the cellTelophaseNuclear envelope reforms and spindle disintegratesCell division than begins (cytokinesis)Myosin and actin interactionMitosis varies somewhat in organismsNot always by the 5 stepsExperiments tell us that the chromosome is pulled away from the spindle from the + end to the poleAnd that the + end disassemblesCytokinesisOnce segregated, need to divide the cytoplasmPlantsCell plate needs to form so that a new cell wall can formAnimal cellsExisting membrane is pinched by actin/myosin contratctionRegulation of the cell cycleSignals are given to the cell to start the cycle2 types of proteins in cell cyclesOnes that are fundamentally required to proceedOnes that are only important if something goes wrong and needs to be correctedMitosis promoting factor (MPF)Induces mitosisCyclin/cdk complexCyclin cyclesCdk remains constantCheckpointsG1SG2MDon’t complete the cell cycle if DNA or chromosomes are damagedMitogensProproliferative growth factorsInfluence a cell’s decision to enter S phaseThe RB protein stops s phase entry until it is deactivated by the mitogenP53Guardian of the genomeActs as a DNA damage sensorCan halt the cell cycle so DNA can be repairedDetects mutations50% of human cancers have a p53 mutationFailing to control the cell cycleCancerunicellularDefect in nutrient sensing allows the cell to be outcompeted for nutrientsDefect in starvation censing leads cells to grow in the absence of sufficient nutrientsMulticellularDefect in sensing signals for growth leads to a failure to developDefect in signals to arrest growth would lead to cancer, which is not a single disease but a collection of different defectsMeiosisSexular reproduction must have some evolutionary advantagePuryifying selectionSexual derived progeny are less likely to have deleterious allelesThey get an evolutionary advantageChanging environment hypothesisSexual reproduction gives greater diversity, and greater chance for adaptationAsexual reproduction is fine when the environment remains stableKaryotype-complete set of chromosomes in a cellDiploid-contains two non-identical copes of each chromosomeOne pair of homologous chromosomesSame set of genes in the same orderContains different allelesHumans22 autosome pairs1 pair of sex chromosomes46 totalMeiosis halves the number of chromosomes to create haploid cellsOnly occurs to create germ cells (egg/sperm)Tetrad of chromosomes formsMeiosis IReduces the number of chromosomesDaughter cells are haploid but with replicated DNAMeiosis IIStay haploidNow have 1 copy of each chromosomeNot replicatedFrom one cell, you get 4 haploid gametesMost important step in meiosis is crossing over that occurs in late prophase IGenetic recombinationProduces new combinations of allelesHeredityMendel’s model peasSelf pollinationCross pollinationTests revealedMathematical rules of heredityPhenomenon of dominant and recessive traitsThe test results contradicted the blending-inheritance hypothesisWhich was just a 50/50 blend of 2 parentsControl experiments needed for conclusionsHe worked with pure line strainsPolymorphic2 or more allelesParticulate inheritanceTrait maintains its integrity through generationsEasy to assessSingle phenotype (in this case) is controlled by a single geneDominantRecessiveRefers to the relationship between allelesDominant is expressed in the heterozygoteWildtypeDefines a reference alleleMutant is a change in function from the wild typePunnet square is based on Mendel’s observationsTest cross determines what the unknown allele isUses ratios of the offspring to tellTraits segregate independently because the segregation of homologous chromosomes during anaphase I is randomThe genotype in a daughter cell depends on lining up and segregation during anaphase ISegregate based on how they line upMendel’s genes were all on different chromosomesSegregated independentlyIf they had been on the same chromosomes they would not have segregated independentlySex chromosomesGenes present on sex chromosomes are sex linkedHuman y chromosome does not have many genes on itMales only have one x and one y, so they only display the defective phenotypeHave no second copy to coverThe closer 2 genes are located on a single chromosome the more linked they areCrossing over is rare between themLinked genes segregate together EXCEPT when crossing