Regulating the transcriptome

RNA turning to DNA
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eukaryotes have more factors to extend jaws etc.basic difference in pol. structure between prokaryotes and eukaryotesmakes pol2 different to 1 and 3 aids nuclear export important for translationpurpose of cappingsource of proteins diversity with alternate splicing and different domainspurpose of splicingpoly A binding protein can protect from degredation prevents circular translation helps polymerase detachpurpose of polyadenylationn and c domain connected by a flexible linkerstructure of prokaryote alpha subunitsregulation under certain conditions structurefunction of omega unitbeta'what unit of prokaryote RNA pol is often used in immunoprecipitation1 brings in new nucleotide 1 displaces the phosphate on the old nucleotidewhat is the function of 2mg in prokaryote RNA polbeta unitwhere is the beta coiled on on prokaryote RNA polbeta' unitwhere is the beta' flap and flap tip helix on prokaryote RNA polbeta coiled coilpart of beta prokaryote rna pol that interacts with sigma unit 2beta' flap and flap tip helixpart of beta' that interacts with sigma unit 4in the active site, it gets in the way of the new RNA strand building tensioninteraction of the sigma fingerforms a holoprotein with RNA pol and directs to promotor may stabilise the first ribonucleotide may regulate elongation initiation factor falls off quicklyfunctions of sigma factorsigma 70housekeeping sigma factorpart of sigma 1 negative charge occupies active cleft prior to RNA protects -54 to -6 DNA may be involved in open complex formation inhibits lone sigma factors binding DNAsigma 1.1discriminatorGC rich region but optimal G on thepart of sigma 2 2 alpha helicies at 90 degrees contacts DNA stand at discriminatorsigma 1.2binds ss DNA at -10 and discriminator 2 bp in the -10 element are melted and buried inside binds non-template stand has 2 elementssigma 2can bind single stranded DNA without other components has a tryptophan wedge where tryptophan 433 inserts into the double helixsigma 2.1-2.4binds non-template during meltingsigma 2.3selects the promotorsigma 2.4sigma 1.2prokaryote part of sigma 2 2 alpha helicies at 90 degrees contacts DNA stand at discriminatorsigma 1.1prokaryote part of sigma 1 negative charge occupies active cleft prior to RNA protects -54 to -6 DNA may be involved in open complex formation inhibits lone sigma factors binding DNAsigma 2prokaryote binds ss DNA at -10 and discriminator 2 bp in the -10 element are melted and buried inside binds non-template stand has 2 elementssigma 2.1-2.4prokaryote can bind single stranded DNA without other components has a tryptophan wedge where tryptophan 433 inserts into the double helixsigma 2.3prokaryote binds non-template during meltingsigma 2.4prokaryote selects the promotora 3 helix bundle binds double stranded DNA at -10, TG consensus stabilises RNA pol on the promotor so that sigma 4 isn't necessarysigma 3sigma 3prokaryote a 3 helix bundle binds double stranded DNA at -10, TG consensus stabilises RNA pol on the promotor so that sigma 4 isn't necessarya loop interacts in the active site channel and RNA exit channel a hairpin/finger binds non template DNA may start RNA synthesis by stabilising the templatesigma 3.2sigma 3.2prokayote a loop interacts in the active site channel and RNA exit channel a hairpin/finger binds non template DNA may start RNA synthesis by stabilising the templatebinds double stranded DNA at -35 in the major groove can change to cope with spacer variance but this is difficult because of movement around the helixsigma 4sigma 4binds double stranded DNA at -35 in the major groove can change to cope with spacer variance but this is difficult because of movement around the helixanti-sigma factors proteolysis for activation de novo synthesis make only when needed eg sigma 32 degradation post translational processing eg modifying an inhibitory sequence chromosomal rearrangement eg in sporulation sigma genes need to be split in 2 parts RNA eg 6s RNA binds RNA pol and sigma 70 making competitionregulation of sigma factorssigma factors holoproteins eg activators and repressors accessory factors like ppGPP, 6s RNA or DKSA elongation factorsthings that can regulate prokaryote RNA polymerasethermus aquatacusfirst discovered prokaryote RNA polymeraseall factors bound including sigma factor, crystalisehow was the structure of e.coli rna polymerase solvedmain, NTP/secondary, exitchannels in prokaryote RNA pol.binding affinity KB and isomerisation rate constant Ki, the speed at which the shape change occursfactors that affect initiation in prokaryotes1. R+P, RNA pol +promotor 2. RPC closed complex 3. RPi intermediate complex 4. RPO open complex 5. RPITC initial transcribing complex and abortive initiation 6. TEC transcription elongation complex - stable with closed clawinitiation in prokaryotesUp unit -35 -10 extension -10 dicriminatorparts of the prokaryote promotorinteracts with alpha subunitup unit elementconsensus= TTGACA not always present binds sigma 4-35 elementbinds sigma 3-10 extension elementconsensus = tAtaaT AT rish allows breathing binds sigma 2-10 elementGC rich but optimal G binds sigma 1.2discriminator elementtrp, close to consensus, controlled by a repressorexample of a strong promotor prokaryotearaBAD, far from consensus, controlled by activatorexample of a weak promotor prokaryoteA-11 is in a hydrophobic pocket T-7 is in a hydrophilic pocket in sigma 2 they are at 180 degrees to eachotherhow does prokaryote RNA pol bind DNAtryptophan 256 in sigma 2 replaces A DNA replaces sigma 1.1how does the prokaryote closed complex formthe template stand interacts with the sigma loop in a positive channel non template is stabilised by sigmahow does the prokaryote open complex formmini transcripts are made until one gets throughhow does prokaryote abortive initiation occurRNA pushes the beta loop which breaks the sigma4 beta flap interaction and removes sigma factorhow is promotor clearance achieved in prokaryotesabortive initiationProcess during initiation of transcription in which RNA polymerase repeatedly generates and releases short transcripts, from 2 to 6 nucleotides in length, while still bound to the promoter. Occurs in both prokaryotes and recognise different promotorswhat is the point of alternative sigma factorsPrincipal sigma factorsgroup 1 sigma factorsrelated to group 1group 2 sigma factorshave no 1.1 region often involved in stress response eg. e.coli sigma 32 in heat shockgroup 3 sigma factorsECF sigma factors have no sigma 1 or 3 units, but do have a sigma 3.2 loopgroup 4 sigma factorsmake flagellawhat does FliA sigma doan accessory factor found in gram + and - bacteria but not e.coli contacts minor groove upstream of -10 uses tryptophan to bindCarDaccessory factor only in gram + or actino bacteria contacts minor groove upstream of -10 binds principal sigma factors and DNA has a backbone of charged residues including Arg79RbpAthey approach from opposite sides of DNA to create the open complex, carD then stabilises this can compensate for bad -35 regionshow do carD and RbpA actrifampicin targets RNA pol in mycobacteriahow do you kill tuberculosisStochastic Releasewhere sigma factors leave at different stages of elongationwhere sigma factors leave at different stages of elongationstochastic release7have many alternative sigma factors does e coli havefunction of discoveryhow are sigma factors namedrelated to iron citrate uptake and didn't originally look like a sigma fcatorwhat is sigma Fecassociated with a 70kDa proteinwhat is sigma 70degrade change shape eg. oxidative stress can make a disulphide bond anti anti sigma factor eg sigma E wraps around RseA to be protected, eg GrY wraps around sigma cnrHhow to remove an anti sigma factorbinding to DNA -7 type1=T, type4=G -11 type1=A, type4=Ghow do ECF and group 1 sigma factors interact in a similar wayN Tag- allows degradation by FtsH transmembrane domain regulatory domain pp2c phosphatase for AA CIIE structureflorescent proteinhow to detect IIEan unstable protease that degrades IIEwhat is FtsHits attracted to the poles the accumulates in the foresporewhere is IIE found in the pre-sporulated cellan anti anti sigma factor that interacts with AB when not phosphorylated AB phosphorylates and IIE dephosphorylateswhat is AAwhen phosphoylated it