BIMM 100 Lecture 15

BIMM 100 Lecture 15 - Lecture 15: Regula/on of gene...

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Unformatted text preview: Lecture 15: Regula/on of gene expression ­ sites and protein binders Pages 281 ­300 Cape (course and professor evalua/ons): comments from you, please! Transcrip/onal regula/on •  How to iden/fy transcrip/onal regulators (aka transcrip/on factors) –  Factor: one or more proteins having a specified ac4vity •  The regulators are modular –  dis/nct func/onal domains –  a gallery of mo/fs •  Combinatorial control •  Mul/protein complexes –  enhancers –  Pol II ini/a/on complex itself •  The mechanisms of repression and ac/va/on –  revisi/ng chroma/n! Anatomy of general eukaryo/c transcrip/onal control sequences ~50 ­200bp long ~10bp long How can we determine what binds the sequences that regulate RNA polymerase and transcrip/on? The general theme: regula/on of ini/a/on by protein ­DNA binding Gene conven/ons: Nega/ve numbers “upstream” mRNA +1 “start” site Posi/ve numbers “downstream” Remember ­ both biochemical and gene/c approaches have elucidated regulator proteins that specifically bind certain DNA sequences Two major techniques for detec/ng protein ­DNA binding: 1. Dnase I footprin/ng 2. Gel ­shi[ assays (retarda/on and EMSA) Dnase I footprin/ng reveals specific DNA ­binding proteins •  Label DNA (P32) •  Add (or don’t add) proteins •  Digest with Dnase I •  Remove protein, denature DNA •  Run on a gel and expose to film Concept? DNAse I cuts randomly. If protein is a_ached to DNA, it protects it! Dnase I footprin/ng reveals specific binding proteins NaCl conc. Looks like it elutes between frac/on 9 and 12 •  Experiment: you are assaying whether a DNA binding protein exists in your elu/ons of a certain cell type’s total proteins. You add these proteins to the DNA probe (last slide) to see if and where it binds! Your protein! Dnase I footprin/ng reveals specific binding proteins •  NaCl conc. •  •  •  •  More about the assay: controls are important! M=marker (sizes!) NE=no extract added to the DNA ­ there should be no DNA binding ac/vity! (nega/ve control) O=onput ­a protein sample that binds to the footprint ­ could just be nuclear extract that hasn’t been frac/onated, showing that the DNA binding ac/vity is present in the sample (posi/ve control) FT ­flow through ­ the solu/on that comes off the column a[er you have eluted the proteins (shows that the elu/ons contain your DNA binding protein) Another way to detect DNA ­protein binding (EMSA or “gel shi[”) •  Labeled DNA probe is added to protein mix •  Then, perform a non ­denaturing electrophoresis. ESSENTIAL! The protein and DNA interac/on would be disrupted otherwise! •  The DNA bound to protein will migrate more slowly through the gel Another way to detect DNA ­protein binding (EMSA or “gel shi[”) •  ON? Aliquot of the protein sample (so you can see where it runs in the gel (especially rela/ve to the probe) Strategies to find transcrip/onal regulators •  Biochemical candidate –  Interes/ng regulatory sequence + cell/nuclear extract •  What specifically binds? –  You can verify this »  in vivo (recapitulate the regula/on) »  In vitro (footprin/ng, EMSA, transcrip/on assays performed in cell extracts) Remember ­ these cells cannot endogenously express your candidate gene, or endogenously regulate the process you are examining! Strategies to find transcrip/onal regulators •  Gene/c: –  Func/onal complementa/on •  You can verify this –  In vivo approaches –  In vitro approaches –  Great example? The story of GAL4 (yeast transcrip/onal ac/vator that binds to the UASGAL site. GAL4 is required for growth on galactose (this is how it was iden/fied)! The paradigm: transcrip/on factors have separable func/onal domains •  Determined that one area was responsible for binding DNA and the other was responsible for the protein’s ac/vity Most eukaryo/c transcrip/onal factors are modular (DNA binding domain and an ac/va/on domain) •  Chimeric, novel factors are possible (combine bacterial and yeast factors, yeast and mammalian factors, etc…) Ac/va/on domains have structural diversity •  The experiment? GAL4DBD –  Random pieces of DNA were isolated from E. Coli, and ligated to the GAL4DBD sequence upstream of a reporter gene. This library was transformed into yeast. •  The result? 1% of ALL sequences could ac/vate the reporter –  All of these ac/ve fusion proteins were acidic in character –  O[en /mes, the rela/vely unstructured random coil regions would induce conforma/onal changes when they interacted with co ­ac/vators Cri/cal ­ this is the basis of the forma/on and func/on of large, mul/protein complexes G F P Repressors are also important! NOTE: no overlap of ac/va/on and repression domains! They have separate binding sites ­ i.e. a repressor doesn’t just “block” the binding of an ac/vator. A great example is WT ­1 (Wilms’ Tumor). It is a repressor. When mutated, it doesn’t repress EGR ­1 expression, which leads to pediatric kidney tumors! •  Same concept, but they are responsible for transcrip/onal repression. •  Repression domains in specific proteins bind specific DNA sites •  Also modular The structural mo/fs in protein that bind DNA are well ­defined •  α ­helix in the DNA binding domain of a protein will bind to the major groove in DNA •  Specific H ­bonding and van der Waals interac/ons occur •  The sugar ­ phosphate backbone