lecture 2 - 9/19?\

recap = talked about gene and gene regulation and differen types of sequence elemtns such as the core promotoers that are in eukaryotic system and multiple types. they are sequence elemtns where genreal tracnriprion factors bind and recruit polumerase enzymes and catalyze the. trancirprion reaction.

There is the tatabox core promotor , strong code promotoer and large number of virual genes utalizes as their core promtooer. the sequence of the tat is 20-30 nucleutides upstream of the trancirpiton start site

we have the intiator element core promtoer where is a sequence next to or overlapping the trancipriron start site, present in multiple genes but not as prevelant as the last one which is the CPG core promotoer which is about 70% of the human genes in our body.

CPG can be up to 1k nucleotides where the GTFS bind to the both sense and anitsense strands and could potenital by ue the core promoteor becuase the. sequence directionaly meaning they can use both direction.

cell known which strand is coding for a protien coding gene and the other side can be degraded because even tho the polymerase can attach and code in the oppoisite direction it isnt protien coding and will be degraded.

the strand will be coded and make a mRNA or a nRNA the anitsensory will produce trancirps that are up 1.5k long (so the other side of the cpg island that is trancriped in the anti sense strand and isnt a coding gene for anything ). and then the tracutp will be degraded fast. so they dont make it past the 1.5 kb.

there are genes in which both strands can code for funcitonal rna moclules. some is just one strand and some is both strands.

enhancers are a stetch of dna sequence present in the sequence that controls the expression of genes. they are regulatory promtoers and enhancers are the same. enhancers are more prevalent in the genes which require differenital expression in repsonse to a specific singal. genes that need to be activated in reposne to a speicifr signal will have have enhancer sequences,… talk about this more later.

since CPG island core promtoeora re housekeeping gene promoters, present in every cell type, scientest though that enhancers may not be that inmporatn for the activting of genes in the CPG island. this isnt true. these core promtoers can also be regulated using enhancers.

this is studied in vitro analysis. sceintest used the tata box as a model system to study trancirption because virual genes use them. we know more about the tata box core promoters. if you look at the sequence you have the dna sequence, and the group of protiens that recognize that region.

Tata bidnign protien TBP. this is the first proteins that recgonize the tat sequence elemtns on the core promtoer that is part of the gtf complex. the. TBP binds to the sequence and then bends the dna / a kink and changes the dna sequence tohelp load other GTF onto those sequences. tagain this TBP is one of the first proteins to recognize the tata box sequence. This is part of the TF2D complex. general tranciprion are named (Tf2a,b,d,c… etc)

TBP is part of this TF2D complex. just one of the many proteins involved. once it is loaded there are other TF2 complex that are recruited, (dont remeber what TF2E or a does not importnat ) … at some point the RNA polymerase 2 will get recruited . RNA polymearse two is a multi protein complex and use the TF2D as a landing pad.

now rna polymerase 2 has to recongize one of the dna strand (cuz is double stranded) and it will attach and open the dna to recongize one of the strand and activate trancipritn. one of the general tranciprion factor complexs(remember this one) is called TF2H which has TWO imporatn funcitons. the first one is to open up the double stranded DNA near the region of the core promoter so it will become two single stranded dna . these are called tracription bubble. so there is no hydrogen bodning between the double strand. TF2H has a helicase activity that will break down the hydrogen bonds, once bubble is formed the poluymerase can recongize the sequence and start trancipriton. The second function is that it will trigger trancirption which is controll by the TF2H. … this has a helixase activity as well as a kinase actiivty that can phorylate specific residuyes within the polymerase 2 complex. this rna pol2 is made of 12-14 poteins togehter and those proteins have a structure called CTD carboxy terminal domain. this is a strecth of amino acids part of one of the subunits of rna pol2. CTD will be phoryalted by the kinase of the TF2H and it will trigger for pol2 to start trancirbing. TF2H has helicase and kinase activity.