activates transcription of sigma F and pro sigma Ewhat is Spo0AN terminus extensionwhy is sigma factor E inactive to begin withit is held sequestered by AB and ATPwhy is sigma factor F inactive to begin withF, E, G, K and Finwhat is the order of sigma activation in sporulationIIE removes phosphate from AA AA binds to AB and ADP, sequestering ithow is sigma F activated in the foresporehomolog of tubulin attracts other proteins to form a divisome makes a Z ring which spirals towards the poles and becomes the septem detectable with flurescence microscopy signals sporulationFtsZ in sporulation2 AB to one sigma F when ATP is present binds AA when ADP is present phosphorylates AAAB in sporulationIIE forms a multimer on the membrane tags are hidden no degradation by FtsHhow is sigma F only activated in the foresporesigma F removes the N terminal extensionhow is sigma G activatedremoval of GIN anti sigma factor mediated by sigma EHow is sigma G activatedsigma fhow is sigma G madebacillus subtilis or anthrax bacillusexamples of organisms where endospore formation happensa region of pro sigma k is removed mediated by sigma Ghow is sigma k activatedectospoulationsporulation outside the organismsigma F and sigma Ghow is Fin madecompetes for active site of RNA pol with sigma Fwhat does Fin do?His tag and nickel column of Fin only pulled out RNA pol, not sigma F two hybrid method using different elements of beta' bound to alpha found that the beta' coiled coil binds Fin no effect on sigma G prehaps because of different affinityexperiments proving function of sigma F6how many sigma factors are involved in sporulationbind between -10 and -35 repositions DNA so RNA pol can bindclass 3 activatorsclass 3 activatorsbind between -10 and -35 repositions DNA so RNA pol can bindCueR promotor is 19bp apart which should be 17 with no silver: twists the DNA 30 degrees bending it away from sigma 2 and RNA pol silver: twists 72 degrees which looks like 17bp, BDNA turns to ADNA and -10 and -35 are on the same sideexample of a class 3 activatora global regulator, cAMP receptor and activatorwhat is CRP/CAPCRP/CAPa global regulator, cAMP receptor and activatorbinds upstream of -35 AR1 residues contact alphaCTD287 determinant minor groove contacts alpha 265 determinant sigma domain 4 contacts alpha 261 determinantclass 1 activatorsbind regions overlapping the -35 region AR1- improves binding affinity of pol, contacts alphaCTD, AR2- improves isomerisation rate of pol, contacts alphaNTD, species specific AR3- contacts sigma4, affects isomerisation rate AR4- affects binding affinityclass 2 activatorstarget the lac operonexample of class 1 activatorstargets galexample of class 2 activatorsAR4 contacts alphaCTD instead of AR1 AR2 definitely contacts the beta flap there is also AR3how is the TAP homology different to other types of class 3 activatorit allows binding to activators/repressors in different wayswhy is the flexible linker useful in the alpha subunitsit recognises an almost palindromic sequence of 22bp it binds at almost the same time as RNA pol it works with cAMP to bend the DNA to help bind to RNA polhow does CRP workbind DNA respond to environment dimeric or multimeric to bind palindromic sequences modular proximal and distal subunits affect frequency of transcription used with weak promotors catalysts contact RNA pol or alter DNAfeatures of activators in prokaryoteshelix turn helix in the major groove, 3-4 baseshow do many activators bind DNA in prokaryotessmall molecule allosteric effectors phosphorylation de novo synthesis/ degredation redox other proteinsfactors that can affect activators binding to DNAcAMP on CRPexample of a small molecule allosteric effector affecting an activatorOxyRexample of a redox molecule affecting an activatorhigh binding constant little control can have an UP element that binds to an alphaCTD until an activator is present eg rRNAfeatures of strong promotorslytic cyclea viral reproductive cycle in which copies of a virus are made within a host cell, which then bursts open, releasing new viruseslysogenic cyclea viral reproductive cycle in which the viral DNA is added to the host cell's DNA and is copied along with the host cell's DNA1. inject DNA 2. circularise DNA LYTIC: new proteins and new bodies till it bursts LYSOGENIC: genes integrated into genome till stress is inducedlamde bacteriophage life cyclePL and PR OFF PRM ONwhich genes must be on/off to be a lysogenbinds OR1 and OR2 to increase the Ki of PRM when there is a lot it binds OR3 repressing itself contacts sigma 4 PC mutants dimerise and only repressC1binds O3 supressing PRMCroPRa switch promoter on the lamde repressorPRMa self regulating promotor on the lamde repressorall the OR sites are empty PRM is deactivated PR and PL are activated Cro is made lyticwhat happens when there is little C1there is cooperation between OR1 and OR2 PRM increases PR and PL decrease c1 increases and cro decreases lysogenicwhat happens when there is some C1they make multiple binding sites for eachother form an asymmetrical tetramer on DNA dimer dimer contacts on 4 CTDs make a twisted beta sheet each CTD stabilises connections between NTD and CTD in other places cooperation between OR2 and OR3 are preventedhow do OR1 and OR2 cooperatealternate pairwise bindinghow does OR2 bind both OR1 and OR3when the OR 1 and 2 on PL and PR combine to make an octamer represses PL and PR stimulates OR3 binding looping helps the alpha unit of RNA pol bind an Up element of PRMwhat is the long range cooperation in the lambda bacteriophage?O1, O2 and O3 are bound O3 has long range cooperation with itself to make a tetramer O3 has short range cooperation with O2 sometimes O3 binding blocks the alpha RNA pol binding site PRM is inhibited less C1what happens when there is a lot of C1p4 binds the alpha CTD of RNA pol represses A2C at -71 because its strong of pol gets stuck activates A3 at -82 because its weak so pol is recruitedphage phi 29 repressor examplesigma 2.4N helix turn helix for DNA binding IPTG binding site tetramerization domain to stabilise Clac repressor structurestable tetramerstructure of lac repressor in solutionCRPactivator of lac operonLac Ilac repressor geneO3 - -82 O1- just after promoter O2-412, after the Lac Z genelocations of lac operatorsZ, Y, Agenes on the lac operonincrease local concentration of repressors assist loop formationfunction of operators1,2,3 1,2 1,3 1 2,3Lac operon mutants in order of most repression causedHuwhich protein can help with DNA bending in the lac operonthe tetramer binds O1 and O2 which hides the promotor and CRP binding sitehow does the lac repressor make a loopatomic force microscopy and single molecule experimentexperimental techniques used to detect looping in the lac operon in vitroadd an ideal operator for the repressor upstream varying the distance shows that loops can only be detected when the operators are on the same face O1 and O2 don't repress much individually but together they doexperiment to overserve lac operon looping in vivodirect competition between RNA pol and repressors making the promotor inaccessible steric hinderance because RNA pol can not fit on the inside of the loophow can a loop repress a gene in prokaryotessteric hinderance-prevention of reactions at a particular location within a molecule due to the size of substituent groupsuncharged tRNA enters the A site and translation is stalled RelA can access CCA on tRNA RelA makes pppGppwhat happens when there is a lack of amino acids in a prokaryote cellthey keep growing so don't retain energy for sporulationwhat happens in RelA mutantsGTP+ATP RelA makes pppGpp in major pathway pppGpp is made to ppGpp by ribosomal proteins GDP+ATP makes ppGpp in minor pathway SpoT makes ppGpp to GDPppGpp synthesis pathwayauxotopha strain that needs nutrients normal bacteria wouldn'tthey are auxotrophicwhat happens to prokaryotes unable to make ppGppbeta', beta and sigmawhat mutant proteins supress RelAbeta, beta' and sigmawhere does RelA bind on the transcription machineryadding an inducible promotor to RelA eg the lac promoterhow can ppGpp be artificially inducedrrn operons that make rRNAwhat does ppGpp downrgulates7 that make 16s, 23s and 5show many rrn operons are in E.colippGpp DKSA and growth rate controlhow are rrn operons regulatedan rrn operon that has an unstable open complex with a short half life and is