Eukaryotic Gene Expression Regulation PDF

unit 5 : regulation in gene expression in eukaryotes * difference between...

unit 5 : regulation in gene expression in eukaryotes * difference between prokaryotes and eukaryotes ↳ prokaryotes gene expression · regulated by operon b of collection controlling sites adjacent to polysistronic protein coding sequence beukaryotic gene expression · regulated in units of protein coding sequence and adjacent controlling sites > - no operon * categories of eukaryotic gene regulation ↳ short term it · depends on when is needed are turned and off in response to the environment and demands > - genes on Glong term · genes involved in development & ex 2"x" chromosomes one is completly turned off until we die e. , copy the when can not working copy works found in cancer cells A transcriptional regulation ↳ cis-acting elements · about DNA > ex -. TATA box , GC box 3 trans-acting factors I attaches to DNA aka cisacting elements · about protein Control gene · expression in several ways : > - expressed in specific tissue > - expressed at specific time in development > - required for protein modification > - activated by ligand binding  eukaryotic gene control * region G consists of a promoter and cis acting regulatory DNA sequence * promoters Crecaps ↳ upstream transcription initiation site ↳ Some show where transcription begin · ex. TATA ↳ activated by specelized transcription factors · specific ifs bind specific promoters ↳ more than one can be present for any given gere ↳ can be positively or negativly regulated & regulatory DNA sequences ↳ upstream or downstream transcriptional initiation site ↳ shows frequency of transcription 3bind specific enhancer eulatoryproteins seesee ↳ loops can form in DNA attached to its - the ability to bend DNA makes the loop ↳ includes · enhancers] cis element/DNA > - promote transcription > - associated with activation ]-> trans element/protein · scilencers] cis element/DNA - decrease transcription rate > - associated with repressor]s trans element/protein ↳ interactions of regulatory protein is what determines if transcription is activated or repressed * mechanisms of action of activators and repressors ↳ activator · shows transcription frequency ↳ repressor · protein that stops expression of a gene > - prevents transcription]> binds to operator site in DNA > - prevents translation It binds to RNA 3 positive control · genes under it can't be expressed unless attached to a positive regulator ↳ negative control · genes under it are expressed but needs intervention to get turned off upstream closer a + t for * gene control region a eukaryotic gene b one or several regulatory sequences can be several down Stream regulate the expression of one single gene between 2 exons · so one promoter and multiple regulatory sequences * locus Control region (LCR) ↳ eis regulatory element 3 one LCR controls several genes · eX : & - human B globic LCR first embrgo's blood is made of epsilon and as it grows only beta remains LCR growth E first > - human hormone expressed in the placenta but over time it's expressed in the petutary glands * mode of action of transcription activators ↳ activators · it allows formation of preinitiation complex faster · how ? > - it assembles faster because the transcriptional factors don't fall off but close to the activator stay very > - it speeds up the transcription · large # of activators bind to Gifs like TfIB , TFIID , TFIll called characteristics A transcription activating mains of activation & ↓ DNA binding aka activator ↳ 3 kinds of protein domains are involved in transcription activation] ↳ these domains interact with components of the transcuption complex I · acidic domains chains · · > - amir acid side are acidic # 2 glutamine rich domains recognition · > - 25% glutamine in the sequance sequance are different 3 proline rich domains ⑭ A proteins regulating eukaryotic mRNASynthasis 3 RNAP binds to promoter region by interacting with tell's ↳ TFs recruit histone acetylase (HATs) · recruits it to the promoter I What # interacts with proteins at the promoter called and > - process stabilize the formation of functional preinitiation complex ? transactivation ↳ ifs binding to other promoter elements and transcription elements what determines the rate of initiation of transcription ? # stability and frequency with which complexes are formed * eukaryotic gene activator proteins 3 promote assembly of RNAP and Gifs at transcription initiation ↳ activators increase rate of transcription · have a bunch of functions attract , position , the GTFs mediator RNAP at promoter > - and modify , and activators #why begin do do what do they ? to transcription # how do