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Lecture 4

BIO240 Lecture 4.doc

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University of Toronto St. George
Jennifer Harris

Lecture 4: Transcriptional Regulation 2 - Last lecture: taken a look at the interplay b/w DNA & its organization & its transcription to RNA & the regulation of those processes. When we did that, started to talk about the components of genetic switches & talked about the role of sequence specific transcription factors/gene regulatory proteins & their interaction with short segments or motifs of DNA. Took a look how one identified the motifs that those gene regulatory proteins are going to bind to – did that through 3 different kinds of experiments: loss of function experiment demonstrating necessity & a gain of function experiment demonstrating sufficiency of a particular segment of DNA to support regulation of transcription. - The loss of function experiment: deleting portions of the gene regulatory sequences (promoter) – call this upstream or 5’ regulatory region – did promoter deletion analysis, removing chunks of it & asking which chunks are necessary for the regulation of the corresponding gene & in this case a reporter gene that is reporting on the activity of these endogenous gene – has been artificially constructed & inserted into the genome & asked when it turns on/off & identified motif/sequence segment that is necessary to drive expression in a particular tissue, at a particular point in time. - Imagine that that promoter is actually chalk-block with different motifs, one that is important for, for example, driving expression in guard cells, another chunk of that promoter may be important for driving expression, for example, xylem cells. Identify therefore this necessary motif & asked is it also sufficient to drive expression from a minimal promoter (by analogy: thought of like being a car that is missing its keys – got everything you need for car to drive somewhere but without that key, it’s not going anywhere) – those sequence specific motifs/necessary sequence that we put in front of the minimal promoter turns out to be the useful car to drive the expression in guard cells, for example – shows that that specific chunk/motif is sufficient for promoter activity – driving expression at a particular place at a particular time. - Then we talked about sort of motifs that one might identify through such a process – these less than 20 in length nucleotides motifs where we can have slight sequence variation in them & nucleotide changing here or there but overall, they are minimally going to be 3 invariant nucleotides. - How do we know that specific proteins interact with these DNA motifs that we identified in the experiments described previously (gain function/loss experiments)? - Way we can look at the interplay b/w a protein & a DNA segment is done very nicely in vitro (outside of the cell) using electrophoresis. - Have indentified target sequence, now we want to ask can a specific protein interact with those sequences. - Just imagine that we’ve already identified a protein that can interact with this target motif. - Going to run motif through electrophoresis gram using PAGE gel electrophoresis – run it from the cathode to the anode – as DNA is negatively charged, it’s going to migrate down the length of the gel & we set the whole thing up so this fragment is going to migrate right down to the bottom. - At the bottom, have the unbound (free-running) motif called the target. - Way in which we’re going to eventually visualize where the migration has occurred in the gel is by making use of radioactivity – the target DNA has been labeled with radioisotope in one of the nucleotide bases that has been added to the double stranded probe target. At the end of the process, if we take our gel & expose x-ray film to it & develop x-ray film, we will see particular bands that correspond to the location of the probe. - We’ve hypothesized that a particular protein can interact with this motif – how can we test that the interaction actually occurs? We either purify the protein through some purification process from the cells in which it’s normally found or we make it recombinantally & purify it from E. coli. In the end you’ve got one protein & you want to ask if it can bind to this DNA motif. - Note what happens is that in addition to the free target running down to the bottom of this particular lane, we have another band that we see on the x-ray film that is higher up the gel, indicating that something has retarded/impeded the progress of the probe of the target through the gel. What could have possibly done that? Well of course it’s got to be the protein impeding the progress – made it for much larger MW complex & through non-denaturing polyacrylamide gel electrophoresis, it’s going to migrate at a slower rate & create another band – that’s our protein bound to the target DNA. - Nice simple method to test the hypothesis that a particular protein acts with a specific DNA motif. - Example of how one actually visualizes this method at the end of looking at the x-ray film. This then is what is the output of such an