highly sensitive to ppGpprrBp1rrBp1an rrn operon that has an unstable open complex with a short half life and is highly sensitive to ppGppmake rRNA -10, -9 and -8 are usually A early experiments show that a -1 to -3 GC rich discriminator is importantrrn operonsamino acid biosynthetic operonswhat is upregulated by ppGppthrABC is upregulated by ppGpp in the absence of threonineamino acid biosynthetic operon exampleppGpp DKSA and amino acid absencehow is the thrABC operon regulatedhave stable open complexes so less affected by ppGppamino acid biosynthetic operon featuredownregulation of other promotors frees up RNA pol for other geneshow does ppGpp activate genes passivelyDKSA bind the secondary channel of RNA pol. using a coiled coil DKSA allows ppGpp to bind RNA polhow does ppGpp activate genes activelythey don't switch off gene expression they are unresponsive to ppGppfeatures of DKSA mutantsDRACULA assay differential radial capillary action of ligand assaymethod for telling if a small molecule binds a residue of a larger protein1. label a small molecule with a radioisotope eg ppGpp binding RNA pol p32 2. drop onto a membrane with RNA pol in the middle 3. a spot forms if it binds because rna pol is too big to moveDRACULA assay2how many binding sites of ppGpp havethey are unresponsive to ppGpp and therefore grow rapidlywhat happens in mutants of ppGpp binding sites?M8 RNA pol mutant delta omega RNA pol mutanthow to make a ppGpp site 1 mutantadd DKSA becausehow can you make ppGpp site 1 mutants active againDKSAwhat is ppGpp site 2 dependant onsecondary channel mutants don't work so beta' is involved it binds between 2 mobile units: the shelf and the core DKSA, ppGpp and RNA pol structure is solved but interactions not fully understoodexperiments that show the structure of ppGpp site 2beta, beta' and omegawhat is the shelf of RNA pol made ofbeta, beta' and alpha NTDwhat is the core of RNA pol made ofat the interface between beta' and omegawhere is ppGpp site 1pyrophosphates on ppGpp interact and are essential for binding in vitrowhat does omega do in ppGpp binding site 1G interacts with aspartate 622 mutant asp622 isn't sensitive to ppGpp in vitro, but it is in vivowhat does beta' do in ppGpp binding site 1mutant asp622 isn't sensitive to ppGpp in vitro, but it is in vivowhat is the evidence for 2 binding sites of ppGpp757how many genes are affected by ppGppchanges structure allosterically stabilises in open or closed statewhat does ppGpp do to RNA polfacultative bacteriaBacteria that can grow either with or without oxygenaerobic, anaerobic or fermentingwhat states can facultative bacteria adoptO2what does FNR detectfumerate and nitrate reductionwhat does FNR stand forbinds DNA to repress aerobic genes or activate anaerobic genes using fumerate/nitrate as an electron acceptorwhat does active FNR do(4Fe+4S)2+structure of active FNA FeS clusterin the presence of O2 (2Fe-2S)2+inactive FNR FeS clusterin the presence of O2 and H2O, no FeSapoform FNR FeS clusterapoformFNR with no FeS clustermolecular oxygen, cofactors, amino acid changeswhat can be detected by redox sensorselectron carriers on the membranewhat does ArcAB detectubiquinol ArcB phosphoylates ArcA phosphotranfers and binds DNAArcAB active statein the presence of O2, ubiquinone binds as an inhibitorArcAB inactive stateubiquinol ArcB is a sensor kinase that phosphorylates histamine on ArcA ArcA is DNA binding and does a phosphotransfer to an aspartatestructure of ArcABbinds DNA in 176 places in 85 operons activates and represses genes activates respiratory enzymes with higher oxygen affinity eg alternate terminal oxidasewhat does ArcAB do when activeubiquitionous signalling molecules/proteinwhat does thioredoxin detect2 SH with oxygen change to a disulphide bind with 2S joined or a sulphenic bind with SOH and SHthioredoxin changeit is detected by amino acid sensors or the can be redox sensitive cofactors that change shapewhat can thioredoxin affecta cofactorwhat does REX detectwith O2 open confromation homodimer with linkers near domain swapped helices NAD+ bound to CTD of one unit because it is charged and they are close aspartate 188 on the linker in one unit makes a salt bridge with the arginine in the other NTD binds DNAstructure of active Rexwithout O2 closed conformation 2 NADH binds each of the the CTD aspartate 188 binds a tyrosine in the CTD of the other, breaking the salt bridge major rigid body rotationstructure of inactive Rexwinged helix turn helix binds DNA helix- major groove wing- minor groove it represses genes including alternate terminal oxidase cytochrome Dhow does Rex actlactose dehydrogenase because it uses NADH as a cofactor with a Rossman foldwhat is Rex similar to?only in gram + bacteriawhere is rex foundthioredoxin, trxhow can one repair thiolsit takes the disulphide bond for itself leaving the protein free TrxB reductase uses NADPH to NADP to turn it backhow does trx workglutathionine gsh repair damaged bases produce resistant enzymes produce antioxidant enzymeshow can one protect thiolsit gets oxidised instead of other proteins 2GSH goes to GSSG which can thiolenate other proteins NDAPH to NADP turns it backhow does glutathionine workendonucleaseshow to repair damaged bases that might make thiolsAconitasesA sometimes have no FeS clusterexample of enzymes resistant to thiol damagesuperoxide dismutase - anti O2 catalase and alkyl hydroxide reductase- anti H2O2example of antioxidant enzymesFes clusterswhat does superoxide attackproteins and lipidswhat does hydrogen peroxide attackDNAwhat do hydroxyl radicals attacksuperoxide superoxide dismutase hydrogen peroxide fenton reaction hydroxyl radicalsthe ROS reactionsthiol to sulphenic acid sulphenic acid to disulphide bond or sulphinic acid sulphinic acid to sulphonic acidhow can thiols be ruined by ROSSH SH to SSG SHhow can thiols be ruined by GSSGSH SH to SNO SHhow can thiols be ruined by RNSROS eg peroxidewhat does OxyR detectOxyR, OhrR and HypRexamples of thiol based sensorsCys199 and Cys208 have a disulphide bondactive OxyR stuctureCys199- SOHstructure of intermediate OxyR structureCys199-SHstructure of inactive OxyRbinds DNA and interacts with alpha CTD to activate genes activates the OxyR regulonhow does active OxyR workbinds DNAhow does inactive OxyR workSOH is not hydrophobic so it comes out of a hydrophobic pockethow does intermediate OxyR workROS or O2how is OxyR activatedGrxA takes the disulphide bond and reduces OxyRhow is OxyR inactivateda ROS regulating protein tetramer its quantity is not effected by the presence of oxidative stressOxyR1. incubate promotors with labelled DNA with protein 2. random cleave with DNAase 3. sequnces can be protected by proteinshow does DNAase1 footprinting workCys199 protein binds and protects DNAexample of use of DNAase1an anti sigma factor that responds to oxidative stresswhat is RSRAROS make a disulphide bond in RsrA so it can't bind sigma Rhow is sigma R releasedTrx removes the disulphide bond by reducing RsrA and putting a zinc between the thiols RsrA binds sigma Rhow is sigma R sequesteredgroup 4 sigma factors often protect against oxidative stress mutants accumulate aggregates so it removes aggregates upregulates Trxwhat does sigma R doFeS clusterwhat does superoxide sensor SoxR detectsuperoxide sensor SoXRwhat detects FeS clustersusually porins which are trimeric pores which retain their C terminus but become misfoldedstructure of outer membrane proteins1. lipopolysaccharides inhibit RseB which protects RseA. Sigma E is wrapped around RseA 2. The c terminal domain of outer membrane proteins is modified and binds the PDZ domain of DegS 3. periplasmic site 1 of RseA is degraded by DegS protease 4. batismastat inhibition of RseP is relased 5. membrane site 2 of RseA is degraded by RseP 6. ClpXP, Lon and other proteases degrade cytoplasm site 3 of RseA 7. sigma E is free activates genesregulated transmembrane proteolyisisstep 1, where lipopolysaccharides inhibit RseB and c terminal domain of outer membrane proteins binds the PDZ domain of DegS both signals are requiredwhich step is limiting in regulated transmembrane proteolysisfusing MicA to a reporter to screen for molecules that decrease expression of sigma Ehow was batismastat discoveredan inhibitor of eukaryotic matrix metalloproteaseswhat is batismastatBamA and DegPwhich proteins does batismastat have no effect