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BIMM 100 Lecture 14

BIMM 100 Lecture 14 - Lecture 14: BIMM 100 ­...

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Unformatted text preview: Lecture 14: BIMM 100 ­ Regula4ng gene expression: the basics and polymerases Reading: pages 269 ­281 Changing gears: moving on to gene expression •  Overview •  Bacterial principles •  The polymerases •  More than one! •  Regulatory sequences •  Finding them •  Building complexity •  Regulators •  Finding them •  Understanding their func4ons RNA polymerase acts in concert with σ (sigma) protein factors Sigma factors: help “find” the promoter and bring RNA pol closer to it… Core polymerase: α, α, β, β , ω = apoenzyme (inac4ve) The apoenzyme + σ = holoenzyme (the ac4ve form) Many other σ factors: see Table 7.1 RNA pol σ factor  ­50  ­35  ­10 +1 +20 σ Factor also separates DNA strands and “feeds” the coding strand into the ac4ve site of RNA pol Ini4a4on and repression of transcrip4on: a look at the lac operon KEY? Mul4ple regulators and mul4ple regulatory sites are important for gene control! OFF ­ repressed! ON ­ not ac4vated, but not repressed! ON ­ ac4vated! Regula4on by distant sequences: enhancers and ac4vators Low glutamine? NtrB phosphorylates NtrC Looping and s4mula4on of bound (but inac4ve) σ54 and RNApol Two components: Sensor: NtrB Response regulator: NtrC We’ve talked a lot about bacterial regula4on… •  Now ­ let’s deal with eukaryotes! –  Same general principles •  Control elements (specific protein ­binding domains) •  Specific proteins binding to these sequences control transcrip4on by repression or ac4va4on Control regions Control of Pax6 expression occurs in this manner! Transcrip4onal control regions are ogen well conserved among different species •  Human SAL1 gene enhancer 500kb downstream from the SAL1 gene is well conserved among mice, chickens, frogs, and fish! Transcrip4onal control regions are ogen well conserved among different species •  Tes4ng it’s expression? Take the conserved sequence, and add the coding region for β ­galactosidase (from E. coli). Make a plasmid, and inject it into a mouse egg. Ager being transplanted into a pseudo ­pregnant mouse, observe the progeny. •  This is a way to figure out where this regulatory region likely directs expression! Do you think the integra4on of this plasmid is homologous? Three dis4nct eukaryo4c RNA polymerases exist Experiment? Make nuclear protein extract Separate by ion ­ exchange chromatography (see page 96) Assay frac4ons for transcrip4onal ac4vity Proteins and mRNA elute at different salt concentra4ons Brief explana4on of ion ­exchange chromotography Separates proteins that differ in net charge in columns that are packed with special beads that have a + (or they can also have a – charge). Proteins that are – will bind more 4ghtly. Then, you elute with increasing concentra4ons of salt, which binds to the beads and displaces the proteins. Three dis4nct eukaryo4c RNA polymerases Pol I: nucleolar, pre ­rRNA (28S, 5.8S, and 18S) Pol II: transcribes protein ­ coding genes (mRNAs) Pol III: non ­nucleolar tRNAs, 5S rRNA, and other small RNAs Pol II: very sensi4ve to α ­amani4n: an 8 amino acid cyclic pep4de found in Amanita phalloides (aka. Death Cap) mushrooms! RNA polymerase structures are similar: conserved over evolu4on •  5 subunits are well conserved (as indicated by their matching colors) •  Eukaryo4c RNA polymerases have addi4onal subunits –  It appears that they are all required for the enzyme’s func4on Schema4c representa4on of the polymerases and their similari4es/differences •  All yeast polymerases have 5 common subunits (homologous to E. Coli) •  The largest (RNA pol II) also has a carboxyl ­ terminal domain (CTD) that is unphosphorylated un4l transcrip4on is ini4ated. The CTD is cri4cal for the func4on of Pol II! Regulatory sequences in protein ­coding genes •  TATA box: conserved sequence 25 ­35 bp upstream of the start site. Present in ac4vely transcribed genes, posi4ons RNA pol II. •  Also: –  Ini4ators: alterna4ve promoter elements ­ have a degenerate ini4ator consensus sequence –  CpG islands: CG ­rich stretch that correlates to transcrip4onal start sites . Less precise “starts,” so you have mul4ple, alterna4ve 5’ ends! Finding regulatory sequences •  Two methods: –  Dele4on mapping (for an overall defini4on of an area) –  Linker ­scanning (higher resolu4on) •  Both techniques u4lize recombinant DNA technologies! –  Subcloning to create variants –  Transfec4on into cell lines –  Assaying transcrip4onal output by some type of reported gene β ­galactosidase: GFP: Luciferase: Is there a transcrip4onal control sequence upstream of your GOI? 1.  Take upstream regions, and make successively smaller and smaller pieces (dele4on series). Can do this with PCR! 2.  Ligate into your vector that has a reporter gene 3.  Transform bacteria, make more plasmid, and isolate it. Is there a transcrip4onal control sequence upstream of your GOI? 4.  Transfect your constructs (individually!) into cells. 5.  