MORE about pol2 =

pol2 is a enzyme, trancribes the gene, multi protein complex, largerst subunts at the c terminal has extended amino acid stretch, it is part of the protien, it is the lat stercht of amino acids called CTD> it is seven amino acids repeated 52 times in humans. 7 × 52. this is a conserved structure present from yeast to human, with the same 7 amino acid sequence, but in yeast its repeated 26 times. if u delte this domain it impact tranciprioal activity, it is unique to rna polymerase 2. CTD isnt there in the pol1 or 3. one of largers poly 2 is called RBP1, CTD is part of the RBP1 which is unique to pol2, but absent in pol1 and 3.

the sequence is YSPTSPS

these can be phoylated . second, fifth, and seventh postiion has a serine. then there is a tyrosine, trionine, and porliens. they can also be depshoylated. the TF2H is a gtf that phroyalted the CTD phoylates the serine 5. which is repeated 52 times gets prhoyalted by the TF2H enzyme. till the time the pol2 bind to the core promoter it will not be active till that phorylateion happens. that triggers and changes confromation of pol2. this is called intation competntn form of pol2 which is serine 5 phorylated.

serine phroylation of serin 2 is done by another different kinase. this i scalled serine 2 phroylated pol2 as elongaiton compentnt, during elongation stages of the polymerase this needs to get phorylated.

they looked at pol2 phroylated over time as it is going through a gene. the serine 5 phroylation statuse declines, and serine 2 increases. so its an inverse corelation between the serin 5 and 2 phroylation. so as the pol goes towards the geene body the serin 5 will decrease.

the core promtoer si sitting next to the gene and essential for all genes, and for GTS to bind.

enhancers are sequences that are similar to core promtoer but they recruit dif family of proteins,

regular promtoers and enhancers are the same and they are 10-15 nuceltodies long and were idetnfied in yeast and located either upstream or downstream of the gene. they are close within 100 to 200 nuceltoide long. when scienst looked at mammalian they saw similar sequences but they are farther, . this makes sense because the yeast genome are smaller so they need to be closer and now enhancers and core promtoers are similar.

what are the genreal geaturs of enhancers, enhancers can either be upstream of the tracnription site or downstream. there are enhancers of a gene can be present several thousand kilobases upstream od downstream of the gener, there is no directionality. so they can be present really far or close and still influcen activity of the gene. enhancers cna infleunce the trancirptioal state of a gene either psotively or negatively. it can activate trancirpion o the gene from the core promoteor or inhibit.

enahncers also recgonize a group fo rpoteins that are Tracnirpion factors. the ones the bind to core promtoers are called GTF’s general. and trancirption factors bind ot he enhancer sequences. the old name is RTF. regulatory tranciprion factors. in humans there are thousands of trancrpion factors, there are thousands of genes that make proteins and all of them funciton as TF’s. a major level of tranciprionl complexiity is the differential expression or bidning to the TF to specific gene enhancers. when a ligand binds to a receptor that activates the signling events and activtin the subset of genes and the product of the geens activate a particular process, most of the time those genes are either TF’s or GTF’s . a group of TF will be made in reponse to a singal and will bind to enhancers of those genes in which those cTF’s can bidnd. and either activte or repress tracurpion. so controlling of TF’s cells can controll expression of genes inside.

Remmeber NFKB. it is a tf, not a gtf, and will bind to an enhancer sequence of specific genes so that it can activate the expression of that genes. P53 is a supressor geen it is a TF that bind to enhyancers o fspecrfic sequences of genes.

TF binds to enahncer sequece.

TF2D is a GTF

TF’s have to have adna bidning domain that is a strecth of amino acids which can bind or recgonzie a dna sequence , dna sequence within the enhancer. so different TF’s will have dif dna binding domain which can bind or recongize dif dna sequneces. it also has to have an acitivation domain. this is the domain that activates funciton of tranciprtion. (some TF’s repress so the activation domain can either activate or represson) it has to have a activation or repressor domain. and also a dna binding domain. many of the tf have multile activation domains. some can have three activations orrrr three repressor domains. but one dna bidnign domainl.

there are dif dna binding domains in dif TF’s.