onupregulate the expression of ClpXP, Lon, BamA, DegP, 1/4 sigma32 promoters lipopolysaccharides are properly maintained outer membrane proteins, OMPs are properly foldedwhat does sigma E dofree lipopolysaccharides and misfolded CTP out outer membrane proteins active sigma E operonwhat can be caused by heat stressonly active in heat stress rpoE makes sigma E rseA rseBstructure of the sigma E operonknockouts are constitutively active only cytoplasmic portion solvedexperiments on RseA structureknockouts have a small increase in activityexperiments of RseB structurein all kingdoms and for ECF sigma factorswhere is regulated transmembrane proteolysis often foundfirst sigma discovered in E.coli mutants are sensitive to heat controls over 30 genes unstable- usually only 20-30 proteins per cell 4 control mechanisms + transcription initiationsigma 32/rpoHmRNA as a thermosensorhow can translation initiation be changed due to heat stressA and B regions interact to make secondary structure which prevents translation at a low temperature the UUUU can cover the shrine Dalgarno sequencehow can mRNA be a thermosensor1. the amount of mRNA doesn't increase in heat 2. transcriptional fusions of regulatory regions don't show increase in heat translational fusions of whole genes do show increase 3. rifampicin inhibitions transcription but it still responds to heat 4. mutants in A and B regions of mRNA don't respond to heat 5. increased synthesis of heat response enzymes continues in the adaption phase 6. compensatory mutants in A and B regions still respond to heatevidence of mRNA as a thermosensor in sigma 32DnaK- DnaJ, DnaK, GrpE GroE- GroEL, GroES2 chaperone systems in E.Coli1. pathogenesis in vibro cholerae host 2. TipA repressor forms a coiled coil when hot and can leave DNA 3. prf mRNA folds when cold 4. CspA allows DNA binding when coldexamples of thermosensor usesDnaK binds sigma32 on hydrophobic sigma domain 3 and one other place DnaK carries sigma 32 to FtsH FtsH degrades sigma 32 in heat: DnaK binds misfolded proteins so sigma 32 is activehow does DnaK system regulate heat stressturnover/degredation translation initiation post translational inhibitionmechanisms of heat stress regulationpathogenesis in vibro cholerae host makes heat in response to pathogens entering, this triggers Tox+, an activator to activate CT and TCP which make virulence factors in bacteria. UUUU mRNA blocks the shrine dalgarno sequencecholera heat shock responsesimple regulationsignal to factor to genesignal to factor to genesimple regulationratio of production and degradationsteady statesteady stateratio of production and degradationwhen it has a strong promoterwhen might a gene overshoot steady state?time to reach 1/2 of steady state= half life of generesponse time=sigma 32 has fast synthesisshort response time examplestable response times like in the cell cyclelong response time examplenegative autoregulationproteins repress their own expression.positive autoregulationexpression of a gene helps to increase its own expression, sometimes by direct feedback onto the promoterif the system is stable then response times are quick because repressors use strong promotersnegative autoregulation response times1. TetR inhibits GFP and an inducer inhibits the inhibition 2. GFP also inhibits TetR and the inducer inhibits that inhibitionexperiment comparing a simple regulation to negative autoregulationslow response time can lead to biostability the promoter requires switching on but may be difficult to switch off start points may differ leading to memory of initial concentrationpositive autoregulation response timewhen response times are slowwhen can positive autoregulation lead to bistabilitysingle input modulefactor x to a group of genes Yfactor x to a group of genes Ysingle input modulejust in time expressionwhen repressor affinity can change the threshold for individual geneswhen repressor affinity can change the threshold for individual genesjust in time expression1. ArgR repressor regulates itself and arginine catabolic genes 2. amino acid biosynthesis where earlier genes are switched on first and in larger amounts to allow the lowest time and metabolic cost eg. ARGA, ArgBC, ArgD, ArgEexamples of single input modulelarge scale promoter GFP fusions with a multi-well flurometer which tell which operons are activated in which order and therefore their relative thresholdsexperiment to demonstrate single input modulesfactor x to factor y to gene zfeed forward loopsfeed forward loopsfactor x to factor y to gene zwhere the overall signal is the samecoherent feed forward loopsthere are conflicting signalsincoherent feed forward loopscoherent feed forward loopswhere the overall signal is the sameincoherent feed forward loopsthere are conflicting signalsx activates y activates z. x activates ztype 1 coherent feed forward loopx activates y inactivates z. x activates ztype 1 incoherent feed forward loopdelay in activation waiting for Y, easy to switch offcoherent and loopcoherent and loopdelay in activation waiting for Y, easy to switch offno activation delay, off switch delaycoherent or loopcoherent or loopno activation delay, off switch delayrequires activation of x and removal of y z is active with no delay but once y reaches a threshold it switches off this makes a pulse generatorincoherent and loopincoherent and loophow to make a pulse generatormake a coherent loophow to filter out random changes in an activator/repressorcAMP activates CRP activates AraC CRP AND AraC activate araBAD genes araBAD genes cause arabinose metabolism in absence of higher quality metabolitesexample of a coherent looppersistent signalhow to get y to the threshold in feed forward loopscAMP activates CRP CRP activates Gals and GalETK galactose inactivates Gals Gals inactivates itself and galETKexample of an incoherent feed forward loopdense overlapping regulonsintegrate different signalsrpos is a sigma factor in stationary phase oxyR is an activator of oxidative stress they both activate KatG which activates catalase catalase degrades H2O2 produced in the stationary phase and oxidative stressexample of a dense overlapping regulonrequire decisions positive feedback is common genes are turned on and off in temporal and spatial wavesfeatures of developmental networksmemory is highdouble positivememory of one or the other gene is highdouble negativestarvation to Spo0A and sporulationexample of developmental decision makingequivalent to beta' forms a side of the positively charged cleft makes a mobile clamp that is larger than in bacteria has jaw and foot domains has the CTDRpb1many conformations made of heptapeptide repeats YSPTSPS 52 repeats in humans hypophosphorylated during initial binding to the promoter hyperphosphorylated by CDKs when transcription starts disrupts binding of factors at the promoter stabilises elongation deletions are lethalCTD rpb1equivalent to unit beta forms a side of the charged cleft has lobe and protrusion domains has 2 more external domains than bacteria C terminus makes a clampRpb2protrusionequivalent to beta domain 3 in eukaryoteslobeequivalent to beta domain 2 in eukaryotesequivalent to alpha has an addition exposed zinc binding loopRpb3only found in the 12 complex the most exposed unit forms part of the wedgeRpb4equivalent to omega stabilises complexRpb6extends the jawsRpb5tip binds pol2 may interact with RNA as it leaves may bind Nrd1 which binds RNA may bind Fcp1 which can phosphorylate the CTD forms part of the wedgeRpb7make a wedge that closes the clamp this suggests that the clamp is closed at initiation so DNA may bind outside firstRpb4/7replaces the second alpha domain of Rpb11Rpb8extends the jawsRpb9fills dips in the surfaceRpb10makes a heterodimer with 3 to bring 1 and 2 togetherRpb11equivalent to the flap blocks DNA so it exits at 90 degreeswallmagnesium ionswhat is found at the active site of RNA pol 2lined by the bridge helix and the active site has a funnel shape where nucleotides enterpore in eukaryotesmake of the NTD and CTD of Rpb1 and the CTD of Rpb2 has switch regions including the zipper, lid and rudder which change conformation when DNA bindsclamp eukaryotestabilises DNA in the active sitebridge helixzipper, lid, rudder they change conformation when DNA bindsswitch regionsnon universal up to 200bp upstream but most at -50 can be downstreameukaryote promotersbind