LEC18 - 11/10/2008 Announcements Regraded exams can be...

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Unformatted text preview: 11/10/2008 Announcements Regraded exams can be picked up in the core office ffi LS3 labs New policy: drop lowest lab score Do not need to make up lab if miss one If you have a midterm conflict with lab this y week, you can drop this weeks lab Monday office hours postponed until Friday 1 pm Epitope tags A short peptide that serves as an antigenic determinant (where antibody binds) can be used to tag a protein Tag can allow simple purification of the protein out of bacteria or organism being studied Tag can allow one to visualize protein in vivo using antibodies 1 11/10/2008 Commonly used epitopes Myc (E (EQKLISEEDL)n EED ) FLAG (DYKDDDDK)n HA (YPYDVPDYA)n Commercially available antibodies Epitope used to purify protein myc9 promoter p Protein X Protein A is a Staphylococcus protein that binds IgG 2 11/10/2008 Another use of epitope p p tags is to visualize location of protein in cell or organism Fluor 3 11/10/2008 Actin: red Microtubules: green Mi t b l Nuclei: blue Tagged proteins can also be detected by western blots Many techniques have been developed b d l d based on the d h following: Separate molecules by size using gel electrophoresis Transfer to membrane f Detect specific molecules with labeled probe 1 2 3 4 4 11/10/2008 Southern blot Named after Edward Southern Separate DNA on agarose gel p g g Transfer to membrane Probe with labeled DNA fragment Edward Southern northern blot Separate RNA on agarose gel Transfer to membrane Probe with labeled DNA fragment western blot Separate protein on polyacrylamide gel Probe with labeled antibody 5 11/10/2008 Southern Blot: - Capillary action - No electropherosis for DNA Northern Blot: - Capillary action Western Blot: - Electropherosis - Protein is in a higher acrylamide gel so can't transfer it well have to use electropherosis to transfer the proteins. SOUTHERN BLOT Nitrocellulose Filter (Membrane): - Very flexible - Set it on top of gel WESTERN BLOT Antibody binds to one particular band Will get a product: -Either a color product (ex: some use a phosphatase that will give some kind of blue band on the gel) -Light Product (Ex: Chemiluminescenc e Substrate. The Enzyme, horse radish peroxidase. Add substrate and some hydrogen Many primary antibodies available commercially already have a conjugated enzyme, s n m so secondary antibody is not necessary Secondary Antibody bond to primary antibody, they don't recognize the Epitope. They recognize the primary Antibody. 6 11/10/2008 -Put an X-Ray film on it, leaves dark mark because where ever light was you converted it to silver molecule. - Out of that mixture we are able to pick out one protein and measure the amount of protein using this Western Blot. If look for labeled DNA and if its binding to protein going to be much bigger, which means that it won't migrate through a gel as far. So it's just a shift of a labeled DNA on a gel b/c it's being bound to protein. Light is detected using X-ray film 1 2 3 4 Horse Radish Peroxidase (enzyme) - Secondary antibody is linked to this enzyme (it is commercially available). - Enzyme converts the substrate to light. All of these 3 Techniques are important for the Process of Transcription. They help to find proteins that Bind specifically to DNA, specifically to promoter regions, identified Repressors and activators... Analysis of DNA-protein interactions 3 Different Techniques Electrophoretic mobility shift assay (EMSA or gel shifts) DNA footprinting Detect protein-DNA complexes by change in migration on gel (in vitro) Protein protects region of DNA from digestion with deoxyribonuclease I (in vitro or in vivo) Immunoprecipitation of protein-DNA complex from living cell This is NOT DNA fingerprinting. DNA fingerprinting is what you use in DNA analysis and crime scenes. This is an Endonuclease, so it will cut all around the DNA, internal Not from the external sites. - Allows to find where in a DNA a Protein is binding. Chromatin Immunoprecipitations - Most common technique - When we use Antibodies to precipitate protein-DNA complexes from living cells. 7 1. What ever sequence you look at you want a very short double stranded DNA. Want to label the 2 5' ends with P32 3. The Control. EMSA/Gel shift Illustration: 3 Diff reactions shown here. 11/10/2008 2. Mixed the protein that binds to this sequence of DNA No SDS, since non-denaturing, don't add SDS because it is migrating as a complex so you don't want to denature your protein or it won't bind to the DNA 3. This complex is even larger, so this proves that the complex from the second lane if it gets shifted to a higher complex, then this shift was due to a protein in a probe b/c Now with antibody it gets even bigger. 2. Bigger, so DNA bound to protein migrates down slower on the gel. This is a Gel Shift, Shifted to a higher complex. 1. Radioactive Oligonucleotide Probe, since they are small they run far down on gel. 1 & 3 are controls, and the one in the Middle #2 is our specific Binding. 