FUNDA BIOMED SCI 1
FUNDA BIOMED SCI 1 GMS 6001
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Date Created: 09/18/15
Eukarvotic Transcription II IDP Fall Semester Dr Robertson Regulation of Eukarvotic RNA Polvmerases and Transcription Pol Transcribes rRNA genes 288 188 588 RNA genes accounts for 8090 of total cellular RNA mass Pol II Transcribes proteinencoding mRNA s and several nonprotein encoding genes consists of 12 subunits that are conserved among diverse eukaryotic organisms Pol III Transcribes tRNA 5S RNA 78 RNA component of signal recognition particle genes and other small RNAencoding genes including genes encoding RNA s involved in splicing tRNA and 58 genes have internal promoter ie within body of gene Transcription Factors Proteins and RNA s that regulate positively or negatively transcription initiation Can act via sequencespeci c DNAprotein interactions or via protein protein interactions Many transcription factors bind to cisacting regulatory DNA sequences regulatory elements in DNA associated With genes Includes general transcription factors upstream activators enhancer binding proteins celltypespeciflc factors coactivators etc The Modular Nature of DNABindinq Transcription Fgctors Yeast Gal4 protein GAL4 AMINO ACIDS L 39 9 5 79 39quot339 Involved in regulation of galactose catabolism N I ll DNA Binding 39 39 39 ran I l lens quota 1 1 m Upstream transcriptional activator 353312quot 39 Rel Sequencespecific DNAbinding protein A 39d39 t39 t I on Io ac Iva 0r spWWVAVN A39AVAV AnnUWK AL I IVATORS v GAL 4 amphipathic I r 11 helix AH 3 ll I n Other ectivetion motifs ynum m m 3 mm W N N A T39VAT RS Prolinerich scrambled Glutamine rich heiix SH Cofactors CoactivatorsCorepressors Not required for basal level transcription Mediate communication between activatorsrepressors and GTF s Do not show specificspecific DNAbinding activity Required for function of DNAbound activatorsrepressors Examples E1A CBPP300 RNA pol ll TFllA 5 E TFIID F G r TGACGTCA TATA Figure 9 Model for Transcriptional Activation by Large E1A Protein Multisubunit coactivator complexes Examples Mediator complex that binds pol ll CTD and activates transcription SAGA CoActivators and CoRe ressors A IINL IESQLJDJWHGCNI B MDNH COFB Tressor coactlvator ACTIVATES p REPRESSES RANSCRIPTION TRANSCRIPTION v IIII EZl GENE ON GENE OFF ACTIVATES coactivator r S ACTIVATE COaCt39Vatm TRANSCRIPTIDWRANSCHIPTION 1 QI jl EI GENE ON GENE ON Figure 7 50 Molecular Biology of the Cell 4th Edition Multiprotein complex facilitates interactions between GTFs and activators Purification of yeast Mediator activity yielded a single complex containing pol ll SRB suppressors of pol ll CTD truncations and many other polypeptides Malik amp Roeder TIBS 25 2777 2000 How the mediator complex works Malik amp Roeder TIBS 25 2777 2000 Al I t H l TFIID b RNA polymerase holoenzyme TFIIA RNA A olvmerase TATA activation mediator domain Bl I i Figure 743 part 1 of 2 Molecular Biology of the Cell 4th Edition Figure 743 nan 2 nf Z Mnlemlar Einlngv nfihe Cell Mh Edition Facilitated Bending of DNA to Activate Transcription DNA bending DNA pmtei n 7 a i r 7W 7 7 r m d activates a I transcriptmr Figure 7 51 Malecular Biology at the Cell 4th Edition Example of Negative Cofactor Model for N02 function N02 binds stably TBP and represses transcription Competes with TFIIA and TFIIB binding to TBP Basal machinery Enhancers DNA sequences ie cisacting that enhanceactivate levels of transcription Function is independent of distance Acts near or far from gene Function is independent of position Acts upstream within downstream of gene Function is independent of orientation Acts in forward or reverse orientation oAbility to enhance transcription of heterologous genes Some enhancers confer tissue developmental stagespecificity to gene expression Immunoglobulin gene enhancer Bcell speci c oAlbumin enhancer Liver speci c Contain