transcription factors and can be very far away from promoterseukaryote enhancers1. recruit pol 2 and GTF 2. form a pre-initiation complex (PIC) 3. double stranded DNA binds the bridge helix and switch regions forming the closed complex 4. when 10-13bp are unwound and the template is in the active site the open complex is formed 5. conformation changes make the initial transcribing complex (ITC) 6. the DNA/RNA hybrid binds the 3 switch regions and the clamp closes 7. DNA exits at 90 degrees after scrunching 8. promoter clearance 9. elongationprocess of transcription eukaryotesbinds the TATA box initiator element that spans the start site, Inr Motive 10, MTE downstream promoter element, Dpe downstream core element, DCE has TBP has 10 associated factors, TAFs which add selectivityTFIIDinitiator element that spans the start siteInrMotive 10MTEDownstream promoter element (DPE)Dpedownstream core elementDCETFIID associated factorsTAFTATA box binding proteinTBPstabilises and activates TFIID from cryoEM we know it makes a subcomplex with TBP and promoter DNATFIIAD, A, B, F, E,Horder of GTF bindinggeneral transcription factorGTFinteracts with BREu and BREd which can be on either side of the TATA box allosterically changes the active site so there are 2 mg lobe and protrusion rotate cleft partially closes tilts RNA towards the exit channel forms a subcomplex with TBP, pol2 and DNA which contacts TFIIE and TFIIF in either side of the cleftTFIIBcore with NTD cyclin fold linker reader ribbonstructure of TFIIBinteracts with wall positions promoter over active site cleftfunction of TFIIB corehas a helix and 2 beta strands helix interacts with the coiled coil on the clamp helps unwind DNSfunction of TFIIB linkermostly in the cleft has a loop and a helix interacts with RNA exit tunnel helps get the template to the cleft R64 and D69 contact DNA at -7 and -8function of TFIIB readercontacts Dockfunction of TFIIB ribbonstabilises TFIIB and TBP by interacting with pol 2 attracts TFIIE and TFIIHTFIIFattracts and regulates TFIIHTFIIEcatalyses ATP dependant DNA unwinding and phosphorylation of the CTD important in promoter clearanceTFIIH1. the nucleotide enters pre insertion 2. the trigger loop closes the active site pushing the nucleotide near where it needs to go 3. phosphodiester bond 4. trigger loop moves backwards 5. trigger loop and bridge helix twist to move the DNA up 6. everything moves back to its original conformationmechanism of the eukaryote pol2 active sitealpha amamititinwhat can block the translocation of the trigger loopblock the translocation of the trigger loopwhat does alpha amamititin do?gating tyrosine recognises internal endonuclease cutswhat happens to fix a small error in pol2other factors are recruited TFIIS inserts a hairpin with charged residues into the pol 2 active site and the internal endonuclease cutswhat happens to fix a large error in pol2mismatch, fraying, backtrackingexample of small transcription errorstightly bound transcription factorsexample of large transcription errorsactivators, enhancers, coactivators, repressors, corepressorsother types of transcripton factorsattach to RNA pol DNA binding domain and activation domainactivator bindingusually separated from the promoterenhancer bindingassociate with main transcription machinery eg TAP or modify chromatin structurecoactivator bindingpassive- block activators active- target corepressors to DNArepressor bindingbind main machinery eg mediator or modify chromatin eg histone deacetylases and some histone lysine methyltransferasescorepressor bindinga coactivator/repessor interacts with GTF and CTD of pol2 to make a holoenzyme conformational change when activators bind transmits regulation from enhancersmediatorhead which binds pol2 middle tail which binds kinase for inhibitionmediator structurenon specific require activators glutamine rich eg Sp1 proline rich acidic eg Gal4activation domain properties25%define richzinc finger, homeodomain, leucine zipper, helix loop helixtypes of DNA binding domainscys2/his2 and cys2/cys2types of zinc fingerN , 2 cysteine, 2 histidine with zinc in the middle, c usually in tandem 23 amino acids long with a 7-8 base pair linker eg Sp1 has 3 fingerscys2 his2 zinc finger4 cystines with zinc in the middle x2 Cys3-5 is where DNA binds Cys 5-6 is the dimerization domain helical fingers work together found in glucocorticoid and oestrogen receptors globular only a few residues are important for specificitycys2/cys2 zinc finger3 helices with helix 3 binding DNA in the major groovehomeodomain structurePitx2 oct1 and oct2 in the pou regionexamples of homeodomains2 helices in a dimer the bottom helices straddle DNA top has a heptad repeat with 1 and 4 being hydrophobic and ususally leucine leucines interdigitateleucine zipper structureamphipathic with a hydrophobic region in the middle 2 helicies and a loop that dimerises basic region bHLH straddles DNA and the other part helps dimerisation repressors may sequester a leghelix loop helix2H3 and 2H4 make a central kernel tetramer 2H2A and 2H2B make 2 dimers which surroundbasic histone structurehistone fold 3 alpha helices with 2 connecting loops have a 'handshake' interaction motifhow is a histone dimer formedcan be extracted with dilute salt without affecting nucleosome structureproperties of histone 1mononucleosome 200bp trimmed nucleosome 165bp H1 loss 146bpwhat happens with increasing micrococcal nuclease7 amino acids on the H2A interact with H4 on the next nucleosome to aid packing coiling may also require H1how is the 30nm fibre/ beads on a string formedcoil/solenoid and zig zagmodels of 30nm fibre formationone start helix experiments with the chicken linker support thissolenoid 30nm fibre model and evidencetwo start helix crystal structure solved but was only 24/25nm and had no H1 crosslinking studies support thiszig zag 30nm fibre model and evidence12 histones of repeat DNA 1. add salt: zig zag 2. add linker histone: tighter zig zag 3. add magnesium chloride: solenoid 4. add more magnesium chloride: too tight to seein vitro experiment for showing nucleosome packingDNase1 hypersensitive sitesanother term of euchromatinonly the promoterwhich part of DNA does pol2 need access to?stability conformation contacts between nucleosomesdirect effects of the histone codewriters leave a mark then other proteins act by adding modifications or altering nucleosome contactsindirect effects of histone codewritershistone modifying enzymes that leave markshistone acetyl transferases HATadd acetyl to histonesGNAT, MYST, CBP/p300HAT familiesacetylate lysines on H3/H4 and H2A/H2B in a non-specific manor may reduce positive charge leaves a mark a readerwhat do HATs do?HAT domain: binds acetyl coenzyme A which catalyses acetyl transfer Bromodomain: binds acetylated lysinestructure of HATsBromodomainbinds acetylated lysinesatypical left handed helix bundle 2 loops: not always conserved like the rest of the structure make a hydrophobic pocket for lysine 26 stabilise contacts between hydrophobic and aromatic residuesbromodomain structureother modifications if the DNA is compactwhat may be required for HATs to actATP dependant remodelling complexcause DNA to loop around DNA differentlySwi/snf2, ISWI, CHD, INO80types of ATP dependant remodelling complexthe largest ATP dependant remodelling complex bromodomain exposes DNA to restriction enzymes before repositioning the nucleosomeswi/snf2restriction enzymes eg. micrococcal nuclease, DNase1 and restriction enzymeswhat can swi/snf 2 expose DNA to?hides DNA involved in chromatin formation and silencingISWIchromodomainCHDchromodomainbinds methylated lysinesATP hydrolysis and nucleic binding domain large multiprotein complexes use ATP hydrolysis contain DEAD/H ATPasesATP dependant remodelling complexslide nucleosomes exchange DNA for part of the octamers remove the octamerwhat can ATP dependant remodelling complexes do?loop recapture and twist diffusionmethods of ATP dependant remodelling complex slidingloop pulled away and dragged alongloop recapturemakes a twisted loop that creates torsional strain that needs resolving it makes small changes and so is good for risky complexestwist diffusionhistone arginine methyl transferasesadd methyl groups to argininesCARM1 and PRMT1types of histone arginine methyl transferasesspecific methylates H3 arginine 2, 17 and 26 asymmetricallyCARM1methylates