over occursIncomplete dominanceIf a genotype is heterozygous, it will appear as a mix of the dominant and recessiveClose to blending inheritance but the second generation ratios were not correctCodominanceTwo alleles are phenotypically expressed in equal measuresExample is blood type ABMultigenic traitInteractions between different genes affect phenotypePedigree analysisVery useful to helping figure out what disease it is and how it is passed onAutosomal-both males and females affectedRecessive-affected children without affected parentsDominant-one parent affected and all children affectedX linkedOnly sons get the disease Daughters are carriersY linkedMale to male (since cannot get a Y from anywhere else)DNA and the geneChromosomes are 60% protein (histones) and 40% DNAHershey-Chase Experiment showed that DNA is the hereditary materialMarked phosphorus (DNA) and sulfur (proteins) and saw the presence of tagged phosphorus in the offspringTransforming principle (Griffith experiment) confirmed DNA is the genetic materialSomething from the dead virolent can transform nonvirolent into virolentDNA structureNucleotide3 componentsSugar (deoxyribose) (5 carbons)Phosphate groupNitrogen base SugarDeoxyribose in DNARibose in RNAPhosphate group is added to the 5’ end Sugar base bound to the 1’ endBasesPyrimidines-single ring structureC, U, TPurines-2 ring structuresGuanine and AdenosineCondensation reaction is a phosphodiester linkageA-TG-CBond between an O and a phosphate group2 strands are antiparallelThe DNA ladder twists to form a right handed helix to optimize the interactions between base pairsThe purines need to bind to the pyrimidines to maintain a constant diameterRNA is a more versatile molecule because it is single stranded so it can form loops and twistReplicationOne strand serves as a blueprint for a second strandAdd to the 3’ endCan only go in the 5’ to 3’ directionPrimaseCreates a RNA primer so DNA polymerase can attach and begin replicationDNA polymerase has a proofread functionReplication is semiconservativeNot conservative or dispersiveFound experimentally with Nitrogen markersHalf and half in first gen, half and half and some completely new in second genReplication begins at replication bubbles It expands in both directionsProkaryotes have only 1 originEukaryotes have multiple replication bubblesLeading strand is in the 5’ to 3’ directionLagging strand is 3’ to 5’ Requires many more primersOkazaki fragmentsJoined by ligase by covalent bondsDNA polym IRemoves the RNA primerDNA polym IIExtends the leading stran/okazaki fragmentProblem when the end of the chromosome is reachedThere is a small end that cannot be replicatedTelomeresAt the end of the chromosome to prevent the real DNA from being eaten awayTelomeraseA protein that can synthesize the repeatsAllows chromosomes to replicate without losing valuable genesNot found in many cellsShortening occurs on the leading strands in addition to the lagging strandChanges to DNAReplication errorsSpontaneous damageChemical/radioactive mutagensTranspositionViral transductionDNA repairNucleotide excision repairBase excision repairMismatch repairDouble stranded break repairExcision repairUsually thymine dimersHow Genes WorkGenes code for proteinsDNA to RNA = transcription by a molecule called RNA polymeraseRNA to Protein = translation by ribosomes3 bases in mRNA codes for a proteinOne gene one enzyme hypothesisProposed that each gene contains the info needed to make an enzymeBeadle and Tatum used bread mold to test the hypothesisThey damaged DNA to create mutants that had new nutritional requirementsSrb and HorowitzTested the one gene one enzyme theory using the Arganine pathwayCentral Dogma of molecular biologyDNA is the information storage materialSequence of DNA calls for Amino AcidsThe base sequence must first go through RNA before it becomes a proteinmRNA carries info from the DNA to the site of protein synthesismutations can occurTranscription identifies the template DNA strand to limit the choices to the three possible peptidesTranslation can identify which frame to readA single point mutation in the DNA can have huge effects on the proteinEx: sickle cell anemiaExceptions to the central dogma revolve around functional mRNA molecules that are not translated and perform other functions in