18.1-18.2, 19.1-19.2 (up to and including “Proteins act in trans…”), 13.3 (“Heterochromatin and euchromatin have different…” only) Flashcards
18.1 The elements of prokaryotic gene expression
gene expression is production of rna and proteins according to instructions encoded in dna
in gene expression, info in dna is trasncribed to rna and rna is a message thats translated into string of aa
trasncription and translation provide opportunities for cells to rgeulate syntehsis of rna’s and proteins so they are present at the times and in the amounts they need them
rna polymerase is key enzyme for trasncription
to start gene expression in proakryotes, rna pol trascnribes a genes dna into rna
rna pol partcpiates in all trhee phases of trasncirption
initiation requires subunit of rna pol- sigma subunit
sigma subunit needed for rna pol tor eocgnzie and bind to specific seqeunce at the promoter
when bound to the promoter, the complete rna pol holoenyzme (care enzyme and sigma) functions as a complex that both initiates trasncription by unwinding the dna and begins polyemriation of bases compelemnatr yto the dna template tsrand
switch from initation to elongation requires moveemnt of rna pol away from rpomoter and release of sigma
elongaton continues until rna pol encounters signal in ran that trigegrs termination (termination seqeunce encoded)
two types of signal found
in rho dpednent terminaton, proten called rho, helicase enzyme that unwidns mrna form dna template helps dissacoiate rna pol from template
in rho indepdent termination, a seqeunce of bases in the rna forms a seocndayr structure- hair pin loo- that signals release of rna pol from compelxed rna
trasnaltion in porkayotes begins before trasnciprtion ends
bacterial chromsome - translation of rna message into polypeetide can happen during trasncription
ribsomes bind to specicial initation site at 5’ end of reading frame (ribsome binding site)
polpetide grows in n temrinus to c terminus accoridng to mRNA codons
rna pol and ribsooems are sometimes close enough to each other on an mRNA so as to infleucne each otehrs actions
unique frature of gene expresion in proakryotes is that ribsomes can initatie transaltion at sevral diff psoitions on mRNa
many mRNA of bacterias are polycistronic - they contain open reaidng frames (section of dna that contains codons that can be transalted - many start and stop codons) for many diff proteins
regulation of egen expression at many steps
many leevls of control dtermine amount of a partciualr polypetide in a bacterial cell at one time
some controls affect aspetc of trasncripton like the binding of rna pol to promoter, the shift from tarsnciption initation to elongation, or rrelase of mRNa at termination of transcirption
otehr controls are postrancriptional and dtermine the stability of the mRNA after its syntehsis, the efficieny with wihc irbsomes reocgnzie transaltional initation sites along the mrNA, or the stablity of atcivty of polypetdie product
18.2 Regulation of Trasncription Initation via DNA Biniding proteins
fundmental mode of rgeulating gene expression involves the binding of regulatory proteins to DNA targets at or near promoters to control trasncription
the DNA biding of these rgeulatory proetins eitehr inhibits or enhances teh efefctivness of RNA pol in nitating trascnirption
inhibition of rna pol atcvty is negative regulation while enhancement of RNA pol activty is pos regulation
catabolic and analbolic pathways requrie diff types of regulation
E.coli used for reseacrh
many pathways are catbolci pathways in which complciated molecules are broken down for the use of the cell
otehr patwhats are anabolic - allow cells to construct end product moelcules they need such as aa and nucleotides froms impler constinuents
logic of regulating each patwhay is diff
catabolic pathways demand inducible regulation - means pathway is truned on - induced- only when complex moelcules need to be broken down (catoblites) are preent in the cell
otehrwise it would be wasting its resoruces to create enzymes when theres no moelcuel to be broke
in anablic pathways tehres repressible regulation - pathway turned on only when cell doesnt have enough of the needed end product like a specific aa
if prsent in suffcent qunatiteis then patwhay should be truned off - repressed- to not waste roeusrces
e.coli lactose as mdoel system of gene regulation
poliferating e.coli can. use any one of several sugars as a source of carbon and energy
examole is lactose made of glucose and gaalctose
memrbane protein, Lac permease trasnprots alctose into cell