2 Proteins: Short Example that Dr. Johnson No Protein name 2 & 9 did in Lab: -Showing that the protein binds to methylated DNA. - Also showing how DNA: unmeth mCG mCHG mCHH different proteins bind different sequences or diff Protein: - 2 9 2 9 2 9 2 9 P t i methylated sequences. Protein-It's a way of studying in DNA the test tube, the binding of complex proteins to DNA. Labeled probe 8 Sort of Similar but only want ot have 11/10/2008 one end of DNA labeled. Don't want 2 ends labeled because running this on a denaturing gel, therefore, want everything measured form the same end. 1. Negative control 2. With Protein DNase 1- kind of base pair specific. - more of an average of about 10 BP's because that is when DNA comes fully around again, so it sort of cuts everything along the same side. With protein bound it protects the DNA from cleavage of DNAse 1. -DNase 1 can cleave before or after but it wont cleave where the protein is bound. Now when running this on a gel, won't see fragments that are result of where that protein was bound, so it's a protection, which is said to be the footprint since it's leaving a footprint on the gel. Real Example: E2 protein, DNA binding DOmain. Negative control: -With out protein, just Dnase 1 Huge protection here, so indicating that the E2 protein is binding to those sequences. 9 11/10/2008 -Basically saying in a living cell, what DNA is the Protein interacting with? Chromatin Immunoprecipitations Use antibodies to isolate proteins bound to t DNA in a cell : Adding proteins to DNA in i ll Determine if specific region of DNA is p precipitated by PCR amplification p y p a Living Cell Antibodies can be to specific DNA binding People have made antibodies that will proteins, or even histone modifications recognize that modification: -Methylated -Phosphorylated -Acetylated (it will only react with acetylated but not unmethylated) Chromatin Immunoprecipitations looks like this: - Crosslink the proteins to the DNA. It Fixes everything,so the proteins don't come off the DNA or the Histones don't come off . - Then break open the Cell. Sonicate it, shearing it into little fragments Incubate Antibody with this Chromatin Basically you know what protein you pulled down because you used and antibody to it but what you want to find out is, where was the protein binding? What DNA Did it bring down? So the next step is to reverse your crosslinking because if there is crosslinking here you can't PCR through the region. 10 11/10/2008 Wanted to see if of Ta3 did she bring down compared to actin? Wildtype Neg control: w/o antibody no precip This is what started with so it's not immunoprecipitate d. Selective enrichment of a specific region (Ta3) compared to the control (actin) she did an Immunoprecipitation, how much Regular house-keeping type of gene that is always expressed highly. Transcription in eukaryotes This Transcriptional Process is much more Complicated than that in Prokaryotes! 11 Allowed one to study promoters and what it took to get transcription going in vitro. Allowed us to study where proteins were binding to the DNA, what sequence. So this identified very many sequence specific DNA binding proteins. Ones that would only bind to some sequences and not others. Allowed us to purify proteins from Eukaryotes very easily Allows one to measure and look at where genes are expressed in diff regions. 11/10/2008 1980's: focus on DNA-protein interactions in vitro Affinity chromatography Reporter gene assays In vitro transcription assays Gel shift assays DNA footprinting TBP-DNA These studies identified many sequence-specific DNA binding bi di proteins t i 1990s: it became clear that in addition to DNA-protein interactions transcription also involved: protein-protein interactions p p Chromatin structure Cellular compartmentalization : That the cell was not just bag of Proteins. 12 11/10/2008 Overview of Eukaryotic Transcription How processed RNA is in Eukaryotes. -No processing in Prokaryotes -In prokaryotes Transcription was coupled to translation, so you were translating before you did anything to the RNA -This is NOT the case in Eukaryotes M h more complex than prokaryotic Much l h k i transcription Primary transcript is processed extensively to yield mRNA mRNA is transported out of nucleus to : So they are not coupled. cytoplasm for translation They are separate & Regulated Each of these steps can be regulated Separation of transcription and translation by nuclear membrane Vital Difference: In Eukaryotes: - Every RNA encodes a Protein so there are no polycystronic operons. - We don't have polysystronic messages, we have multiple ORF's - We don't have operons, every gene is controlled separately. Modifies the 5' end 3' end and removes all of the introns. SO we get a single RNA that encodes one Open reading Frame (ORF). RNA gets transported out of the nucleus and is translated in cytolasm. 13 11/10/2008 Prokaryotes Don't have Nucleosomal DNA which inhibits In vitro, RNA polymerase and the general Transcription. transcription f t t i ti factors are sufficient t ffi i t to : in a Test Tube. So give it a DNA molecul initiate transcription Polymerase & transcription factors=initiatio Chromatin inhibits transcription in eukaryotes have Nucleosomes, but all Eukaryotes In vivo, additional proteins are necessary to deal with the inhibitory nature of nucleosomes M di t complex:Mediates activation and repression Mediator l DNA-binding regulatory proteins chromatin remodeling and modifying proteins Remodeling Enzymes: - Either slide nucleosomes out of the way or change the DNA wrapped on nucleosomes so it's looser. - These are critical for the initiation process Modifying Enzymes: - These modify the N-termini of Histones, which alters their ability to bind other proteins or to condense the DNA. Eukaryotic RNA polymerases (also plant specific RNAP IV) : 1 Polymerase :3 Polymerases rRNA mRNA tRNA 14 ...
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