The electrophoresis gel shown in part (a) is from a DNase I footp...

Question

The electrophoresis gel shown in part (a) is from a DNase I footprint analysis of an operon transcription control region. DNA sequence analysis of a 35-bp region is shown in part (b). The control region, labeled with ³²P at one end, is shown in a map in part (c). Separate samples of control-region DNA are exposed to DNase I, and the resulting DNase I–digested DNA is run in separate lanes of the electrophoresis gel. Unprotected DNA is in lane 1, DNA protected by repressor protein is in lane 2, and RNA polymerase–protected DNA is in lane 3. The numbers along the electrophoresis gel correspond to the 35-bp sequence labeled on the map in part (c). Use the information provided to solve the following problems.

Locate the regions of the sequence protected by repressor protein and by RNA polymerase.

Accepted Answer

Welcome back, everybody. Here's our next question. DNA S one ft printing is a method used to identify protein binding sites on a DNA sequence. It involves treating the DNA with DNA S one enzyme to cleave it at random sites and then analyzing the resulting banding pattern by gel electrophoresis to identify the regions of DNA that were protected by the protein in a DNA S one ft printing analysis. What is the difference between the banding pattern of protected DNA and that of unprotected DNA choice? A the banding pattern of protected DNA will be a smear while the banding pattern of unprotected DNA will have sharp bands choice B, the banding pattern of unprotected DNA will be a smear while the banding pattern of protected DNA will have sharp bands choice C both protected and unprotected DNA will show a smear of bands choice d both protected and unprotected DNA will have sharp bans. Well, to think about these banding patterns. And again, when we look at our answer choices, we just have a choice between a smear or sharp bands and whether there's a difference between protected and unprotected. Well, if we run a test and in a hurry, you could probably eliminate right away choices C and D which describe both unprotected and protected DNA as being the same choice. C having them being a smear and choice D being sharp. Just because logically what would be the purpose of doing this technique if we can't distinguish between protected and unprotected DNA? When we see that, that's part of the whole purpose of this is to identify protein binding sites and analyzing the resulting banding pattern. There's no difference in the banding pattern. There's no use in analyzing it. So just by pure logic, if we were in a hurry on a test, we'd cross those out. So we just have to have to determine which scenario will show a smear versus sharp bands. Well, to think about this, we know we are taking our DNA sequence and treating it with DNA S one enzyme. Well, this is a restriction enzyme. So it cleaves at specific sites. But it's important to note that there are many of these DNA S one sites in a given DNA strand. So there's a whole lot of places where this enzyme is going to cut. And when you do these types of essays, you use a low concentration of the enzyme. So that on average each DNA molecule present is cut only once. So even though there are many different sites where it can cut, if you use a low enough concentration, just on average, you'll just cut each molecule once? Well, what will that end up doing when you run these fragments on a gel? So you've cut it at many different places. But each one only once, well, you will end up with, we'll say, therefore, a gel will show Asmir because you're going to have so many bands that differ by just, you know, a little bit and they'll just run out just really close to each other in a big smear. However, when we look at our DNA mixed with a protein that binds to different sites on the DNA. Everywhere there's a binding site. So everywhere protein binds, we know that the DNA S one can't cut as a result that will end up giving us sharp bands. Because instead of this continuous bunch of fragments differing by very little, we're going to have whole regions where we have no cuts at all. So fragments, gaps in our fragment pattern. So that will lead us to our answer. Choice. That choice B the banding pattern of unpredicted DNA will be a smear while the banding pattern of protected DNA will have sharp bands again because you'll have whole regions where there are no cuts at all. So choice B is our correct answer. And then we can eliminate choice A because it's the opposite from what it should be saying, the banding pattern of protected will the protected DNA will be a smear while unprotected DNA will have sharp bands. But we've explained why that's not the case. So again, our answer here will be choice B see you in the next video.

Key Concepts

DNase I Footprint Analysis

Electrophoresis

Transcription Regulation

Watch next