enzyme b galtosidase splits lactose into two monomers
(catabolic)
the two proteins Lac permease and b gaaltosiade both required for lactose utuliztaion, are rpesent at low levels in the cells grown without lactose
the cell doesnt need for the proteins is lactose is basent - but is lactsoe aded then protein production inc
the process by whcih a aspecific moelcule stimulates synthesis of a given protein is knonw as induction
moleucle repsosnible for stimulating production of the protein is inducer
lactose mdofiied to derivative knwon as allolactose is inducer of genes for lactose utilization
possibility of cultring large numbers of bacteria made it easy to capture rare muatnts
once isolated the muations responible for latered pheontyeps can be mapepd
anotehr adavanteg was that the lactose utilization genes arnet essential for survival
bc proetin production inc in induced clels it makes it easy to see affects of mutant and wild type
chemisst syntehsized compoudns otehr than lactose that can be split by b gaalctosidase into products that are easy to assay
one product of ONPG splitting has yellow colours hwose inetsnity is propsotional to amount of prodcut made and thsu reflects level of atcity of b gaalctosae enyze
operon tehroy expalins how singel subsatnce can regualet many genes
Monod with regulation of lactsoe utilization
operon tehroy which suggests that a signle signal can regulate simulatenosuly the expression of serval genes that are clsutered toegtehr on a chromsoem and are involved in the same processes
genes form a cluster so they can be tarsncirbed into one mRNA and thsu anything that regulates tarscniption of this mRNA affects all genes in clsuetr
clusters of egnes regulated in this way are operons
three sttuctrual genes (lacZ, lacY and lacA) encoidng proteins needed for lactose utulziatin, togetehr with two regulatory elements - promoter and operator make up the lac oepron
single dna unit enabling simulatenosu regulation of trhee strcral genes in repsosne to envriment chnages
moelucles that ienrtact with operon are repressors which binds to operator and the inducer which bidns to represor and rpevents it firom biding to promier
the represor is an allosteric protein - protein that undgroes a revrsible chnge in conformation when boudn to anotehr moelcule
lactose operon:
- clsoely linked clsuter of trhee genes (lac Z,Y and A) that encode enyzmes atcive in split of lactsoe
-rpometr site form which rna pol initates traxncription of polycistronic mRNA - promoter acts in CIS affecting expression of only downstream strcural lac genes on same DNA moelcule
- a CIS acting dna opertor site lying very near lac oepron proometr on teh same DNA moelcule
- TRANS acting repressor protein that can bind to oeprator - rpressor encoded by lacl gene which is sep form operon and unreglated
- inducer that prevents the rpesors biding to teh operator - althoygh early expriemeters thought lactose was the idnucer, we now know that the idnucer is atcually allolactose
repression - in absence of lactose, represor protein bidns to dna of the oeprator and this binding prevents trancription - rperesor is neg rgeualtroye lent
induction - when lactsoe is present, the idcer allalctse binds to the repressor - bidning changes shape of repressor making it unable to bind to oprtor - with releas eof reprrssor form oeprator, rna pol cna iniaet transcirption
genetic analysis for operon hypothesis
operon tehroy of gene regulation
Lac- mutants are bacteiral cells unable to utilize lactose
using compelemattion anaylis of many lac- mutants, research showed that tehse cells inability to rbeak down latcose resuletd form mutation to either lact Z (b gaaltosidae ) or lacY (lac peromease) genes
they also disoevred lacA gene for trasnaetlase enzyme that adds acetyl to lactose and otehr sugars
these genes ar elnked clsoely on chrosmome in order of Z,Y and A
bc lacA isnt rruired for latose breakdown, most studies dont followA
loss of function mutations in a gene caleld lacI located nearbut not within the lac operon produce constitutive mutants that synethsize b gaalctoside and lac permease even in absence of lactose
contitutive mutants synethsize gene products all the time, irrepstive of envrimental conditions
the existence of tehse contsituive mutants sugegst that lacI encodes a neg regulator or a repressor
cells would need repressor to prevent expresison of Y and Z lac’s in absence of inducer
in cosntituive muatnts, muation to I gene generates defect in reprosor protein, stopping neg regulatory function