H4 arginine 3 asymmetricallyPRMT11. convert arginine to citrulline using PADI4 but this only antagonises the effect of mono methylation 2. lysine specific demethylase LSD1 removes active methyls 3. Jmj-c domains remove by hydroxylationhow to undo methylationLysine- acetylated, methylated, ubiquitinated Arginine- methylated Serine and Threonine- phosphorylatedcovalent histone modifications1. activator: p160coactivator and p300HAT. coactivator recruits PRMT1 which does H3R3 methylation. HAT recruits CARM1 and H3R3 methylation promotes H3R17 methylation and H4K8 and 12 acetylation 2. H3K27 acetylation promotes H3R17 methylationexamples of interactive chromatin modificationon the same histone tailwhere do interactive chromatin modifications usually happenDNA methyl transferase DNMTEnzyme that catalyzes the addition of methyl groups to bases of DNA. Functions to prevent restriction endonuclease from cutting up cellular DNA.5 positions of C in CpG doubletswhere is DNA methylatednon methylated CpG regionsCpG islandsto switch off genes, islands can be methylated in cancersfunction of CpG island methylationmethyl binding domain MBDrecognises methylated DNAmethylates DNA which is a tag to be recognised by MBDDNA methyl transferase DNMTMECP2, MBD1examples of proteins with MBDin fungus Neurospora H3K9 methylation signals DNA methylationexample of a signal for DNA methylationhas an MBD recruits HKMT and Suv39 possibly also HADCsMECP2recruits HKMT which helps with H3K9 methylation in DNA replication Suv39 and Hp1MBD1histone lysine methyltransferase HKMTmethylates lysines for positive or negative modificationmethylates lysines for positive or negative modificationhistone lysine methyltransferase HKMTset1: H3K4 set2: H3K36 Dot1:H3K79 which is almost at the coretypes of HKMT and what they methylatechromodomain, Tudor, PhD, MB+royal family domainsroyal family domainsrecognise methylated lysinemethylation, phosphorylation and ubiquitinationtypes of positive histone modificationkinases and phophataseswhich enzymes affect phosphoylationmitosis different contextswhen is H3T3 and H3T11 phosphorylated?1. development/heat shock 2. Ras/ERK1/MAPK and TNF alpha 3. MAPK signals MSK1/2 kinase TNF alpha signals IKK alpha 4. MSK1/2 kinase does H3S28 phosphorylation and IKK alpha and MSK1/2 kinase do H3S10 phosphorylation 5. H3S10 stimulates H3K14 acetylation by HATs 6. H3S28 and H3S10 phosphorylation is recognised by a 14-3-3 domain which stimulates c-fos- a growth factorexample of histone modification in development/heat shock pathwayrecognises phosphorylation14-3-3 domain14-3-3 domainrecognises phosphorylationH3T3 and H3T11what is phosphorylated during mitosisE1- activating E2- conjugating E3- ligaseubiquitination enzymespoly ubiquitination of lysinehow can cell death be signalledcell deathwhat does poly ubiquitination of lysine signalnot affect nucleosome structure but does other thingswhat does monoubiquitylation do?activators stimulate E3 and E2 H2BK120 is ubiquitinated di/trimethyl H3K4 and H3K79 by HKMT possibly required for elongationubiquitination exampleup or downregulate geneswhat can ubiquitination of histones doubiquitination, deacetylation, methylationtypes of negative histone modificationco repressor complex with E3 eg polycomb repressor causes H2AK119 ubiquitination this causes repression, assisting with H1 binding and an inactive x chromosomeexample of negative ubiquitinationPolycomb repressor complex Prc1a repressive complex a co repressor with E3a repressive complex a co repressor with E3Polycomb repressor complex Prc1histone deacetylation complex HDACdeacetylates histonesdeacetylates histones found in complex with Sin3 repressor enhance associations between histones and DNA not very specificHDACclass 1, 2 and 3- the NAD dependant/Sir familytypes of HDACUme6 repressor Sin3 RPD3 deacetylase H4K5 deacetylatedexample of a HDAC pathwaySUV39: H3K9 EZH2: H3K27 SUV4: H4K20types of repressive HKMT and what they methylateposition-effect variegation PEVvariable expression of a gene in a population of cells, caused by the gene's location near highly compacted heterochromatinmethylation with HKMT SUV39 methylates H3K39 methylation is detected by the bromodomain of Hp1 which increases SUV39 expressionhow can PEV be causedheterochromatin protein 1reads histone code and binds to trimethylated H3 to begin condensation1. the E2F activator is repressed by RB 2. RB activates HDAC 3. HDAC deacetylates H3K14 which promotes H3K9 methylation 4. HDAC deacetylates H3K9 5. a repressor recruits SUV39, a HKMT 6. SUV39 methylates H3K9 7. H3K9 methylation recruits Hp1 which upregulates SUV39 8. H3K9 methylation downregulates cyclin E 9. ultimately this leads to upregulation of RBexample of crosstalk in histone modification in the RB generetina blastomaRB gene1. pho repressor upregulates E3 and EZ H1/2 units of the polycomb repressive complex 2 2. PRC2 di/tri methylates H3K27 3. methylated H3K27 is recognised by the chromodomain of PRC1 4. PRC1 compacts chromatin and increases RING1A/1B which increase mono-ubiquitination of H2AK119 5. E3 helps mono-ubiquitinate H2AK119example of crosstalk in histone modification in the polycomb repressordone by CDK7 and cyclin H and TFIIH common at early elongation due to many TFIIH common at 5' endS5 CTD phosphorylationimportant in cell cycle CDK7+cyclin H + MAT1 makes CAK which regulates cell cycle CDKs but not using the CTDCDK7peaks at early elongation not always in the repeat sequence required mediator or other kinases discovered in snRNA genes U1 and U2S7 CTD phosphorylationcommon in the middle done by CDK8 and cyclin c and mediator can phosphorylate before the initiation complex is formed which can prevent transcriptionS2 and S5 CTD phosphorylationwith mediator it tends to be repressive it phosphorylated cyclin H which leads to inhibition TFIIH associated with viral activator proteinsCDK8common at 3' end done by CDK9 and cyclin T1 and pTEFb common in late elongationS2 CTD phosphorylationpositive transcription elongation factor pTEFbtranscription factor involved in phosphorylating CTD S2travels with pol2 essential for elongation overcomes negative effects by NELF and DSIFpTEFbDRB which inhibits pTEFb and therefore also inhibits CTD phosphorylation and the elongation complexpTEFb nucleoside analogueartificial tethering co activators DNA/RNA bound activators chromatin bound activators eg Brd4 bromodomainhow can pTEFb be recruited?1. HEX1M1/2 and 7SK snRNA sequester pTEFb 2. the starter RNA recruits trans activator protein TAT which activates pTEFb 3. pTEFb with CDK9 and cyclinT1 phosphorylate DSIF and NELF 4. DSIF becomes a positive regulator 5. NELF falls off 6. elongation happensexample of pTEFb pathway in HIVphosphorylation: serine glycosylation: serine and threonine isomerisation: proline 3 and 6how can the pol 2 CTD be modifiescis to trans peptidyl prolyl bonds are formed with peptidyl propyl isomeraseisomerisation of pol 2 CTD1200-2000 bases/minin vivo reconstructed elongation system rate100-200 bases/min pol 2 pauses and falls off quicklyin vitro reconstructed elongation system rateremains at promoterTFIID in elongationstill associated with pol 2 increases the stability and rate of nucleotide additionTFIIF in elongationleaves 10 bases in helps TFIIHTFIIE in elongationleaves 30-60 bases in its helicase activity helps to make the open complex before the first phosphodiester bond is formedTFIIH in elongationTAFan example of an activator that is an elongation factorprevent arrest promote reactivation of elongationfunctions of general transcription factorsTFIIS, Elongin complex and ELLtypes of general transcription factorsalways present triggers cleavage of RNA for a new 3' OHTFIIS elongation factormade of elongin A,B and C stops pausing VHLelongin complexvon hippel lindau tumor suppressor proteinprotein that interacts with the elongin complexinteracts with elongin B and C regulates A because mutants prevent interractionsVHLprevents pausing common in acute myloid leukemia homologs=ELL2 and ELL3ELL elongation factortranslocation of MLL means that when ELL is transcribed so is MLL MLL recruits sec to switch on Hox geneshow is ELL involved in acute myloid leukemiac-myc, c-fos, heat shock proteinspromotor proximal pausing exampleswhen Pol 2 is always bound to the gene and only needs to be unpausedwhat is promotor proximal pausingpromotor