and the minor groove also contribute to these interac/ons Example: bacteriophage 434 repressor protein (green is one monomer, yellow is the other). Helixes (*) bind in the grooves. The other helixes are also important for stability of the DNA recogni8on helixes! The structural mo/fs in protein that bind DNA are well ­defined •  There are other structural mo/fs that help the α helix get to the DNA in the proper orienta/on. –  They are defined by consensus aa sequences –  Examples: •  Homeodomains •  Zinc ­fingers –  Many types! •  Winged ­helix (Forkhead) •  Basic Helix ­loop ­helix (bHLH) –  O[en heterodimers Leucine zipper (GCN4) •  Leucine zippers B ­HLH –  O[en heterodimers Heterodimeriza/on offers more gene control possibili/es •  Three different factors can create 6 different combina/ons •  CRITICAL for combinatorial gene control! There are only about 2000 transcrip/on factors in the genome, and this is how they exert their control! Heterodimeriza/on offers more gene control possibili/es •  Introducing an inhibitory factor gives even more control ­ these factors can prevent binding, repressing normal ac/va/on •  Remember ­ all of these factors have specific DNA binding sequences ­ they can just pair up with different partners to control gene regula/on Example of this control? IL ­2 transcrip/on control Another level of control? IL ­2 transcrip/on only happens when NFAT is ac/vated (it gets transported from the cytoplasm into the nucleus) and AP1 subunits are synthesized. There is separate control of these two steps! •  Both monomeric NFAT and heterodimeric AP1 transcrip/on factors have low affinity for their respec/ve sites in the IL ­2 proximal promoter region •  Together, they stabilize each other, and can bind more efficiently. Another great example? β interferon enhancer •  4 control elements exist in this enhancer (70bp) –  Bound by 2 heterodimeric regulators –  Bound by 2 monomeric regulators –  HMGI actually bends the DNA •  All of these interac/ons greatly strengthen the interac/ons between the proteins themselves and the protein ­DNA complex Transcrip/on ini/a/on: RNA Pol II needs other factors! Looking at this in vitro… TATA binding protein (TBP) binds the minor groove and bends DNA •  General transcrip/on factors ­ important for transcrip/on of all mRNAs! •  Highly conserved throughout evolu/on •  This is a stepwise process ­ proteins bind sequen/ally TBP is used in these experiments because the protein complex it is a_ached to (TFIID) is too difficult to purify! Also, TFIIA is required in vivo. Transcrip/on ini/a/on: RNA Pol II needs other factors! Looking at this in vitro… TFIIH has helicase ac/vity •  Forma/on of preini/a/on complex (in vivo this is 60 ­70 different polypep/des, and is 3MDa!) Transcrip/on ini/a/on: RNA Pol II needs other factors! Looking at this in vitro… •  ATP hydrolysis is required! •  The CTD is phosphorylated, a[er transcrip/on begins. •  All of the TAFs are released (except TBP) •  Elonga/on occurs (as long as there are NTPs present)! The whole complex… Yeast RNA pol II Transcrip/onal regula/on •  How to iden/fy transcrip/onal regulators (aka transcrip/on factors) –  Factor: one or more proteins having a specified ac4vity •  The regulators are modular –  dis/nct func/onal domains –  a gallery of mo/fs •  Combinatorial control •  Mul/protein complexes –  enhancers –  Pol II ini/a/on complex itself •  The mechanisms of repression and ac/va/on –  revisi/ng chroma/n! Mechanisms of regula/on •  Remember: the in vivo template for transcrip/on is chroma/n –  Chroma/n is comprised of DNA + histones (and other proteins) •  Histones: have lysine ­rich N terminal tails that are subject to modifica/on –  –  –  –  Acetyla/on Methyla/on Phosphoryla/on Ubiqui/na/on •  These modifica/ons affect transcrip/on! –  Deacetyla/on (nega/ve affect) –  Acetyla/on (posi/ve affect) –  Methyla/on (nega/ve affect) Heterochroma/n is correlated with silenced transcrip/on •  Stains darkly with DNA dye ­ is more condensed, and physically inaccessible! HML or HMR is transcribed and then transferred to the MAT locus ­ this determines the cell’s ma/ng type Cell ma/ng type control is performed by chroma/n modifica/on in this example (the silenced gene is in a condensed chroma/n structure that sterically prevents transcrip/on factors from interac/ng with it) Proof of chroma8n involvement? 1. All genes in this area are silent, even unrelated tRNA genes transcribed by PolIII 2. E. Coli methyltransferase couldn’t methylate sites within the loci (they were inaccessible) 3. Muta/on of histone tails could de ­repress, restoring the methyltransferases accessibility Silencing of genes at yeast telomeres •  Repressor ac/vator protein 1 (RAP1) and silent informa/on regulator (SIR) proteins work together •  SIR2 is a histone deacetylase! •  SIR3 and 4 bind to the deacetylated tails to generate large, inaccessible condensed SIR1 (not shown), binds DNA regions to the HML and HMR •  This all prevents other loci to s/mulate this proteins from binding to ac/vity at genes to be start transcrip/on! silenced! Visualiza/on of these complexes by confocal microscopy Probe for telomeres An/body for SIR3 ...
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This note was uploaded on 10/12/2011 for the course BIMM 100 taught by Professor Pasquinelli during the Summer '06 term at UCSD.

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