evidnce that acI encoded this neg rgeulator: matinsg in which chromsomal DNA of an Hfr donor cell is transfered into an F- recipent cell served as basis of the reseacher study - they trasnfered lacI+ and lacZ+ alleles into bacteral cell devoid of LacI and lacZ proeins in medium containg no alctose
shortely after tarnsefr of the genes then proetin was produces but stoped later on
inrpertation: when dodnor DNa is tarsnfered, no reprssor (lacI proetin) is in the recipnets cell cyoplasm bc the cells chromsome is lacI- - in absence of repressor teh Z+ gene is rexxpresed so reprossor is made
when sufficnet lac repressor accualmtes in cell (bc of mating) then furtehr prodction of enzyme decreases
prpsoed that teh repressor protein prevents tarcnription of lacZ by binding to opertor site - dna sequnce near promoter of the gens
repressor bidning to operator blcoks promoter and this bnding only happens when lactsoe not present
researchers then added alctose - precusor of inducer to medium
syntehsiz of enzyme resumed
indicuer bidns to wild type repressor - changing shape of repressor so it cant bind to DNa
when inducer removed, repressor binds again stoping the production
inducer is an efefctor - small moelcuel taht binds to allosteric protein,c ausing confirmationalc hange, that release repression without itself binding to DNA
operator muatnts
while lacI- muattion in repressor gene erase respsor atcivty, muattions that alter nucelotdie seuqnce of operator recognized by repressor can have same affect
result is constiyutive synstehsis of lactose utlizaton proetins
researches have isolated constitive muatnts whose egnetic defects map to lac oeprator site which is adjecent to Z gene
constituve oeprtaor DNA alteractions oC muations
bindign of inducer to repressor prtein stop repressor
so muations taht prevent reprossor form inertatcing with indcuer that results inc ells that dont turn on operoon eevn when idncuer was added to medium
reserachers isolated nonidncuble mutations in repressor genes and desginated them lacIS or suppersrpesor muattions
mutants cant bind to inducer but can to DNA and repress trascnription of operon
3 muations that allow expresion of lac oepron even when inducer not tehre - constituive oeprator muatnts and constitutive lac I- muatnts
elemnts that act in trasn (action of protein or RNA that can bind to tagret siets on any DNA or RNa) can diffuse trhough cytoplasma nd act at trrget sites on any DNA moelcuel in the cell
elements that act in cis can ifnelunce expression only of adjenct genes on same dna moecule
studies of merodiploids (partial diploids) in whcih second copy of lac gene introduced helepd show muations in oeprtaor site are in cis and muation in lacI are trands
monod staretd with lac I- lacZ+ and lacY- constitive for b gaaltosade prodiction bc it couldnt synetshzie repressor
inttroduction of an F’ (lacI+ lacZ- and lacY+) palsmid into strain created merodiplod thats phentcally wild type for Z an dY
widl type penotype for meropplic shows that lacI+ is dom to the muatnt
indicuability of not only lacY+ but also lacZ+, menat that lacI protein rpodcues from lacI+ gene on palsmid can bind to opertor on its own chromsome and also to the oeprtaor on bacterl chromsome
so prodyc of lacI gene is trasncacting protein able to diffuse inside teh cell and bidn to any oeprtaor it encounetrs
in anoethr expeirmnt they introuced lacIS plasmid into wildtype I+ that was orgnally both represible and inducible created bacteria that were still represisble but no longer inducible
occured bc lacIS repressor while able to bidn to oeprtor cant bind to inducer
allele encoding noninducible superrepresosr ws dom to wildtype repressor allele
reseachers then egan with lacI+ oC lacZ+ lacY- constituive for b galtsoside bc wildtype reprresor they prdocued coulnt bind to altered operator
introduced lacI+ o+ lacZ- lacY+ plasmid didnt change the affect - cells remained constiytive for b galatcoside prodction even though now inducible for permease
expalntion is that o+ opertaor on plasmid had no afefct onf laxZ+ gene on chromsal dna bc operator atcs in cis
bc it was able to ifnleuce gene expression of lac z- and lacY+ egenes on its won dna moelcue, wild type oeprator on palsmid couldnt override the mutant chromsoal oeprator to allow rpression of geens on abcteiral chromsome
oC muatnt oeprtor on bacteiral chromsoe couldnt act in trans on lacY+ gene on plasmid and so permeas ynsteis was idnculeb ratej tahn constiive
general ruels: if gene specifies a diffsuble eleemt like a protein, that can bidn to target sites on any dna moelcle in cell, the dom allele will ecnode protein that acts in trasn
