A 2-kb fragment of E. coli DNA contains the complete sequence of a gene for which transcription is terminated by the rho protein. The fragment contains the complete promoter sequence as well as the terminator region of the gene. The cloned fragment is examined by band shift assay. Each lane of a single electrophoresis gel contains the 2-kb cloned fragment under the following conditions:
Lane 1: 2-kb fragment alone
Lane 2: 2-kb fragment plus the core enzyme
Lane 3: 2-kb fragment plus the RNA polymerase holoenzyme
Lane 4: 2-kb fragment plus rho protein
Explain the relative positions of bands in lanes 1 and 3.

Question

A 2-kb fragment of E. coli DNA contains the complete sequence of a gene for which transcription is terminated by the rho protein. The fragment contains the complete promoter sequence as well as the terminator region of the gene. The cloned fragment is examined by band shift assay. Each lane of a single electrophoresis gel contains the 2-kb cloned fragment under the following conditions:
Lane 1: 2-kb fragment alone
Lane 2: 2-kb fragment plus the core enzyme
Lane 3: 2-kb fragment plus the RNA polymerase holoenzyme
Lane 4: 2-kb fragment plus rho protein
Explain the relative positions of bands in lanes 1 and 3.

Lane 1: 2-kb fragment alone

Lane 2: 2-kb fragment plus the core enzyme

Lane 3: 2-kb fragment plus the RNA polymerase holoenzyme

Lane 4: 2-kb fragment plus rho protein

Explain the relative positions of bands in lanes 1 and 3.

Accepted Answer

Welcome back everyone. Let's look at our next problem in the singer sequencing procedure. And then we have two statements here. Statement A when too little D D N T P is applied, bigger fragments are not easily seen on the sequencing gel. Then statement B, so that's statement A statement B says when too much D D N T P is added, shortened fragments are not easily seen on a sequencing gel. And then our question is which of the following options is correct. Choice. A only statement A is correct. Choice B only statement B is correct. Choice C both statements A and B are correct or choice D both statements A and B are incorrect. So we need to evaluate our two statements but to do that, we should recall what's involved in the Sanger sequencing procedure. We have the creation of many copies of the DNA to fragment to be sequenced in a procedure similar to PC R. We've got a primer, we've got our template and we've got nucleotide bases in there that can be added on to form a new copy. However, the difference is we have a small amount of labeled di deoxy N T P S or D D N T P S, each one of the four kinds is labeled with a different color as we can recall. And these di deoxy N T P s lack a three prime O H group. And therefore, when they are added, it terminates the DNA segment since there's nothing for the next base to link on to. So by adding the proper amount of these D D N T P s and making enough copies, you can end up with a fragment that stops at every single base of your segment. You run them on a capillary and can watch the different colors come off the gel, reading off a sequence of the DNA segment. That's our quick ser sequencing review. So let's evaluate our statements here with this process in mind. Statement A says when too little D D N T P is applied. So what happens in that case? So we'll put a too little D D N T P. Well, when there's not enough of these sequence terminating Diy N T P s, then our chains are going to grow bigger or our DNA segments are going to grow bigger before they get finished. So we'll end up with more of the bigger fragments and fewer of the little fragments. So when we look at statement a, what does it say? It says bigger fragments are not easily seen on the sequencing gel. Well, that is not correct as we saw that is the reverse. So statement A is not correct. So we can eliminate. So I'm gonna cross to sort of an X over statement A, A red X so that I remember that's not correct. And I can eliminate choice. A only statement A is correct and I can eliminate choice C, both A and B are correct. Now, let's look at statement B and what it's telling us. Statement B describes the situation when too much D D N T P is added. Well, if there's too much there, those are going to get added more frequently. And therefore you'll see more of the smaller fragments because our segment won't be able to grow as far before being terminated by the addition of a di deoxy N T P and we'll see fewer of the larger fragments. So let's check on statement B here and it's saying that when too much D D N T P is added short and fragments are not easily seen and just like with statement A, it's exactly the opposite. So there's red X over the term statement B and I know that that, that means choice. D, both statements A and B are incorrect is my answer. And of course, choice B only statement B is correct is not my answer. So again, which of the following of our answer choices is correct. Choice. D both A and B are incorrect. See you in the next video

Verified video answer for a similar problem:

Key Concepts

Transcription and RNA Polymerase

Band Shift Assay

Rho-Dependent Termination