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RNA Transcription

RNA Transcription. Pre-Initiation Complex (PIC) binds promoter PIC recruits RNA Polymerase II Pol2 transcription elongation complex (TEC) transcribes sequence 7-methyl guanosine cap Spliceosome ribozymes remove introns Polyadenylation factors recruit poly-A polymerase.

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RNA Transcription

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  1. RNA Transcription • Pre-Initiation Complex (PIC) binds promoter • PIC recruits RNA Polymerase II • Pol2 transcription elongation complex (TEC) transcribes sequence • 7-methyl guanosine cap • Spliceosome ribozymes remove introns • Polyadenylation factors recruit poly-A polymerase

  2. Transcription regulation • Initiation • Chromatin structure • Promoter elements • Enhancer elements • Elongation • Stability • miRNA

  3. Conformational control of transcription • Transcription initiation depends on • DNA accessibility • Affinity for Pol2 • DNA structure • Supercoiling • Kinks • Nucleosomes • Matrix association Transcription Bubble Nucleosome Core Particle Transcription Elongation Complex Promoter MAR Enhancer Core Intron Exon Intron Exon Intron MAR -4000 -500 -40-+50 10000+

  4. Chromatin remodeling • Nucleosome remodeling • SWI/SNF , ISWI, CHD • Displace nucleosome relative to DNA • Displace histone proteins • Steric regulation of transcription factor binding • Histone modifications • Methylation • Acetylation

  5. Histone code • Histone tails have many lysines • Terminal amine H-bonds with PO4 backbone • Subject to multiple methylation/acetylation • Interactions between nucleosomes • Interaction btw histone & DNA • Histone acetyltransferase (HAT) • p300/CBP,P/CAF • Transcribed genes • Histone deacetylase (HDAC) • Sin3, NuRD • Silenced genes

  6. Histone structure • Amino terminal of H3/H4 link adjacent nucleosomes • Acetylation (-CO-CH3) blocks this association • HATs activate genes • HDACs repress • Epigenetic inheritance

  7. Gene specific transcription • Promoter proximal region • Reporter construct • Canonical response elements • Remote enhancer/insulator elements • Clusters of regulatory elements • Chromatin loops of 50+kb Promoter MAR Enhancer Core Exon Intron Exon Intron Exon MAR -4000 -500 -40-+50 10000+

  8. Gene specific transcription factors • TFs may be as much as 6% of the genome • Specific regulation requires teamwork • Combinatorial control • Synergistic, greater than additive interaction • Common features • Basic, DNA binding domain • Phosphate binding • Dimer/oligomer-ization • Options • Major groove • Over the top

  9. Characteristics of Gene-specific TFs Transcriptional activators ConstitutiveSp1CCATNF1 Conditional DevelopmentalGATAHNFPit1MyoDBicoidHox Signal Dependent Steroid ReceptorGR ER PR TR Surface Receptor Internal SignalsSREBPp53ChoREB NFkB Nuclear ResidentETS CREB SRF FOS-JUN Latent Cytoplasmic SMAD STAT NFAT Brivanlou & Darnell 2002

  10. DNA binding • Charged AAs • Positive (blue N) groups (lys, arg, his) • Negative (red O) groups (asp glu) • O+N (asn, gln) • Protein a-helix 3-4 NTPs c-jun jun side-chains compliment nucleotide polarity to give sequence recognition

  11. Jun binding Jun (leucine zipper): 1 mtakmettfy ddaLnasfLp sesgpygysn pkiLkqsmtL nLadpvgsLk phLraknsdL 61 LtspdvgLLk LaspeLerLi iqssnghitt tptptqflcp knvtdeqegf aegfvralae 121 lhsqntlpsv tsaaqpvnga gmvapavasv aggsgsggfs aslhseppvy anlsnfnpga 181 lssgggapsy gaaglafpaq pqqqqqpphh lpqqmpvqhp rlqalkeepq tvpempgetp 241 plspidmesq erikaerkrm rnriaaskcrkrkleriarl eekvktlkaq nselastanm 301 lreqvaqlkq kvmnhvnsgc qlmltqqlqt f Jun DNA binding: kaerkrm rnriaask DNA Consensus: TCA Dimer palindrome: TGA X tca Other side chains interact with DNA PO4 backbone