the cellSome viral genomes also contain reverse transcriptase, which takes RNA and inserts it into the DNA of the host cellMostly retroviruses such as HIV20 total AA64 possibilities based on the 3 codonsRepeats (redundant)Nirenberg and Leder Cracked the codons by creating RNA moleculesFound that the start codon is AUGThere are 3 stop codonsUGA, UAA, UAGMany organisms can incorporate a 21st AA SelenocyteineAnd some Archaean bacteria have a 22ndPyrrolysineCodeRedundantUnambiguous (only a few exceptions)Nearly universalConservativeUsing the CodePredict codons and AA sequence encoded by a particular DNA sequenceApproximate the mRNA and DNA sequence that codes for a particular sequence of AAEngineer specific changes to a protein in order to determine the consequences of such a changeMutationsCan be point (one change)Missense-one AA replacedSilent-no change to the AANonsense-premature stop codonFrameshift-shift the whole frame + or – 1Or can be chromosome levelPolyploidy-increased number of chromosomesAneuploidy-addition or deletion of chromosomesInversion-within the chromosome it breaks off and reattaches somewhere elseTranslocation-moves to a different locationMutations can be:Beneficial to increase fitnessNeutral (no effect)Or deleterious and decrease the fitness of the organismTranscriptionDNA to RNAGene expression describes the production of a functional product (RNA or protein)Different processes regulate gene activityRNASynthesisProcessingDegredationProteinSynthesisModificationLocalizationDegredationTranscription is the synthesis of a single stranded RNA complementary to a DNA template strandH bond between basesPhosphodiester bonds between sugar and phosphate backboneRNA is synthesized in an antiparallel complement to the template strandRNA bondsPhosphodiester bond between OH on 3’ carbon and the phosphate group on the next nucleotideORFOpen reading frameStretch of sequence starting with a start codon and ends with a stop codonProkaryotes1 RNA polymeraseEukaryote3 types of RNA polymerasesI-most rRNA genesII-protein coding genes, miRNA, plus some of the small RNAsIII-tRNA and some other types of RNAProkaryotesPromotor=start signalWhere RNA polymerase starts the process of transcriptionAttaches to a TATA boxFound on the nontemplate strandA preinitiation complex assemblesRNA polym IICTDOther factors (transcription factors)Required for transcription to initiateProkaryotic TerminationThe presence of the 2’ OH in RNA allows greater flexibility, this allows greater flexibility to generate hairpin that triggers terminationA hairpin loop forces a termination that yanks the RNA out of RNA polymeraseEukaryotic terminationProcessedFactors recognize that a sequence is telling them to stopSplicing activity needs to occurExonsCoding (expressed)IntronsNoncodingSome DNA does not have complementary RNA (introns)Introns are removed by splicing and the exons are spliced together to create one RNA strandCap and tail are still includedSpliced by a spliceosomeSmall nuclear ribonucleoprotein (snRNP)snRNP ID’s the ends of introns and binds to them3 other things happen to RNA5’ end is capped for protectionSplicing of introns out and exons togetherPolyadenylation –add a poly A tract to the 3’ endEukaryotes tend to only code for one proteinSome exceptionsAlternate patters of splicing can produce multiple forms of a protein from one transcriptEnhancersCis acting that bind to trans-acting factors in Eukaryotic cellsProkaryotes translated right as its being transcribedSouthern blots=DNANorthern blots=RNATranslationalSynthesis of proteins from an RNA templateTake the RNA molecule outside of the nucleus to the ribosomes in the cytoplasmRibosome needs to recognize mRNA as a substrate and reading ittRNA3’ end is the amino acid acceptor arm and has an internal anticodon base pairs with codons in the mRNABinds to and brings a tRNA to the ribosome3 loops for a tRNA2nd loop contains the anticodonNet result-AA is selected by its codonCreates a high energy bond between 3’ and AAtRNA becomes charged when bound to an AAsome tRNA can wobblemeans that there can be a mismatch in the 3rd position of codon-anticodon pairexplains why there are fewer tRNA than codonsInosine5th Nucleic AcidModified derivative of GuanosineRibosomesEukaryotic49 ribosomal proteins, 3 different rRNA molecules