if a muation is cis atcing, meaing that it oafefcts only the expression of adjacent genes on the same DNa moelcule then the muattion does this by altering a dNA site sucha s the one that bidns a protein retaher than by aletring pretine codng gene
biochem exp3orments for operon thypothesis
monod showed that represson and the idncytion of Z Y and A occur in unison
to explain the coordinate expression of the genes for lactose utliztain, tehy prsoed thatthe trhee genes are tarscnirbed as same polycistronic mRNA
bichem studies showd taht rna pol did intiate trasncirption of linked lac gene clsuetr from a single promoter
during tracnription, the pol produces a single polycistonic mRNa ocntaing lac gene order Z,Y and A
mutatons in promoter affect trasncirption of all three genes
rmbr for eahc gene tranclate form polycistronic mRNa, each open reading frame must be precceeded by its own ribsome bidning site
clusterin genes with simalr functons into oepraonsallows coordinate gene expression
sicnetisst prufied lac repressor portein - veryfy that it does bind phsycally to dna operator
radiactive labeled represso rprotein and bacterial dna with lac oepron
centrifguding showed where the wre
if viral dna contained lac oeprton with oC muaton, pritin didnt cosediment with dNa bc repressor couldnt bind to laetrted opertor site
purified repressor proetin is a dimer of two idnetcial lacI encoded subunits; in some situatns, two dimers of lac repressor can asocite to form tetremaer
eahc subunit contains 3 istinct domains
one rgeionbidns to inducer hwile second reocgnzies and binds to dna at oeprator sites
lacI mutations encoding proetins that couldnt bind to oeprator affected aa in porteins dna binding domain
in constastm lacI S superrepresor muations, encoding porteins that couldnt be idnuced were clustered in codons for aa in indcuer binding doamin
third domain of lac repressor is found at c terminus - inetract siwth the same domain - allows formaton of dimeric and tetrameic rpetisn
dna binding domain of lac repressor has trhee tsrcures - two alpha helcial regions - helix turn helix motif in proteins fit into the major groev of dna
HTH motif found in hudnreds of dna bnidng proetins, not bacteria but also in eukartoic
alpha helixes in hth containing rracnirpton factor carry unique aa that reocgnize a speifc seqeunce of base pairs
so arious hth contsing oroetins that bidn to unique dna seqeunce
in batria it means that diff hth conating tancription factors inratc with diff operators to rgeulate genes and expresson
in vitro technique for dtering wehre regulatory proetins bind tothe dna
purified proetisn that bind to dna proetct the rgeion to whcih tehy bind from digestion by enzymes such as DNase I that break prhsopdiester bodns bw nucelotides
gel eletcropheris of DNa and audroatdgraphy revela bands at positions corresponding to cleavage ebween each case excpet in region where bound proetin proctec the dna
portions of the gel without bands are thus footprints indictaing the ncueltdies of dna frag were proetced by dna binding protein
dna footrpiniting expeirments shoed that some of the ncuelotdies in lac operon opertaor are part of lac operon promoter (wehre rna pol bidns to gene)
means that if oeprtaur site is cocupeid by repressor then rna pol cant reocgnize the pomoter and bind to it - explains why theses a blck in expression of the egens
lac oeprator seuences revelaed by dna fottpronting expeimrnst using lac repressor protein disply the proeprty of rotational symmetry
the two dna stand have idnetcial sequnce when reading in 5’ to 3’ diretcin
makes sense when u condisder that one form of the lac reprrssor proein is a dimer
one usbunits of the diemr formstight conatc with bases aing up half of the oerator seuqnce
the oterh subunit of lac repressor dimer makes face and asociated with the otehr roetaonllys mettcial half of oeprator sequnce
dna footspirning with tetramric lac repressor with lrger frgaments of lac operon dna that icnlde more sequnces upstream of promoetr, revealed that the lac represor protein can bind to three sites
one site is just caleld O1 (site orgnally deincie dby oC muations)
site o1 has tsrngest affinity for repressor and one of simple dimers making oup tetramoer always bding to rationally smetic csuqnces atthe site
otehr dimer in termaer bidns to eitehr o2 or o3
for maximal represison, two dimers so all usbunits of tetrmaer should bnd dna simlutanoesly
looping needed for bidning of repressor to two oeprator sites
