A 3.5-kb segment of DNA containing the complete sequence of a mou...

Question

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay (see Research Technique 8.1), and the following gel bands are observed. Match these conditions to a specific lane of the gel. 3.5-kb fragment plus TFIIB

Accepted Answer

Hi, everyone. Welcome back. Here's our next problem. It says a DNA segment containing a gene including the promoter and terminator regions was studied by band shift assay. The following image shows the gel bands that were observed and we have a picture of a gel with five different lanes. Each one contains a single band two of which look like they're the same size. Lane one and lane four and then the other three, each band is of a different size. Mhm Then it says lane three will correspond to which of the following choice. A naked DNA B naked DNA plus R N A polymerase two choice C naked DNA plus T F two B, choice D naked DNA plus T F two B plus T F two D and choice E naked DNA plus T F two B plus T F two D plus T F two F plus R N A polymerase two. So let's remember, we're going to be trying to identify what is in lane three here. When we look at our gel, we see that uh lane three is sort of in the middle in terms of size. And when we're trying to analyze a band shift essay. We want to remember that we'll be looking at what happens to a DNA sample when proteins are added to the mix, do those proteins bind to the DNA? And we remember that the more proteins bound to the DNA, the less mobility that complex has because it's a larger molecule. And so therefore the higher the band because it can't travel as far as a smaller molecule. So higher the band, more proteins bound to the DNA. And the other key thing to remember here, we have proteins, the proteins in our samples are R N A P polymerase two and then various transcription factors as indicated by the initials T F. And when we're looking at these proteins, we need to recall that R N A polymerase two will not bind to DNA without the help of transcription factors. So if we just mix R N A polymerase two with our DNA, and there's no transcription factors present that R N A polymerase will not bind to the DNA. So let's look at our gel and try and match lane 3 to 1 of our answer choices. Well, we can go ahead and eliminate choices. A and B. They are a naked DNA DNA by itself. And then B DNA plus R N A polymerase two, as we discussed that R N A polymerase two by itself will not bind to the DNA. And therefore, those two should be about the same size since the DNA won't have any protein bound to it and choice B and these would be the smallest bands because it consists of DNA with no protein bound to it. So when we look at our gel, we see that lanes one and four have bands about the same size and they are the smallest bands. So A and B are going to correspond to lanes one and four and therefore, are not our answer choices. So now we have three choices left and three lanes left, which would be lanes 23 and five. When we look at choice E, we see it has the most proteins in it, it has R N A polymerase two along with transcription factors. So we know that that's going to be bound. So in total, there's three transcription factors plus R N A polymerase two, that's compared to choice C which has one transcription factor and choice D, which has two transcription factors. So choice E will be the largest band and therefore correspond to lane two, which is our highest band and therefore cannot be our answer. So we just have to pick between choices C and D and we're looking, we're left with lanes three and five. And we see that lane three has a larger band than lane five. So our answer choice must be choice D naked DNA plus transcription factor two B plus transcription factor two D because that's two proteins bound as opposed to choice C which has just one transcription factor, which would give a smaller band. So lane three, in this case must correspond to choice D that DNA plus the two transcription factors. See you in the next video.

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