activators act ? ↳ by : these > - acting directly on components > - changing the chromatin structure around the promoter activators Some other * bind directly to one or more * interacts with mediator and GTFs attract it to DNA this accelerates their this starts of > - > - assembly promoter RNAP and Gifs at promoter assembly on activator proteins * gene ↳ they work Synergistically RNA when 2 activators are added togather and have more Altogether than the amount original * mutations affecting promoters -example discussed is hemophelia B sex linked disease so in boys (on x chromosome only · · transcribed region is less than 32 , 700bp with 8 exons · caused by factor a (X) gene > - it is responsible for coagulation] > - blood clotting ↳ factor has Sites for AR HNF4 IX gene promoter overlapping binding and AR · = androgen receptor > - binds androgen > - androgen levels increase at pubirty · HNF4 = hepatocyte nuclear factor 4 known > doesn't - have ligand so called an orphan receptor development adult liver > - expressed early in and in ↳ site forHNF4 blue block is the binding mutations at -20 : > - result in Memophelia Bleyden > - hemophelia improves at of androgen pubirty via increase + A before e Is after pubirty pubirty mutations at -26 : > - result in hemoprelia B brandenburg > I factor after - stays low pubirty  mechanisms of inhibition of transcription & by eukaryotic gene repressor protein # what does the eukaryotic gene protein do ? repressor regulate transcription of their genes 3 mechanisms ↳ · competitive DNA binding the activation surface · masking · direct interaction with GTFs * methodologies used to study promoter ↳ promoter analysis : progressive deletions · analyzing an unidentified promoter by using betagalactisidase activity (la(2) usually in plant genes affected by light and the results > - and are viewing general element cut > a is region affected by light element is cut represor present & repressor basel is removed promoter which is is removed why that's why the region affected there is no activity is back ↳ promotor analysis heterologous promoter : · conforming the promoter by removing/chopping 1/2 of the sample and how observing is it affected by light ↳ aftern - I the gene new with new promoter is not affected by light promoter fused with the last part of heterozygous promoter and response to light element ↳ electrophoretic mobility shift assay called shift · also get assay · used for detecting protein nucleic acid interactions > - not labeled Je shows that there is DNA protein interactions bind to DNA meaning a protein will it - [ ~ TyDNA means is radioactive so it is seen and no protein 3 deoxyribonuclease 1 (Drasel) footprinting · deoxyribonuclease Cuts DNA ↳ · Dnasel ↳ doesn't cut low concentratin where protein is present one ↳ an enzyme in large amounts cuts everything so just a is needed for it to cut in each DNA we needit to cut lbp at a time to find the PBS > - protein protected sequence so it is easy to identify the protein binding sequence (PBS) ↳ analysis of protein-protein interactions 2 hybrid assay : called yeast two hybrid of distinctive features like · assay because : ↳ Gal4 BD (binding domain) and Gal4 AD (activation domain) ↳ proteins are labeled as "bait" and "prey" ↳ standard terms in the yeast two hybrid assay ↳ reporter activation gene ↳ if the bait and prey are interact , then the interaction dependent transcription of a reporter gene is activated ↳ use of plasmid constructs Step 1 Synthesizing constructs from library : · a proteome I paint construct means structure - ↳ first the stuck to DNA Gal4 BD bait protein is binding domain - ↳ next this structure is introduced into a plasmid that acts as the "bait" in the interaction I it draws the plasmid in prey construct 2 ↳ first proteins from the proteome prey proteins the library are used as ↳ next each prey protein is fused to a transcription activation domain > - Gal4AD ↳ these structures are introduced in the plasmids to make prey # and #2 prey Go Step proteome for potential interactions 2 : screen library shows if there interaction between beit and is an prey protein ↳ works by monitoring the transcriptional activation interaction Ibait-prey is present mean reconstituted ↳ if they interact the Gal AD and GalvBD are brought together ↳ next the reconstituted Gal4 transcription factor binds to promoter and activates the transcription of reporter gene Limactivation shows positive interaction 2 no interaction present ↳ if there is interaction between bait and the GalyBD and no prey Gal4 AD stay separate ↳ because both domains are not working together the promoter is not active and no transcription of reporter gene occurs ↳ this shows negative result