bc many dna bidning proetins ar multimers eahc subunit of what has dna bidng domain is assmebled tarcirpton fatcor often has mutple dna bidning domains
if sites towhich mutimeric proteincna bidn to regultroy rgeion fo cluster - many conatcts can be made
increaes tsbalisty of these inertatcins
lac operon and pos control
while glcose is rpesnet,c ells dont turn on expressin of lac proteins een if lactsoe present
lactose doesnt act as idncuer - bc trasncirptonal initation at lac oepron is a complex event
initation deprdents on pos regylator proetin that assist rna pol in start up of arcnripton
nucelotdie kown as cAMP bidns a protein called cAMP recpetor portein
binding of cAMP to proetin nble protein to bind to DNa in rgeualtory region of promoter i
when bound to dna, portein hlps recurit rna pol
when bound to dna, protein hekps recruit rna pol to promoter by making conatct with pol enzymes - the dna binding protein inc ability of rna pol to trancirbe lac gnes
proten acts as pos regulator enhaicng trancirptonal atcivty of rna pol at lac promotor while cAMP is an efefctor whos ebinding to prtein enables his phone
protein ascoiated with cAMP bidns to dna as dimer
dna seunce to which protein cAMP compelx bidns has rotatnal symmetry so protein bidng sites has two reocg sequnces poining in opp directions,e ahc able to bind one subunit of crp dimer
glucose controls amount of cAMp - whne glucose presnet, cAMP is low - but inc when glucose gone
glcuose is presen, little cap availbel for protein
oervall efefct of glcuose in preventin lac gene trascnription is catbolite repression bc presence of prefefrec substarte for ctaoblism (glucose) represses tracnripton of oepron
crp portein works as a regulator for many otehr systems like gal oprson and ara operon
muaton in gene encoding crp that alter dna bidningdoomain of proteinreduce ttacnription in mnay oeprosns
having oeprosn relies on atcivty of two diff regulators - lac repressor and cpr in cas eof lac oepron - that respond diff to envrimental cue inc range of gene regulation
pos and neg regylatros have opp efefct of translation - most rgeualors of both type work through afefct on rna pol
neg regulatros prevent initaion by blocking fundonal binding of rna pol while pos rgeulatros etsblish conatct of rna pol to prmoetr
repressor.efefctor inetratcion enables represisble regulatoion of trancirpon initaion
muktple genes in pathwya clsuetred togetehr in operons in bacteria - true for ctabloic and anabolic
catbolic lac oerosn repodns to rpesence of lactorse by inducing expresison of lac gene
anabolic operosnr espond to rpesence of patwhay end prdict by shutting down expression - repressible regulation
many anabolic operons involved in production of aa
e.coli trp oeprosn - 5 ggeens E,D ,C, B and A neede dto produce tryptophan - max expression when its absent in growth medium bc anabolic repressible
trip oepron contorlled at level of transcipt iniation through action of reprsor portein that sprodict of trpR gene - on difif chromsome
trypotphasn functons as effector for trpR repressior an allosteric protein
biinding of tryphan to trpR causes trpR to change shape biding to oeprtor and stoping trancirpon of trp oepron
mutatio in trpR that changing bidning domainto dna or triphan stops trpR represor ability to asosciate with dna causing constitive expresison of trp gene even when its rpesnet in meidum
19.1 Overview of Eukaryotic Gene Regulation
trasncriptional regulaton occurs through atatchment of DNA binding proteins to specific dna seqeunces that are in vaicnity of trasncirption unit itself
but more leevls of compexity are presnet in controlling expresison in eukaryotes bc:
chromatin stcture makes dna unabilble to transcirption mahcinery, additional rna processing occurs, transcription occurs in nucleous while transaltion in cytopalsm, polysisctronic trasncripts dont exist, gene regulation needs to control cellular differentiation into hudnendrs of specilzied cells
gene expression in eukyrotes can be regulated at time of tranxirptional initation, when rna pol mkes transcript
many steps in rpcoess of expression exist beyond inttaion
trascnirpt processing, export of mRNA, trasnatibilut and stability of message and mdos to protein after are all things that can be afefcted to change amount of active protein
19.2 Control of Transcription Initation through ehhancers
three types of rna pol trasncribe genes
rna pol 1 tradcnirbes genes that encode ribsome rna
rna pol 2 transribes genes that encode proteins
ran pol 3 transcirbe trna and otehr small noncoding rna
promoters and ehnancers are major cis acting regulatory elements