binding sites for multiple sequencespeci c DNAbinding transcription factors Enhancer Promoter gene control regions RNA start polyA addition site I exons l 1 DNA 1 mtrons 400 nucleotide pairs CP 1 cn h N1 39 EKLF TATA GATA 1 GATA1 GATA1 GATA1 GATAl GATA 1 GATA l 220 30 2200 2400 Figure 7 59 Molecular Biology of the Cell 4th Edition Enhancer element factor RNA DNA TATA polymerase I o 0 p binding protein TATA initiation site The Constellgtion of Cis and Trans cting Regulatom Elements general transcriptim RNA polymerase gene gene reguiatory proteins factors regulatory proteins In ene X I reQUNBIGW spacer DNA sequence prognoter RNA transcript thegene enntr ni regime rer giene XL Figure 7 41Mniecuiar Biaiogy ofthe Celi 4th Edition Enhance 0 reginn Pmnm taraprqximalnagian TATEIL m TTR germ ii k HM N THE TBFquot 39 Q TFID39 Paill HHFE gt HHH Eue mivalmg g F1 I kIHIF TFIIE TFII L r Srmmed imm k J if 1 5 a liyl t fg m m I D m General tra nscrimim Emma nI DNA J a Enhancer I a x are DNA I39linding Pmmntm f39 NW maximal 5 5 7 Lodish et al Fig 1061 Chromatin Insulators and Boundary Elements inquot 3 quotHem I heterechrematin gene A i SUIIEITDIT39 enhancer gene B insulator element element domain ef activer transcribed chromatin Figure LE1 Mnleculer Binlegy nf the Cel 4th Editien Chromatin and Transcription Nucleosomes are inherent repress ors of transcription Transcriptional activation requires alteration of repressive chromatin structure Opening of higher order chromatin structure Moving nucleosomes Remodeling of nucleosomes Systems for chromatin remodeling nucleosome remodeling complexes Systems for histone modi cation acelylalion methylalion phosphorylation ubiquitination ADP on amp sumoylation Particle he DNA makes 17 mm around thy hlvmna comma110mm an overall pnnlcle Wlth Figure 2 Nucleosome Remodelinq Complexes SWISNF ISWl ISW2 Yeast oAll contain an ATPase subunit hSWVSNF NURD RSF A exhibit ATPdependent chromatin perturbation activity Human SwiSnf best characterized Regulates 56 ofyeast genes Both a and regulator dSWISNF NURF CHRAC ACF o O SWR1 complex involved in replacement H2AHZB dimer with th lHZB dimer in nucleo somes thH2AZ is a variant of core histone H2A Nucleosome Remodeling Occurs over critical regulatory sites Confers access of regulatory DNA sequences to DNAbinding transcription factors Facilitates formation of PIC Various chromatin remodeling complexes can A1ter DNAhistone interactions to disrupt or open nucleosomes Transfer intact hi stone octamer to DNA and or Slide nucleosomes along DNA ATP ADP 0 2 RSCEl zl umm Transler A 4 00 0 I39ilt 0000 ATP ADPOPI 1L 1L g Nucleosome Array N gmw m M Two normal nucleosome cores A are bound b SWlSNF Ind w n ym 3 Figure 7 Two Models for Nucloosorne Remodeling Figure 6 Model of SWIISNF Action species C before misuse D The raaction also works in m establlshing an equilibrium between species A and D Remodeling complexes appear to be recruited to sites of action by transcription factors Histone Modi cation Histone ace lation Occurs on lysine side chain of nterminal tails of histones Changes charge of histone tails becomes less basic May weaken histoneDNA interactions and open nucleosome May alter histonehistone interactions May alter interactions between histones and regulatory proteins May facilitate binding of regulatory proteins to cisacting elements in DNA Effects could be on individual nucleosomes andor higher order chromatin structure Makes histone tails more ahelical Catalyzed by histone acetyltransferases HAT s ReVersible by histone deacetylases HDAC s Histone methylation Does not alter net charge of histone tails Mono di trimethylation of lysine side chain Catalyzed by histone methyltransferases HMT s Methylation of histone tails thought to be stable but histone demethylase identi ed in 2005 called LSDl lysinespeci c demethylase Histone Acetvlation B C O sine NH y e 3 CHggCOA Ac HAT CoA Ac lysinesEH 8CH3 