experiment where what we have on the lane on the far left hand side is the labeled target alone – can see that it runs right down to the bottom. Then what we have is our labeled target (our DNA sequence motif) + recombinant protein that’s in the next lane & what we observe is a shifted or retarded band in the gel indicative then of binding of the motif by the protein. - Now what we do to show the strength of the interaction is to add increasing quantities of unlabelled competitor & as one adds increasing quantities of unlabelled motif, it should outcompete the labeled motif & that we will see is a decrease in the amount of radioactive in the band as we go across. Increase concentration of unlabelled target DNA that functions as a competitor & again, instilled with the protein present – note how the amount of radioactivity in that band decreases – that’s b/c we’ve just displaced the radioactively labeled with non radioactively labeled competitor – this shift is still there – the protein is still binding to the DNA but b/c we’ve added cold unlabelled non-radioactive competitor, it’s bound to that preferentially over the radio-labeled & therefore when we visualize using x-ray film, the band diminishes. - By taking a look at how much the concentration of the unlabelled target is required to get rid of the band, we have a measure of the affinity b/w the protein & its target. - This method has tested the hypothesis that a specific protein has bound to the DNA target motif. What if we don’t know anything about what is important to bind to this sequence that we’ve identified as being necessary & sufficient. What if we’re just interested in identifying the proteins that can bind to it in a mixture of all proteins that are in a given cell type? - Have again our target sequence that has been radioactively labeled & it’s going to run to the bottom of the gel but in contrast to what was shown previously, now what we have is a mixture of proteins – some of them are going to bind to the motif, others will not, there may be multiple proteins that can bind to the motif & in the example provided, that is precisely what occurs – multiple proteins can bind to this DNA sequence region. - B/c the proteins themselves all vary in either MW or iso-electric points, they’re going to migrate differentially in the gel so as opposed to seeing just one shifted band (one shift corresponding to one protein), we’ll see multiple that correspond to the different proteins binding. - There’s the unlabelled probe at the bottom & when you run it together with the mixture, you end up with 4 bands – the unbound that is down at the bottom, then the 3 shifted bands representing the sequence being bound by the different proteins. - What if the red & green bound simultaneously, would I end up with a shift that is higher or lower than that? Higher up the gel – that can occur. - Now we’re interested in identifying what those proteins are. - Start with a column that has a matrix (very small gelatinous beads) with beads that have DNA bound to them of many different kinds of sequences. Now we flow through that column/matrix total cellular proteins. The only things that are going to bind to the column are going to be those that are able to bind to DNA & so at low salt what will happen is that those proteins that have very poor affinity for DNA will flow right through the column & those that are able to bind will remain bound. Then add medium salt wash (increase salt concentration) & that will displace through ionic competition the interaction b/w the proteins that had specifically bound & the DNA that is on the column – wash all the DNA-binding proteins. - Recall we’re after a protein that binds to a specific motif so now we take our general DNA binding proteins & we ask which ones bind to the motif that we’re interested in so we run them through a column using the same method again but in this instance we’ve replaced the generic sequences with our specific sequence. Now bound to the matrix we have this specific sequence GGGCCC & its complementary strand. Then we take our DNA binding proteins, allow them to flow through the column & now we start with the medium salt wash & anything that just binds DNA generically will come off with the medium salt wash. - In order to get the proteins that specifically bind, they’ll remain bound to the column/DNA sequences there. Now we need to use a high salt wash & that will remove the proteins that are specifically bound to the specific motif. - Now what we can do is go forward & characterize those particular proteins & their interactions with their DNA binding partners. **Don’t need to know anything besides the specifics in this lecture** - Binding site selection assay is a specialized electrophoretic mobility shift assay conducted in multiple steps. So far we’ve identified a protein that we know can bind to a motif but we don’t know if that’s the motif that it preferentially binds to. - In this instance we take a pool of all possible radioactive oligonucleotides & we mix it & conduct an electrophoretic mobility shift assay that’s going to run right down to the
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