although each regulatory region of the tjosudands of pol2 transrvibes genes in eukaryogic genome is unique, genes that are trasncirbed in cell ttpe specific manenr contains two essnetial dna sequences
the first is promoter with TATA box of seven nucelotides located upstream fo trasncription initation site
as it atatracts rna pol weakly on its own, tata box allows a low, basal level of trasncription
second type of dna seqeunce elemnt improtant for trasncirption in eukayrotes is enhancer: regulayory siste thats distant from promoter
in specific cell types, bidning of proteins to ehancers cna augment or repress basal levels of trasncription
ehnacers located at either5’ or 3’ end of tarscnirption strat site - some even in introns
single gene can have multipel ehancers or just one - one has multipel bidning sites ofr diff trasncirption fatcors
scinetist use gene from jellyfish that encode green fleurocent protein GFP as reproter to dineify enhancers in eukaryotes
contrustc a recomb dna moelcule with regulatory seqeunce of gene of interest is fused to GFP gene coding sequence
this recomb dna then used to geenratore trasneginc organism
only if the DNA frag contains an enhancer that directs trasncription in a partuclar type of tissue reports express detetable leevls of GFP
GPF flueorexes green
tissue specific enhancer can be idnified if that tissue glows when the trasngenic animal iluminated witg light
one interesting outcome of reporter gene expeirments is teh finding that ehnancers can function even if orientations or psoitions relative to promoter are changed
proteins act in trans to control trasncirpton initation
binding of proetins to a genes promoter and enhancer control frequency of trasncirptonal initation
diff types of proteins bind eahc of the cis acting regulatory regions - basal factors bind the promoter while actviators and repressors bind the ehnacers
we use term trasncripton fatcor to descrie only seeunce dspecific dna binding proteins that fincue trasncripton (basal fatcor, actvitor or repressor)
one tracnripton factor binds to dna they recruit additonal proteisn that can inflcune trasncirption
basal factors
basal factors assisst binding of rna pol 2 to promoter
component of basal factor complex that forms on most promoters is tata box binding protein or tbp
this trasncirpton fatcor interacts with the tata box at prmter
TBP recruits other proteins caleld TBP ascoiated factors to promoter
once the basal fatcor complex is formed, rna pol can initate low level trancirption
riamry sequences and trhee dimensional structrues of basal fatcors are conserved in eukarotes
mediator
trasncirpton of many eukaryotic genes needs mediator which contains 20 promteins at least
mediator doesnt bind dna directly but serves as bridge between rna pol2 complex at promoter and actovator or repressor proteins at enhancer
activators
although similar basal factor complexes bind to all promoters of the many genes of eukayrotic genomes, not all genes are transcirbed in a cell type
a range of trasncirptonal rgeulatoon occurs trhough binding of diff trasncirpton factors to distinct enhancer elements asociated with diff genes
when bound to ehnancer element, trasncirptonal ACTVTORs inc trasncirpton above basal leevl
actvrs can inetract directly or indriectly with basal factors at promoter in trhee dimensonal protein/dna compel;x to inc tarsncirptonal activty
bc ehnacers can be far from promoer, ena between enhancer and promoetr loops as consequnces of inetractions between basal factors, mediator and actvators or repressors
long stretches of dna within loops are flexible expalining why ehancer sequences can function if moved to a diff spot
actoavtor proteins bind their target sites on dna and inc rna synethsis by two ways:
- activatros help recurit basal fatcoes and pol 2 to promoter sqeunces by inetracting directly or indetcly through meidtor with components of basal rna pol 2 complex
- activtors recruit coactviators- these are proteins that open local chromatin strctrue to allow gene trascnirpton - promoter dna thats covered in ncuelosomes (closed chromatin) is inaccesible to basal fatcors - for gene to be rrancribed the prmoter dna has to be freed of ncuelosomes (open chromatin)
two types of coacvtiatoes open chromatin - histone mod enzymes and chromatin remodelling complex
histone mod enzyme include HATs, HMTs and histoene dymethylases
chromatin remodelers contain ATPase subunits and use enegry of atp hydrolysis to dispalce nucelosomes from promoter
actvitro proteins can help attarct eitehr type of coavtiator to promoter