proximal pausingwhen Pol 2 is always bound to the gene and only needs to be unpauseda checkpoint inhibitorNELFmade of spt4 and spt5 pauses elongationDSIFsuper elongation complex switches on genes contains pTEFb, ELL and the AF family which helps assemblysecassemble and disassemble histoneshistone chaperone function in elongationFACT, ASF1, spt6examples of histone chaperones in elongationmade of spt16 and SSRP1 binds H2A/H2B dimers, disassembles them then reassembles themFACTreplaces H3/H4 travels with pol2 binds PAF1 complexASF1binds TFIIS and H3 to reassemble nucleosomes after elongationSpt6spt16, spt6 and ASF1which mutants don't have properly assembled chromatinbinds phosphorylated S5 in the CTD methylates H3K4 which acivates Chd1 ATPase binds methylated H3K4 and allows HATs to be recruited near the promoterSet1 modifications within genesrecruited by phosphorylated CTD S2 and S5 methylated H3K36 causes repression HDAC recruited with chromodomain methylates recently transcribed genes then deacetylase Rpd3 binds and prevents initiation againSet 2 modifications within genesSet2 and HDACwhich mutants make cryptic transcriptionubiquitin on H2BK120 causes a conformational change on Dot1 and activates it H3K79 is methylated this inhibits Sir3 binding and inhibits telomeric silencing activates genesDot1 modifications within geneshypomethylation of oncogenes and CpG island shores hypermethylation of tumour suppressors and CpG islandswhat types of DNA methylation are there in cancerschromosome instability transposable elements loss of genetic imprinting more expression of geneswhat does hypomethylation of DNA lead toswitching off genes stabilises repetitive DNA sequences stops transposable elements movingwhat does hypermethylation of DNA lead tomutants in acute myeloid leukemiaDNMT in cancertranslocations in leukemia Deletions/ null mutants in myeloid cancerTets in cancerCytosine DNMT adds methyl 5'methyl C which is repressed Tets adds OH 5' hydroxymethyl C poised for activationDNA methylation cycle5' hydroxymethyl Cfound in haematological solid tumours downregulation of them varies in different types of cancernucleosome remodellersATP dependant remodelling complexes can also be calledtumour suppressor mutants inactivate the complex usually increase: interferon beta signalling, embryonic stem cell programming, nuclear hormone receptor signalling, proliferation usually decrease: BRB pathway/ retinoblastoma these are undone in cancerSwi/SNF A or B in cancerRhabdoid tumours and familial Schwanomatosis which affect kidney, brain and soft tissue inheritable and if it is inherited then the other allele loses its function mutants in mice get sarcomas mutants activate growth genes and activate checkpoints to trigger the cell cycleSNF in cancerRhabdoid tumoura type of aggressive and lethal tumourthe ATPase in SWI/SNF inactivated mutants are in small lung cancerBRG1inactivated in renal cell carcinomaBAI80SWI/SNF, SNF, BRG1 and BAI80nucleosome remodelers involved in canceroverexpressed global reduction of H4K16 acetylHDAC in cancertranslocation and mono allelic loss reduction in H4K16 acetylHAT in cancerH3K4 methyl differs with different cancers eg MLL1, EZH2HKMT in cancermyeloid lymphoid or mixed lineage leukemia often translocated may be amplified or tandem duplication fusions possible upregulates HOX genesMLL1 in cancerSEF domain lost fuse with other proteins to affect their functionfusions in MLL1a HKMTEZH2H3K27 demethylated is active gene EZH2 is the HKMT and UTX is the demethylase EZH2 is overexpressed in breast cancer leading more more inactivation EZH2 mutants in the SET domain are in large diffuse B cell lymphomas leading to overexpression bi allelic UTX mutants are in multiple myeloma UTX may regulate the tumour suppressor networkEZH2 in cancerUTX JMJD3 is upregulated in prostate cancerexamples of demethylases in cancersupregulate lineage specific genes downregulate alternative lineage genes and pluripotency genesregulation of genes in developmentBivalent marks can keep genes poisedactivating and repressing histone marks in the same locationtelomerase promoter or colon cancerexamples of mutant promoters and enhancers promoting cancersnormal tissue hyperplasia neoplasia in the vasculatureprocess of cancer developmentcan't target mutants or deletionslimitations of epigenetic drugsDNMT inhibitors and HDAC inhibitorstypes of epigenetic drugs currently in useBET inhibitors and IDH1 inhibitors for brain tumorsepigenetic drugs being developedscreen a library study genetics and proteomics model organisms safety studies, small trial, large trialhow to develop a genetic drug1. RNA 5' triphosphatase cleaves the terminal phosphate on usually an A or G 2. RNA guanyltransferase adds a terminal G in reverse 3. RNA methyl transferase methylates G at the 7 positionprocess of cappingmethyl 7 Gtype of cap in eukaryotesCet1 = 5' triphosphatase Ceg1 = guanyltransferase they are in a complex that binds phosphoylated S5 CTD Abd1= methyl transferase also binds the complex enzymes are all released by dephosphorylationyeast capping enzymesKin28CDK7 in yeats5' triphosphatase N and guanyltransferase C are on the same gene guanyltransferase binds phosphorylated S5 on CTD the CTD allosterically activates it to have a high GTP affinity guanyltransferase is activated by binding SPT5, a DSIF which inactivates NELFmammal capping enzymestwo step transesterificationwhat reaction takes place in splicingAG|GU...C,T, A/G, A, C/U, C/U...|AGCconserved splicing elementsmall nuclear riboprotein snRNPmade of snRNA and 7 proteins makes up spliceosomemade of snRNA and 7 proteins makes up spliceosomesmall nuclear riboprotein snRNPoccurs during transcription which affects the rate CTD may be involved in recruiting splicing factorssplicingAAUAAApoly A signal1. CPSF bind the poly A signal 2. CSTF binds the GU/U rich site and enhances CPSF binding 3.endonuclease assembles in complex with PAP 4. RNA is cleaved 5. PAP adds a short oligo A sequence 5. PAB binds the As and elongateshow is the poly A tail addedCleavage and polyadenylation specificity factor, CPSFan RNA binding factor that binds the poly A signalCSTF cleavage stimulation factorBinds U-rich sequence, required for cleavage at the poly(A) signalpoly A signal, cleavage site, GU/U rich siteparts of the end of the transcriptendonuclease subunits CF1 and CF2cleaves the 3' end of the transcriptpoly A polymerase PAPThe enzyme that adds the stretch of polyadenylic acid to the 3' end of eukaryotic mRNA. It does not use a templatepoly A binding protein PABelongates the poly A tailGTF like TFIID recruit CSTF and CPSF at initiation and they are transferred to the CTD for elongationwhat is the CTDs role in polyadenylationCDK9 deletion in yeast and inhibition in drosophila reduces polyadenylation suggesting phosphorylated S2 is important S2 phosphorylation in yeast increased cleavage factor PCFIIevidence for CTD being important in poly adenylationallosteric/ anti terminator modelmodel for termination that suggests the release of a factorTorpedo modelmodel for termination that polymerase is physically removed from the DNAtranscription of the poly A tail causes a conformational change that releases an anti termination factorallosteric/ anti-terminator modelRNA falls off DNA without cleavage in drosophila the elongation PAF +TREX is lost after the poly A site the elongation complex and cleave complex are dismantled in yeast RNA binding protein/elongation factor mutants often lead to termination defectsevidence for the allosteric/ anti-terminator modelafter cleavage the left over 5' end is cut with endonuclease until pol2 falls offthe torpedo modelphosphorylated S2 on the CTD is associated with Rat1 endonuclease which is at the 3' end of the gene Rat1 or Xrn2 mutants lead to termination defects truncated transcripts destabilise terminationevidence for the torpedo model1. allosteric switch decelerates pol2 2. enzymes in capping and poly A complexes slow it down further 3. there is a dephosphorylation of the CTD and other modifications 4. pausing sequences and R loops make RNA vulnerable to endonucleasesthe mixed termination modelR loopa DNA loop that is formed because RNA is displacing it from its complementary DNA strand3 strand nucleic acid which is most at the start and end of genes can be caused by pausing elementsR loopsRNA pol starts using the RNA