  12. TF Mechanisms • PIC interaction • Mediator interaction • Target histone modifiers CREB Activation domain DNA binding GMSA shows increase in PIC assembly with CREB activation domain (CRG) but not CREB DNA binding domain alone (DBD) Felinski et al., 2001

  13. MyoD • “Master control switch” for myogenesis • Associates with muscle specific promoters • Recruits P/CAF HAT • Trigger differentiation • Recruits HDAC1 • Represses myogenesis Growth Differentiation Dual specificity chromatin-IP shows MyoD complexed with HDAC1 during growth and P/CAF during differentiation

  14. Cooperativity • Most promoters contain dozens of TF domains • Most TFs capable of binding dozens of cofactors • Hydrophobic protein-protein interaction • Combinatorial/network control of transcription OtherTF HATs TFII Protein-protein binding map for MyoD Other TF

  15. Kim & Maniatis (1997) Enhanceosome • Combination of GTFs & GSEs • Family of xscription factors produce specific molecular surface • Highly nonlinear • Interferon-beta • ATF+jun • IRF-1 • p50+p65 • HMG-1 • Only effective as combined group

  16. Mediator • Helps recruit Pol2 to PIC • GTF cofactor • Integrates GTF & gene specific TFs • CTD kinase • Many subunits (30+) • Head • Middle • Tail Chadick & Austurias, 2005

  17. Mediator • GTF interactions • TBP • TFIIH • Cdk activity targets Pol2 CTD • CDK8/CDK11 + cyclin D • Gene specific activation/repression • Gene specific TFs may act through mediator to recruit/activate pol2 and through HDAC/HATs to recruit GTFs

  18. Regulation of elongation • TEC velocity • Pause/arrest • Negative Elongation Factor (NELF) • Binds DNA & blocks PolII progress • NELF-B = Cofactor of BRCA1 (COBRA1) • Reversible Block • Binds PolII/DSIF complex • Released by P-TEFb phosphorylation of PolII • NELF-P-TEFb competition

  19. P-TEFb releases pause • Cyclin dependent kinase • Cdk9 • Cyclin T/K • Phosphorylates DSIF • Phosphorylates CTD • Stimulates elongation • Blocks NELF binding Pol II DSIF Transcription Blocked NELF P-TEFb PO4 Transcription Allowed Yamaguchi et al., 1999 Cell 97:41

  20. Chromatin remodeling Set1/PAF methylate histone, loosen structure Chd1 displaces H2A/B, allowing transcription Competition displaces Set1, downstream histones less methylated Turner 2003

  21. miRNA • Short, noncoding RNA • Drosha forms hairpins • Dicer form short dsRNA • Steric translation block • Slicer dsRNA degradation Drosha Slicer Dicer Mature slicer (Ago) with ssRNA

  22. Regulatory Circuits • Cascade • Feedback/Regulatory Inhibition • Combinatorial activation External signal External signal One or more external signals allow activation External signal Product inhibits expression of promoter

  23. Modulation of TF activity • Many TFs are constitutively expressed • Secondary control mechanisms • Phosphorylation (phos-myoD binds HDAC) • Ligand/dimer binding (steroid) • Inhibitor dissociation (NF-kb) • Subcellular localization (MEF2 dephos) • Some TF form cascades • “IEG”s • Delayed Early Genes • Cyclins/cdks

  24. Example: c-Jun • Activator Protein-1 component • Phosphorylation by JNK/ERK • Ser 63, S73 (protein binding domain) • Required for activity • Phosphorylation by GSK • Thr231, Thr239, Ser243, Ser249 (DNA binding domain) • Prevents DNA binding • Association with JNK • Facilitates ubiquitination • Binds TATA-binding protein-Associated Factor-1 • Cell cycle regulation • CyclinD/cdk4 transcription after mitogens • Repression by GSK during starvation

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