scinstist think that actoravtros bind ehnancer dna just after dna rep or that actvitros can bidn to dna even when bound in ncuelosomes
trasncriptonal actvior proteins bind enhancer dna in sqeunce specific way and afetr binding they inetract with otehr proteins to atcviate trasncirton
two structrual domain of actvitaor protein is the dna binding domain and actviation domain - mediating the two biochem functions
trascnripton factors belong many prtein families with siimalr dna bindin domains distinct from those in otehr fmailies
two characterized dna bindng strctures are ZINC fingers and HElix turn helix domains
zinc finegrs found in eukarytic rpotein but HTH factors in prokaryotes too
dna binding domains fit within major groove of dna
subtle differences in aa asequences among actvitors of same fmaily allow tehse proteins to reocgnize specific dna seuqnces at diff enhancer elements
actvitaion domains of trasncripton fatcors are less characetrized than dna binding domain and elss strcutured
aa sqeunce of actviation domains depend on whetehr activator inertcts with basal complex or with coactvtaors
many actvitor polypetides also have a third domain - dimerization domain - enables them to inetract with otehr copies of same polypetide or with trasncripton factor subunits to form multimeric proteins
one strcytrual motif in dimerization domains is a leucine zipper - a helix with leucines at regualr inetravals - abiluity of two elcuine zipper proteisn to interlock depdens on species aa between leucienes
best charctized trasncription factor along iwth lecuine zipper is Jun, a protein impprnat for cellular polfieriton and otehr pcoresses like loss and regen of euterine endrometrum duirng menstrual cycle
JUN can form dimers with istelf, Jun Jun homidmer or with otehr proetisn called Fos making JunFos heteroimzers
JunFos system can produce only two types of tarsncripton factors - jun jun protein or jun fon proteins
these two dimers bind to various enhancer sequnces with diff afinities
repressors
eukartic trasncritoon factors that bind specific dna sites near a gene like ehancer, and prevent initation of trasncirtpon are repressors
primary function of repressors is to recruit corepressor proteins to enhancers
corepressor can bidn to dna on its own and needs to repressor
corepressors have two functions:
- some inetract directly with rna pol 2 basal complex and prevent it form binding to promoter
- otehrs are enzymes that modify histone tail aa, resulting in closec chromatin so HDACs and HMTs and histone demethalses
repressor strcutrues are simialr to actviators: repressors have DNA binding motifs, repression domains for inetracting iwth corepressors, and some dimerization domains
certain trasncirpton fatcors can act as either activtor or repressor depending on teh context
ex. dorsal factor is actvitor when buoudn to ehancer in some geens and repressor at enhancer of otehr gens - dorsal is intsrinclly actviator that bidns to coactiavtor some at some enhancer it inetracts with otehr tarsncirtponal fatcors resulting in recrutiment of corepressor Groucho
indirect repression
many regulatory proetins called indirect repressors, prevent trascnripton initation indrectly, not by recrutiing corepressors but instead by inetrfering iwth the function of actviators
in such a mechnais, some repressors can compete with actvuatros for acess to an enhancer bc teh bidning sites of repressor and activtaor overlap
in anotehr form of indrect repression - quenching - protein can bind the actvtaion domain of actovator bound at enhancer and prevent actvitor form functioning
some infdirect repressors bind to actvtaors and hold them in cytoplasm - post trasnaltional mods of indirect repressor causes it to release actvuator which can then enetr ncuelos and bind to target enhancer
some indiretc repressors can form heterodimers with activators - if only actvitor homodimers can bind dna, the idnirect represosrs can titrate actvitros so that few homodiemrs are able to form
histone mods
correlations exist between gene tarsncirtpon and various covalent mods that can be added to specific aa in N terminal tails of hsitone proteins in nucelosomes
for trasncirpton, most imprtant mods are acetylatio and methylation - addition of those groups
acetyation of partcular lysine aa within histone tails by enzymes called histone acteylatransferass HATs favors gene expression by he;oing clear prmoters of ncuelosoems -mkaing open chromatin
many trascnirton factor coavtitros are HATs
HATs help open chromatin in two ways
- lysine actetlaton reduces the pos charge on histones and lessen interactions with the neg charges dna