TF’s are modular in funciton meaning they have two important domains , if you take the activation domain and then put it with another dna bidng domain of a protein and can funciotn indepently. so the activatio domain and the dna bidnign domain are not interdpeendet when it ocmes to a funciton. o u can cut the activation doman and the dna domain and make chimerase of different comination and they will work without any problms. if u take the activatio domain with the repressor domain and have dif dna bindign domains, the new chimera the repressor will activate and the activator will repress… ppl use this emcahnism in flies and other systems to control the expression of genes.

some factors have multiple activation domains like p65 which is a componenot of NFKB. two activation domains and onw dna bidnign domain. the activation domains role. activation domains can help recruit GTF”s onto the core promoteors. it interacts with GTS and brings high ocncentration of them to the core promoters. it increases associateion of GTS . it can also recruit other protein that influence the expression of the gene. So now we know when it comes to differential egene expression enhancers play a key role.

many of these enhancesr allow the bidning of multiple TF’s, so enhanvers can have ten different tracnirption factors. this is called combinatorial control. if it brings ten TF to the same stetch which is small , lets say 50 then each TF will have 5 nucleotide binding domains. now each of these TF have its own actiaion or presorrs. so i t can recruit more factors to taht region and control expression of the gene domainstream gene or expression gene from the core promtoer.

Scientst hav learedn that many times cell may require. more tha one tF to ocme to one play in order to activate the expression of a gene. u have multiple TF come together to stabilize eachother to bind to different sequences within the enhancer and bidn to eachother aninteract witheacother through proteo tein. interaction and stabilize achother. . cell may activate the expression of one tf which can bind and actiacte the expession of a downstream gene, maybe i would say activation level is one and then in repsonse to a dif singal it can recuit. two TF there and the level of the activation of the downstream may go from one to five or one to three so you can not only activate or inactation expresssion by controlled the levels and ocntrolling the type of TF which can be syntheized or brough into the cell . the experssion and the elevels of the expression can be controlled insise the cell. Its like a clock system by adding more TF u can chance expression levels of a speifci gene.

same thing to repressor.

example of enhancer - of a gene which is immune gene caleld interfrom INFB . this gene contains enhancer tha tis 50bp long . in order fo the gene to be activated it need specific TF to binda nd stabilize the GTS to load and activate pol. this is a immune reponse gene, it also activated by NFKB, it is a NFKB target.

it goes into the nucleus and binds to the enhancer and then it will get stbalize with the help of other TF and activates the trancirpion oft the gene INFB. It needs 8 TF’s, some sequences can overap but not compeltely. when it comes to say in the case of human cells mamilain cells, you have a core promoter clsoe ot the trancirpiton site, wehter its tata, intaition, or CPG island. in the ase of the CPGN and intation element they are basically overlapping with the sstart site . and TATA is 30 nucleotides further but still close. enhanvers can be closer upstream or downstream. there are enahncer sequences that recruit TF and then the TF funciton to stbaiize teh FTS on the cor epromtoer, a protein cannot be that long or big that TF sitting in 1k away from the core promtoer and the TF can phsycally interact. thats too long. its so far it cannot phsycially interact with the GTFs. so there are other mechanisms that. are mediators. there are two major things that cell does, is a group of proteins called mediators. are a conserved group of rpoteins from yeast to humans that are 30 protein complexes. its a specific type of structure, meditor complex is always physcially attaced to the RNA pol2 .

mediator and RNA pol2 are called holoenzyme , mediator and pol2 togehter. mediators and pol2 are always together as hollow enzymes. mediator , 30 protein complex, cna specifically itneract with specific domains within the activation domains of the TF’s. TF bound to the enancer sequence whihc has an activatior/repressor domain, and some of those rregions can bind to regions of the mediator complex. TF1 activationdomain may bind to MET6 or head group fo the mediator complex. others can bind to MET11 or other part of the mediator complex. medator is essential factor, not every 30 members may be funtion the activatio of ever gnee each mediator can be controlling88 the expression of specific genes by recruiting or stabilizng interaction with specfiic subset of TF so u can delete on of the mediato complex form the 30 of the them and. cell will be okie and reugaltes subste of genes not all of them so many genes are expressed normally but some genes will not be express becausei t bacially connects a TF factor a speifici tintersin that conect right remember that .l

idk

this sytem regulates expresion of a subset of genes but not all of theme so many genes can be expressed nromally btu some wil not be expression because it basically connects a TF of a spefici interst in that contect .

you have a group of a enhancers or TF havtors bind but hte dna sequence can look,

several enhanvers for one gene and each one can control expresson of the core promtoer postively or negatively and the distance can be really far away so the dna can loop and the enahncers are now sitting clsoer to the core promtoer and the mediators are interacting to different TF with different mediator compoennts. each emdiator compoentn can itnearct with speficity type of tf.