AC Histone Acetylation and Methylation H G 0 w N a l H my v gm 1 c c c c man coo HgNHCOO HaNHCOO39 HanHcoo cN lysine Lysine c N Elmlysine c Wyshe a N mysine y Hydrophobicin and basicity m Ac Mr Act Ac Ac w in Human H3 N ARTKQTARFSTGG HAPRKQLATKAAR RSAPATGGVKKPHR 4 9 14 1e 23 27 3637 lbs Ac rAc M w Mr methy1 HumanH4 AcN SGRGKGGKGLGKGGAKRHRKVLRDN E 5 8 12 16 20 quot gtacetyl Cum Opinion in can Biology Hi stone hyperacetylation usually correlated with transcriptional activation Histone methylation at lys 9 of histone H3 correlated with transcriptional silencing Histone methylation at lys 4 of histone H3 correlated with transcriptional activation transcriptional activator a a Transcription factors and repression by historic H3H4 deaceryiarion Repressors include Mad Mm interacting with Max UMEG or heterodimerized nudeor hormone receptors The repressor recognizes its DNAbinding site and imerum directly with sma The intention oi swa with RPD3HDAC maynorbs direct Proteins such as SMRT and NOoR iorm pan oi rho compiex at oatin promoters and other proteins such as SAP18 SAP30 W W math pan 01 me complex ham or sitar it interacts with a DNMmund worm Deaoetyintion would hawk L L termini red dots to bind DNA preventing access in transcription 19mm ii is L u L uu urnu alue ur repressor is no upstream of lhe transcription sum sire E E box Model for Heterochromatic Silencin b Histone Meth lation Mediated by heterochromatin protein 1 HPl 4F Step 1 Establishment of w HBKEI methylation Actlve Step 2 Recruimment of HP 0 Step 3 Propagation of heterochr nn a dn TRENDS I39I39I Serpents EPIGENETIC S The genetic info i e DNA provides the blueprint for synthesizing the molecules of life while the epigenetic info provides instructions on how where and when this genetic info should be used Nterminal ta modification state 910 14 18 23 28 unmodified gamesile39nci g acetylated ggjve exprefsiun l acetylated methylated phosphorylated phosphorylated acetvlated higherorder combinations un modified acetylated acetylated E Figure 4 35 part 2 of 2 Molecular Biology of the Cell 4th Edition gene activator protein hietone eoetyleee HAT chromatin remodeling complex 4 l l remodeled nuoieoeomee specific peem of histone eoetyletion general transcription factors and RNA polymerase holoenzyme temeoelerloxe AETIVATl N Figure 7 45 Molecular BiDIDg f of the Cell 4th Edition RNA Polymerase II Elongation Processivity is a particular problem in higher eukaryotes Dystrophin gene is gt2 Mb and requires 17h to transcribe Elongation factors Function in vitro to promote elongation by pol 11 through direct interactions with the elongation complex PTEFb positive transcription elongation factor b Cyclindependent kinase phosphorylates CTD CSB Cockayne syndrome complementation group B Yeast homologue is RAD2 6 transcriptioncoupled DNA repair DNAdependent helicase activity chromatin remodeling ELL 1 119 lysinerich in leukemia ELL chromosomal translocation in acute myeloid leukemia Suppresses transient pausing also inhibits initiation by disrupting TBPpol II interactions Regulation by premature terminationpausing of elongation cmyc cmyb ADA etc Transcription Elongation Through Nucleosomes FACT facilitates chromatin transcription a complex of SSRPl and pl40hSptl6 interacts with hi stone H2AH2B dimer Required for P0111 elongation thru nucleosomes Destabi1izes nucleosomes by displacing one H2AH2B dimer Octamers lacking one H2AH2B dimer are bound preferentially by P0111 FACT holds onto displaced H2AH2B dimer and is also required to restore chromatin structure after P0111 passes by Works with other chromatin remodeling complexes like CHDl an ATPase U Eff 3 1h H3 H4 F334 w is w I 7 m5 w f 42A 423 A114 429 SW 1 EENAP Ii gt EMPIV139 b 139 3 A 39 Belotserkovskaya amp Reinberg Curr Opin Genet amp Dev 14 139146 2004 Contact information Of ce ARB Rm 113226 Of ce hrs Tues and Thurs 12quot00 or by appointment keithru edu