make up the large (60s) ribosomal subunits33 ribosomal proteins, 1 different rRNA creates the small (40s) subunitCreation of ribosomal subunits takes place in the nucleolusAssemble into a full ribosome in the cytoplasm when bound to an mRNArRNAdon’t code for proteinstranscribes from genesrecognize and bind RNAs and proteinscatalyze the peptide bond between AA80% of RNA is ribosomalPeptide bond formation lengthens AA chain in the 5’ to 3’ directionE, P, and A sites in the ribosomeA is for aminoacyl site where the tRNA entersP is for the peptide site where the bond formsE is for the exit sitePeptide transferase reactionAccomplished by the transfer of AA from tRNA in P site to the A siteEukaryotesThe 5’ cap binds to the 3’ tail and creates a loop, this allows the binding of the small ribosomal subunit to the 5’ end Shine dalgarnoRibosome binding site in bacterial RNA (mRNA)Specifically tells what reading frame (since there are multiple onesTerminationRelease factor binds to stop codonNo charged tRNASpecial release factorWill cause a release of polypeptideLarge subunit is first to leaveIn bacteria, transcription and translation are tightly coupled allowing for a mode of transcription regulation based on availability of certain tRNACan transcribe and translate at the same timeEukaryotes separate transcription and translation in time and spaceMolecular chaperonesHelp proteins find their correctly folded shapeBind to hydrophobic surfacesHSP70 (heat shock protein)Helps the protein refold after it denaturesGene Expression ProkaryotesGene expression can be regulated at any stage from mRNA synthesis to protein activityTypes of regulationTranscriptionalSaves the cell the most energyDon’t transcribe all the genesOnly the ones that need to be used are transcribedTranslational controlLeast commonUsed to control levels of some ribosomal proteinsControls whether or not ribosomes will translate mRNACharge mRNA life spanChange translation ratePost translationalSpeed of response is of the greatest importanceEnergy is expensive but creates a fast responseProteins are activated/inhibited by chemical modificationsProteins get made in the cell but the activation signal usually comes from outsideThere are many control points of gene activityTranscription controlledRNA abundance is alteredProtein abundance is alteredNo change in other aspectsTranslationalRNA abundance is not changedProtein abundance is altered (does not translate)Other aspects unchangedPost translationalRNA no change (already a protein)Protein abundance changesIf RNA levels change…Can figure out if it is reduced transcription or increased degredationTo differentiate, we block transcription from taking placeLearn about how fast RNA is being degradedTranscriptional regulation: Protein-DNA interactionsA transcription factor is a DNA binding protein that influenced the level of transcription of a gene, either positively or negativelyThey bind to promotorsSpecific minor/major grooves that recognize specific binding sites on DNAConsequences of protein binding to DNAHinder RNA polymerase from recognizing the promotor regionIf a repressor is bound to an operator region no transcription occursAn activator may bind near RNA polymerase promotor siteThis draws the RNA polymerase to the promotor regionRepressors or activators are referred to as trans acting factorsSite where they bind is a cis acting elementCIs mutations2 mutations on the same DNA moleculeTrans mutation2 mutations on different DNA moleculesUse cis/trans test to determine whether or not mutations are present on the same gene or notCis dominant mutations only affect the function of the DNA molecule on which they are locatedHaploid-automaticDiploid-have another to cover, can be recessiveOn the same strandPhenotype is wild type and the function is limited by the genes acitivity that is mutatedTransOn different strandsElevates phenotypeMutation on upper chromosome appears to be dominantConstitutive mutationsAlways onExpress the gene even when the promotor is not being targetedCatabolite repressionForm of feedback inhibition, the product of a pathway inhibits upstream components of the pathwayBack to translational controlN-end ruleThe amino end of the AA determines the half life of the proteinSynthsismRNA binds to small subunit via the ribosome