Read out transcrip4onal ac4vity with your reporter! Is there a transcrip4onal control sequence upstream of your GOI? 1 2 Interpreta4on? 2 important control elements! Looks like construct 1 and 2 s4mulate the most expression. So, there is something in the sequence that they share! Looks like 3 and 4 also have some expression, so there is likely something in the sequence that they share as well! Check your understanding of this experiment! •  How was the dele4on series constructed and characterized? •  You are given a cloned gene in a vector •  You know the restric4on map of the vector and the insert •  How could you generate the pieces in step 1? •  How could you confirm what plasmids you isolate in step 3? How about looking more sensi4vely? •  Remember the differences between: they are CRITICAL defini4ons! –  Enhancers: transcrip4on control region over 200bp from the transcrip4onal start site –  Promoters: TATA box (or the ini4ator sequences that determine the ini4a4on site on the template strand) –  Promoter ­proximal elements: control regions within 100 ­200bp upstream of the start site Iden4fying promoter ­proximal elements by “linker scanning” mutagenesis (more sensi4ve technique) 1.  Generate a set of constructs with con4guous overlapping muta4ons (not dele4ons!) 2.  Assay their effect on a reporter gene’s expression (in the same manner that we just talked about!) 3.  Look for overlap of the regions of importance upstream of the TATA box Used to iden4fy control regions in the tkHSV upstream region! Finding regulatory sequences •  What about enhancers? –  Discovered in SV40 virus •  S4mulates transcrip4on in most mammalian genes if inserted upstream or downstream in either orienta4on! •  Seems to be a model of how most enhancers work (can basically be anywhere in the sequence!) –  Ogen they are cell ­type specific! SV40 enhancer increases mRNA produc4on 1.  Make plasmid with (and w/o) SV40 enhancer & transfect cells 2.  Isolate mRNA that was made, and probe with a labeled, complementary piece of the mRNA DNA in this case! SV40 enhancer increases mRNA produc4on DNA in this case! 3.  Treat with S1 nuclease ­ this digests dsDNA, but not the DNA/RNA hybrid (so you are only looking at gene expression!). The ~340bp nucleo4de probe is protected by the hybridiza4on to the mRNA. 4.  Run out these probes on a gel (can determine size and amount). Conclusion? The SV40 element greatly increases synthesis of β ­globin mRNA Anatomy of general eukaryo4c transcrip4onal control sequences ~50 ­200bp long ~10bp long How can we determine what binds the sequences that regulate RNA polymerase and transcrip4on? The general theme: regula4on of ini4a4on by protein ­DNA binding Gene conven4ons: Nega4ve numbers “upstream” mRNA +1 “start” site Posi4ve numbers “downstream” Biochemical and gene4c approaches have elucidated regulator proteins that specifically bind certain DNA sequences Two major techniques for detec4ng protein ­DNA binding: 1. Dnase I footprin4ng 2. Gel ­shig assays (retarda4on and EMSA) Dnase I footprin4ng 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 on DNA, it protects its diges4on Dnase I footprin4ng reveals specific binding proteins NaCl conc. Looks like it elutes between frac4on 9 and 12 •  Experiment: you are assaying whether a DNA binding protein exists in your elu4ons 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! Similar concept: electrophore4c mobility shig assays (EMSA or “gel shig”) •  Labeled DNA probe is added to protein mix (again, in this case it is frac4ons from lysate). •  Then, perform a non ­denaturing electrophoresis. •  The DNA bound to protein will migrate more slowly through the gel Strategies to find transcrip4onal regulators •  Biochemical –  Interes4ng regulatory sequence + cell/nuclear extract •  What specifically binds? –  You can verify this »  in vivo (recapitulate the regula4on) »  In vitro (footprin4ng, EMSA, transcrip4on assays performed in cell extracts) Strategies to find transcrip4onal regulators: in vivo •  Does protein x affect transcrip4on of your reporter gene? •  Remember ­ the cells you perform this assay in must be lacking protein X! Strategies to find transcrip4onal regulators: in vitro •  SP1 was purified by column chromatography. To test whether it has ac4va4on ac4vity, it was incubated with template DNA, a lysate of RNApol and transcrip4on factors, and labeled rNTPs. SP1 has no effect on adenovirus promoter DNA transcrip4on (there are no SP1 sites in that promoter), but it does increase transcrip4on from the SV40 promoter! Strategies to find transcrip4onal regulators •  Gene4c: –  Func4onal complementa4on •  You can verify this –  In vivo approaches –  In vitro approaches –  Great example? The story of GAL4 (yeast transcrip4onal ac4vator that binds to the UASGAL site. GAL4 is required for growth on galactose (this is how it was iden4fied)! The paradigm: transcrip4on factors have separable func4onal domains •  Found in a func4onal complementa 4on screen, GAL4 was suspected to bind to UASGAL. •  To test this, a reporter construct was made The paradigm: transcrip4on factors have separable func4onal domains •  Determined that one area was responsible for binding DNA and the other was responsible for the protein’s ac4vity Most eukaryo4c transcrip4onal factors are modular (DNA binding domain and an ac4va4on domain) •  Chimeric, novel factors are possible (combine bacterial and yeast factors, yeast and mammalian factors, etc…) Not just ac4vators: repressors are important, too! NOTE: no overlap of ac4va4on and repression domains! They have separate binding sites ­ i.e. a repressor doesn’t just “block” the binding of an ac4vator. A great example is WT ­1. 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 transcrip4onal repression. •  Repression domains in specific proteins bind specific DNA sites •  Also modular ...
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