strand as a template leading to uncommon double stranded RNAR loop formationDICER complex is recruited triggers Hp1, repressive marks and RNAi factors which degrade RNAgetting rid of R loopsCCP1 phosphatase docks onto pol unit 12, CTD and TFIIF TFIIF activates CCP1 CCP1 dephosphoylates S2 specificallymain CTD dephosphoylationsmall CTD phosphatases less specificother types of CTD phosphorylationSSU72 dephosphoylates S5 RTR1 dephosphorylates S5 and helps transitionsexamples of small CTD phosphatsesSR, U1snRP and U2AFthe E complexSR, U1, U2, U2AFThe A complexspliceosome SR, U1, U5, U6, U2 and U2AFthe B1 complexSR binds ESE SR recruits U2AF and U1 U2AF recruits U2how is the A complex formedubiquitinous regulated by phosphorylation have a RNA binding domain RRM or KH has an RS protein, protein interaction domain binds eseSRexonic splicing enhancers esesequences within the exon that promote exon joining during splicingbinds GU at the 5' end of the intronU135s unit binds AG at 3' end of intron 6s unit binds the polypyrimidine tractU2AFbinds the branch site/ AU21. E complex SE, U1 and U2AF 2. A complex, U2 3. B1 complex/ spliceosome, U5 attached to U4 and U6 4. B2 complex, U1 leaves so U6 can bind GU 5. ATP hydrolysis releases U4 6. U6 base pairs with U2 7. U6/ U2 do transtenification to make the lariat 8. C1 complex, U5 cleaves the 5' end and moves to the 3' end 9. C2 complex, 3' is cleaved an exons ligate 10. U6, U2 and U5 remain on the lariathow splicing happensbinds the 5' boundary of the intron responsible for cleavageU5strong interaction with U6U4binds strongly with U4 binds GUU6splicing in the order of transcription common because splicing factors are often associated with the CTDconstitutive splicingmost common= exon skipping alternative promoters alternative splicing sites can cause early stop codons causing non-sense mediated decaytypes of alternative splicingESE, ESS, ISE, ISScis splicing motifsdegenerate, overlapping and cluster around splice siteseseif it is in an exon it will remove that exon, if it is in an intron, it will inhibit the closest intron recognised by the knRNP familyESScan be ubiquitinous or tissue specific have an RNA binding domain eg RRM or KH regulatory proteinstrans factorsHeterozygous nuclear RNP hnRNPexpressed everywhere RRM domain KH domain tissue specific can prevent splicing factors bindingexpressed everywhere RRM domain KH domain tissue specific can prevent splicing factors bindingHeterozygous nuclear RNP hnRNPKH domain arginine glycine rich domain for RNA/protein bindinghnRNP homologyhnKNP or FOX a tissue specific factor that prevents splicing factors binding the poly pyramidine tractexample of hnRNP1. X chromosomes: autosomes determines SXL expression 2. SXL inhibits male default splicing of SXL and TRA 3. TRA expression promotes female splicing in DSX 4. DSX suppresses other types of differentiationdrosophila sex determination pathwaySR proteins work with splicing co-activators 1. T cell restricted intra cellularantigen- TIA1 binds U rich ISE downstream of the 5' splice site which helps U1 bind 2. CUGBp and ETR3 like factor proteins CELF. ETR3 binds similar sequences to hnRNP/PTB to compete so exons are includedexon inclusion examplesFOX 1 and 2 inhibit inclusion on CALCA exon 4 they block SF1 from the branchpoint they block tra 2 and SRP55 from the ESEexon exclusion examplesinactivates cis acting elementswhat does loss of splicing doenhances a splicing elementwhat does gain of splicing do1. Becker muscular dystrophy is exon 37 exclusion 2. Breast cancer susceptibility gene, ESE mutant causes aberrant exon skippingexamples of loss of splicing1. beta thalassemia makes a cryptic splice site 2. spinal muscular atrophy mutant in exon 7examples of gain of splicinga cryptic splice site is activated in the beta globin gene C to T change is 654 of IVS2 intron beta globin is non active SSO has been tested in human mouse model for treatmentbeta thalassemiamutant in exon 7 exclusion of SMN2 gene which leads to an ESS loss of SMN1 gain of SMN2 cant compensate SMN2 mutants allow more binding of hnRNP to an ESS causing exon exclusion hnRNP can also block tra dependant ESE and block U2 this all causes a truncated proteinspinal muscular atrophy1.splice switching oligonucleotides, SSO 2. spliceosome mediated RNA trans- splicing technology SMART 3. ExSpeU1 exonic specific U1snRNAtypes of splicing problem treatmentsmakes anti-sense oligonucleotides to RNA restores normal splicing can block elements which stop splicing can also block silencers to increase functionSSOartificial pre-mRNA trans-splicing molecule, PTM it has a binding, splicing, and coding domain that replaces the mutated splice siteSMARTintroduce consensus U1 snRNA which binds with greater affinity than regular U1 to upregulate splicingExSpeU1no poly A no TATA transcripts extend past 3' essential proximal sequence element no splicing 3' box only found here integrator complex plays a roletranscription of snRNA1. DSD binds STAF and OCT1 2. PSE binds PTF 3. pol2 is recruited 4. GTF bind: TBP instead of TFIID, TFIIB, TFIIA, TFIIE, TFIIF, prehaps TFIIH. S5 on the CTD and integrator element is involved 5. 3' box makes extended RNA and helps termination 6. transcription and processing are coupledhow snRNA is transcribed3' box makes extended RNA cleavage occurs the CTD is required for 3' formation the integrator complex has a CPSF unit which is involved in processing, but further trimming may be required s7 phosphorylation is required for the integrator complex to be associated so it can recognise the 3' boxtermination of snRNA transcriptionx ray crystallography and homology modellinghow were pol 1 and 3 structures determinedthey have intrinsic RNA cleavage activityhow do pol 1 and 3 differ to pol214 units compact extra units 34/49 stick out and are similar to rap74/30 of TFIIF and are used for elongation rpb 14 and 43 make the stalk which is more anchored wider DNA binding cleft extended DNA loop which looks similar to DNA backbone A12.2 subunit extends the active site RNA exit channel has a A190 lid loop clamp is more closed and has unique helices bridge helix has a kink and is more unwound additional 4Fe4S domain is used to help structure a zinc ribbon like TFIIS enters the nucleotide entry pore to prevent amanitin bindinghow is pol1 structurally different to pol2in the nucleolar organising regionwhere are rRNA genesgrowth phasewhat do rRNA genes indicate-156:-107 USE -45:18core this is an intergenic spacer which can make its own transcripts with unknown functionparts of pol 1 promoter1. SL1 binds cooperativly with UBF 2.pre-iniciation complex: TFIA and pol1beta are recruited, many at once 3. initiation and promoter escape: UBF1 is used but remains at the promoter 4. elongation: loss of TFIA makes pol1 epsilonpol 1 initiationselectivity factor, SL1 with its TBP and 3 associated factors upstream binding factor UBPfactors that bind upstream of pol1 promotera single 14Kb/47s transcript processed co-transcriptionally to make 18s, 5.8s and 28s RNA methylation and pseudo uridinylation is done laterpol 1 rRNA transcription1. TTF1 bends DNA using PTRF, a transcription release factor and a T-rich stretch 2. pol1 epsilon is converted back to pol1 betapol 1 termination3how many types of pol3 promoter are there?TATA intermediate element and C box within the gene poly T for 5s RNAtype 1 pol 3 promoterTATA box A box and B box within the gene poly T used for tRNAtype 2 pol 3 promoterDSE, PSE, TATA poly Ttype 3 pol 3 promotertRNA 75lRNA for membrane insertion U6, MRP,H1RNA for RNA processing 75K, AUM, B2RNA for pol 2 transcriptionwhat does pol3 make17 subunits extended stalk and other bits subcomplexes made of RPC53/37, RPC17/25 and RPC31/34/82 RPC31/34/82 can make it specific to promoterspol 3 stucture1. polymerase recruitment type 1: intermediate element recruits TFIIIA which recruits TFIIIC others: promoter elements like A and B boxes bind TFIIIC 2. TFIIIC binds along the gene and recruits TFIIIB with a TBP to the upstream region 3. TFIIIB recruits pol3 and interacts with DNA at -30, the TATA boxpol3 initiationhas 6 subunits used in pol 3 inititaionTFIIICnon template has poly T that causes termination other bases can affect termination TFIIIC binds the terminator region which allows genes of different lengths and brings on other genes that help with terminationpol3 termination