binding siteF-Met tRNA binds (initiator codon)Large subunit bindsSecondary structure of the mRNA can hide the Shine Dalgarno (SD) sequence so that the small ribosomal subunit does not bind, and translation is not initiatedBasically the RNA itself is regulated instead of the ribosomesThe SD sequence also can form a stem loop to block accessCalled riboswitchesThermosensativeCould respond to proteins tooFeedback regulation tells the cell to stop making a certain product if there is enough of itSaves the cell energyAlso a regulation to the presence of ribosomal subunitsNeed an equal amount of the small and large subunitsPost translationalProtein phosphorylationphospho-relay (cascade)Protein methylationDetect post translational modificationsWestern blots can detect a mobility shiftIf a mobility shift is observed one can use an enzyme to remove the modification and see if the mobility ceasesMutating the predicted site of modification should alter the mobilityTag the proteins with antibodiesAnalyze using mass specGene expression EukaryotesVariety of mechanisms to control gene activityChromatin (unique)Protein/DNA complexNucleusChromatin remodelingEuchromatin-activeHeterochromatin-inactiveTranscription – pre mRNARNA processingCap and poly-A tailCytoplasmmRNA stabilitytranslationpost translational modificationsevery step from heterochromatin to active protein can be modifiedchromatinhistone protein and DNA8 subunits of histones with DNA wrapped = nucleosomeOrganized DNANucleases can take DNA off for translationChromosome is interspersed with hetero/eu chromatinCondensed uncondensedHAT (Histone acetyl transferase) adds acetyl groups to decondense chromatinUncondensed condensedHDACs (Histone deacetylases) remove acetyl groups to condense chromatinEnhancers in a gene are binding sites for transcriptional activators that are not near the promoterCan be found upstream or downstreamCore promoter binds to general transcription factorsPromotor proximal elements are binding sites for transcriptional activator or repressors that are near the core promotorIf an enhancer is translocated to a different place, it can enhance a different geneTranscriptional control allows cells to respond to environmental changesEpigenetic inheritanceInherit the same patterns of histone modificationsHave same hetero/euchromatinX chromosome inactivation in women creates 1 barr bodyTranscription initiation depends on an initiation complexThe TF binds to DNAChromatin loosensTF bind to enhancers and promoter proximal elementsBasal transcriptional complex begins transcriptionDNA is dissociated from the nucleosomes by RNA polymerasePost translational controlOnce an mRNA is made, a series of events occur if the final product is going to affect the cell4 major control pointsAlternative splicing of exonsTakes place in the nucleusBenefit is that different forms lead to different domains that can lead to different functionsAlter the rate of translation initiationAlter mRNA stabilityPost translational modifications to alter protein abundance, localization, or interactionsRNA stability and RNA interferenceIn the cytoplasm, there are mechanisms to control gene expressionRegulate the amount of DNA translatedBlock access to mRNA by ribosomesAlter stability of RNAHighly variableSome degrade rapidly, others are very stableLife span of mRNA is controlled by RNA interferenceSpecific mRNAs are targeted by microRNA (miRNA)Binds to parts of the RISC protein complexDegrade the mRNA or prevent translation20-23% of all animal and plant genes are regulated by miRNAmi vs si RNAmiRNAdsRNA from an endogenous geneno perfect base pairingmRNA level is unchangedthere is a reduction in proteinsiRNAdsRNA from a foreign sourceperfect base pairingleads to RNA being broken downmRNA levels reducedprotein levels reducedtranslation is controlledmiRNA blocks translationmechanisms can control the time and rate of translationtranslation can be slowed or stopped by phosphorylation of ribosomal proteinspost translationalspeed of response is fastcells can respond to new conditions rapidly by activating or deactivating proteinstypeschaperone proteinenzymes can modify proteins by adding groupsProteins can be activated/deactivatedTargeted protein destruction ................
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