- histoen tails with acetylates lysins act as landing pads to recruit proteins - cna be HATs that acteylate other tails or DNA remodelling proteins that use enrgy from atp to rmeove histoens from dna
methylation of lysines or arginines in histone tails by histone methyltrasnferases HMTs can actviatr or repress trasncirptn depnding on partcular proteins that the methylated site recruits to ncuelosome
certain methylated aa bind factors that open chromatin while otehrs bind proteins that close choramtin
so some HMTs are coactvitors otehrs are corerpessors
histone actetlation and methylation mods can be added rapidly by HATs or HMTs and taken off rapidly by histone deacetylases or histone demthylases
speed of adding and rmeoving is key to dynamic repsonse of trasncirton regulation
histone mod enzymes cant bind dna sites in genome on their own so histone mods initated by seuqnce specific dna proteins - tarscnirton fatcors
this has consqecnes when cells in tissue divide and duaghetr cell needs same histone mdos are parent
most mods ar elost during dividion bc nucelosomes are rmeoved at replciation fork
but trancirpton fatcor canquickly bind to newly replciated dna and they can recruit hsitone mod enzymes that estabslih patetrn of hsitone mods
indentfying tracnritpn factors
scinetsit often use GFP reporter genes to very trasncirton factor syspected of regulating expression of target gene plays that role
two reequriements:
- researcher must constrct organism with trasnegen containing enhancer of target gene fused to GFP reporter - GFP expressed at high level in approp cells then
- second reuqiment is that mutations in gene encoding the trasncirtpon fatcor has to be availble - if anymal with GFP reproter has loss of function muation in gene encoding actviator, reproter will no longer be expresed if actvitor in fact inetracts with that enhancer
by contstarct loss of function muaton in gene encoding a repressor may yield higehr leevls of GFP than normal or cause it to be in annaprp cells
13.3 heterochromatin and euchromatin have diff histone mods(only)
several inetrdepdnet mechnisms govern distinction between active euchromatin and silenced hetrochroamtin
mods to histones in ncuelosomes ar eone thing
the N terminal regions of the four core histones form tails that extend outward from ncuelosome
enzymes can add serval diff kinds of chem groups to various aa along tails while otehr enzymes can remove groups
such mods of histone tials can infleunce packing of nucleosomes and thus the mod tails can also serve as platforms to hwich chromatin modfiier proteins can bind
the histone tals of ncuelosome core potentially can be mdofiifed in 100 ways
two best dunerstood histone tail mdos are acetyl group to lysisne (acetylatin) and addition of methyl groups to lysine and argainines (methylation)
lysisine acetylation accomplished by family of enzymes known as hustone acetyltransferases HATs ooen chromatin by preventing close ppacking of ncuelosomes
histone acetylation favours the expression of genes in euchromatic regions bc promoetrs in open chromatin which is accesible to rna pol
acetylated lysine on hsitone tuals serve as bding sites for HAT enzymes thus faciliating spreading of hsitone acetylation to neighboruing ncuelsomes
Histone deacteylases HDACs are enzymes that remove acteyl groups resulting in closed chromatin and repressed tarsncirtion
methylatoon of histone tails is complex and can clsoe or open chromatin dpending on partcualr aa methylated
enzymes that methylate histone tail aa are histone methyltransferases HMTs and enzymes that revese it are histone demthylases
one of the geens whose loss of functionmutant alleles act as PEV suprressors in drophila code for HMT enzyme that adds methyl groups to specific lysine in H3
this methlation marks chromatin for aassembly into hertochromatin by providing binding sites for hertochromatin specific proteins
methylation of hstone H3K9 is comon feature of chromsal rgeons with clsoed chromatin that are trasncirtonally silent
diff drosphila gene whose loss of function allele also surpsress PEV encodes HP1 a key hetrochromatin protein that binds to histone H3 tails contaiing methylated K9
HP1 protein promotes chomatin compacton into hertochromatin in two ways
it self ascoiates and this helps bring adjecent methylated ncuelsomes toegtehr
second, HP1 bidns same HMT enzyme that adds methyl groups to H3K9 - teh recruite dHMT can methylate k9 on histone H3s of adjcnet nucelosomes
this autcatlysic affect gives epxlnaton of linear spreaidng of hertochromatin in PEV
